Ent. mundtii CRL35 was incubated as individual and co-culture with E. coli O157:H7 NTCC12900, as described before, in 100 ml of the sarcoplasmic model system to achieve sufficient amount of cells for proteomic analysis. Cells from co-cultures were harvested at 6 h (T6) and 30 h (T30). Cells from single cultures of Ent. mundtii were also harvested at 6 h (T6) and 30 h (T30). As mentioned before, cells from E. coli growing alone were collected at 6 h (T6) and 96 h (T96). Cells from different cultures were harvested by centrifugation at 8,000 × g for 10 min at 20◦C and twice washed with 40 ml 0.1M Tris-HCl buffer, pH 7.5 and centrifuged at 8,000 × g for 10 min at 20◦C. The resulting pellets were stored at −20◦C until lysis for protein extraction. Three independent biological replicates were performed for each condition.

### Preparation of Cell Free Protein Extracts for Proteomic Analyses

The cell pellets from the co-cultures were mixed with glass beads (150 ± 212µm diameter, Sigma-Aldrich Co., St. Louis, MO, USA) and further re-suspended in 0.1 M Tris-HCl buffer, pH 7.5 in a 1:2:1 (cell:buffer:bead) ratio. Then, cells were disrupted using a Mini-BeadBeater-8 cell disrupter (Biospec Products Inc., Bartlesville, OK, USA) at maximum speed for 10 min (10 cycles of 1 min each, with 1-min intervals on ice among cycles). To remove cell debris, unbroken cells and glass beads, samples were centrifuged (14,500 × g, 5 min, 15◦C). The supernatant constituted the cell free extract. The protein concentration of this extract was estimated according to Bradford assay using bovine serum albumin as a standard. The whole process is described in **Figure 1**. Aliquots of 600µg of protein were finally stored at −80◦C, until further analysis.

Ent. mundtii and E. coli cell pellets from individual cultures at T6 were mixed before lysis and the same was performed for T30/T96 (see schematics in **Figure 1**). This procedure was carried out to standardize and avoid differences in cell lysis efficiency between mono and co-cultures, as well as problems with differences in protein enrichment of each microorganism in 2DE gels, that could affect proteome comparisons. The cell proportions of each microorganism used for T6 and T30

mixtures coming from single cultures were established according to the cell counts obtained in the respective co-culture in order to have in the mix a similar ratio between the two microorganisms. This way, assuring to have 600µg of proteins placed in the IPG strip, with the same protein proportion of each microorganism than in IPG strips with samples from the co-culture. Each of these synthetic mixtures (SM) will constitute the respective controls (single culture) at T6 and T30 respectively. The SM were mixed with glass beads and subjected to lysis as previously described for cell pellets originating from co-cultures (see **Figure 1** for details).

### Two-Dimensional Gel Electrophoresis

Sample preparation and 2DE gels were carried out according to Bustos et al. (2015). Isoelectrofocusing (IEF) was performed in IPGphor (GE Healthcare, Uppsala, Sweden) at 53,500 Vh, using the immobilized pH gradient (IPG) strips (Immobiline DryStrip Gels, linear pH 4–7, 18 cm, GE Healthcare; Uppsala, Sweden). For the second dimension, IEF strips were equilibrated at room temperature in 6 M urea, 2% (w/v) SDS, 30% (w/v) glycerol, 50 mMTris-HCl, pH 8.0, containing alternatively 50 mM DTT (15 min) and then 400 mM iodoacetamide (15 min in the dark). Second dimension was performed on homogeneous 12.5% (w/v) polyacrylamide gels at the constant current of 15 mA/gel at 15◦C (∼16 h) using an Ettan DALTsix Large Vertical System (GE Healthcare, Uppsala, Sweden). Gels were stained with colloidal Coomassie blue Stain according to Candiano et al. (2004), distained with distilled water. The 2DE maps were digitalized using Image Scanner III LabScan 6.0 (GE Healthcare, Uppsala, Sweden).

### Image Acquisition and Data Analysis

Volume spot quantization and normalization were performed on digitalized gel images (600 dpi) using the software Prodigy SameSpots version 1.0.3400.25570 (Totallab, Newcastle, UK). The volume of each spot was calculated and normalized by referring the values to the sum of total spot volumes within each gel. Student test for unpaired samples was applied. A protein was considered differentially abundant if the mean normalized spot volume varied at least 1.5-fold between compared spots. The effect was confirmed by analysis of variance at a significance level of p < 0.05. Protein spots showing significant variation between studied conditions were manually excised from the gels using a scalpel blade and identified using Mass Spectrometry.

### Mass Spectrometry Protein Identification

Selected spots were excised from the corresponding gel, digested with trypsin and, submitted to tryptic digestion and then to mass spectrometry analyses as previously described (Nally et al., 2017). Tryptic peptides were subsequently ionized using α-cyano-4-hydroxycinnamic acid as matrix. Mass spectrometric analysis of the peptide solutions was carried out on a MALDI-TOF/TOF tandem mass spectrometer ABI 4700 proteomics analyzer (Applied Biosystems, Foster City, USA) according to Grosu-Tudor et al. (2016) or on a MALDI 5800 (Sciex, Foster City, USA) and performed at CEQUIBIEM (Facultad de Ciencias Exactas y Naturales, UBA, Buenos Aires, Argentina) and at LIST - Luxembourg Institute of Science and Technology "Environmental Research and Innovation" (ERIN), respectively. MASCOT search engine (Matrix Science Inc., Boston, MA; http://www.matrixscience.com/search\_form\_ select.html) was used to identify proteins from peptide mass fingerprint data based on the annotated genome of Ent. mundtii CRL35 (https://www.ncbi.nlm.nih.gov/nuccore/JDFT00000000). All proteins were identified using BLASTp in NCBI database (Altschul et al., 1990).

### Functional Analysis and Interaction of Proteins

The functional study of identified proteins and their classification into functional categories were performed using the databases Universal Protein Resource (UniProt) (UniProt Consortium, 2015) and COGNITOR to identify the Clusters of Orthologous Groups of proteins (COGs) (Galperin et al., 2015).

To explore the interactions between the proteins that have shown differential expression, we conducted an insilico analysis using the publicly available STRING version 10.05 (database Search Tool for the Retrieval of Interacting Genes/Proteins) (Szklarczyk et al., 2015). For the differentially over expressed proteins, the number of protein-protein interactions documented in the database were determined. For visualization purposes, a graph was constructed linking proteins represented by nodes with known interactions with the identified proteins. All available prediction methods on STRING were used and 0.4 was select as confidence level (Szklarczyk et al., 2015).

### Statistical Analyses

All experiments (growth inhibition, growth kinetics and differential protein expression assays) were done three times, and the values and the standard error were calculated from the data with three repetitions. One-way analysis of variance with t-test was conducted, and a p value of less than 0.05 was considered to indicate a statistically significant difference. The hypergeometric distribution was assayed to evaluate the enrichment of COG categories of the proteins encoded by Ent. mundtii CRL35 related to the ones differentially expressed by it in co-culture or alone (CC T30 - SM T30) and in co-culture during the time (CC T6 – CC T30).

### RESULTS

### *E. coli* Growth Inhibition Assay

The E.coli growth inhibition assay was performed as a first approach to determine the antagonistic potential of LAB strains toward the pathogen (**Figures 2A–C**). By means of this fast and simple method the inhibitory capacity of each LAB strain on E. coli NTCC12900, as well as the nature of the inhibition effect were evaluated. The three assayed LAB showed a similar inhibitory pattern, although they presented low variability in their inhibition halos. In general, halos of inhibition were registered in those conditions where LAB cells remained viable and not seriously damaged; regardless of the presence of the culture supernatant (spot #1 and 4). When cells were treated with lysozyme, minor inhibition halos were present (spot #3). In contrast, no inhibition were observed when lysed cells or cell free supernatant (untreated, heated or heated and neutralized) containing metabolites such as organic acids, heat stable bacteriocins and different soluble factors were spotted (spot #5, 6 and 7). The absence of inhibitory halos when 4% lactic acid solution was spotted (spot #8) confirmed the known acid resistance of E. coli (**Figure 2**).

### Performance of LAB and *E. coli* NCTC 12900 When Grown Individually Or in Co-culture in Sarcoplasmic Model System

When grown individually, LAB presented an adequate growth in the sarcoplasmic model system, achieving a maximal growth approximately at 24–48 h depending on the strain (2.0 × 108 – 9.8 × 108 CFU/ml) (**Figures 3A–C**). They reached the exponential growth between 3 and 8 h and the stationary growth phase around 24 h. Afterwards, LAB strains maintained approximately initial bacterial counts until 96 h of incubation (1 × 106 CFU/ml). On the other hand, a significant pH drop was observed in all LAB strains growing alone. When E. coli was inoculated alone, it also achieved an optimal growth presenting a traditional sigmoid kinetic curve, the exponential growth was attained between 4 and 6 h with the maximal cell viability at 24 h during the stationary phase (1.8 × 108 CFU/ml) (**Figures 3A–C**). When LAB-E. coli co-cultures were analyzed, a different growth kinetic was achieved by both type of microorganisms compared to its individual growth. A decreased growth rate of LAB in the presence of E. coli was observed, achieving 1–2 logarithmic units less of cell viability than in the single culture condition, depending on the strain. However during co-culturing, all LAB were able to keep the steady state until the end, with a population almost similar to the beginning. It is worth noting that the acidifying potential of LAB was not affected by the presence of the pathogen, reaching similar values than those observed in pure cultures (final pH between 4.5 and 3.7) (**Figures 3A–C**). On the other hand, the growth of E. coli in coculture was affected considerably by LAB, specifically after the first 8 h. For instance L. curvatus CRL705 (bacteriocin producer), showed a slight decrease of E. coli population, ∼0.6 log units (**Figure 3A**). Whereas the bacteriocinogenic strain Ent. mundtii CRL 35 (**Figure 3C**) and L. plantarum CRL 681 (non-bacteriocin

Co-culture and individual culture of Ent. mundtii CRL35. The red arrows indicate the time of sampling for proteomic assays.

producer) (**Figure 3B**), showed higher inhibitory effect. In fact, their presence produced a significant reduction of E. coli viability after 8 h of growth, accelerating the entrance of E. coli into the death phase. At 96 h a significant decrease of E. coli counts was observed reaching more than 2 and 3.5 logarithmic units of decrease in the co-cultures containing Ent. mundtii CRL35 and L. plantarum CRL681, respectively (**Figures 3B,C**).

### Differential Protein Expression of *Ent. mundtii* CRL35 in Co-culture With *E. coli* NCTC12900

L. plantarum CRL681 and Ent. mundtii CRL35 resulted the most effective strains to fight against E. coli in co-cultures. However, we decided to select Ent. mundti CRL35 for the proteomic analyses due to the availability of its genome, partially sequenced and annotated, which is essential for protein identification during proteomic studies. Additionally, this strain is able to produce the Enterocin CRL35, a very effective bacteriocin toward Listeria monocytogenes. The well-established bioprotective activity of Ent. mundtii CRL35, offers an extended range of bioprotection, not only against E. coli O157:H7 but also against other food-borne pathogens of concern to meat industry (Salvucci et al., 2007). Finally, Ent. mundtii CRL35 possess complete biochemical and genetic studies that guarantee its technological and bioprotective features that make this strain an interesting candidate to be used as adjunct culture in food (Saavedra et al., 2004; Pingitore et al., 2012). Nevertheless interaction studies between E. coli and L. plantarum CRL681, as well as, the effect of both LAB toward E. coli O157:H7 are focus of future experiments.

In order to study the interaction between Ent. mundtii CRL35 and E. coli O157:H7 NCTC12900 and to identify the molecular mechanisms underlying the antagonistic action carried out by Ent. mundtii in a meat environment, differential protein expression during the growth of Ent. mundtii alone or in co-culture was evaluated by 2DE. Two key moments of the growth were analyzed: 6 h (T6), when both microorganisms grew exponentially, and 30 h (T30), when Ent. mundtii achieved the stationary phase and E. coli entered in death state during co-culture (**Figure 3C**). Differential protein expression was evaluated according to the following comparisons: (i) Ent. mundtii growing in co-culture vs. Ent. mundtii growing individually at T6 (CC T6 - SM T6); (ii) Ent. mundtii growing in co-culture vs. Ent. mundtii growing individually at T30 (CC T30 - SM T30) and iii) Ent. mundtii growing in co-culture at T6 vs. Ent. mundtii growing in co-culture at T30 (CC T6 - CC T30). Representative 2DE maps of the bacterial proteomes when grown alone or in co-cultures are depicted in **Figure 4**.

In the three proteomic analyses, the most significant differentially expressed proteins (p < 0.05, fold > 1.5), 106 spots, were submitted to MS identification. Of these, a total of 91 proteins were successfully identified, 50 belonged to E. coli and 41 to Ent. mundtii, according with protein databases. In the present work, we focused in proteins related to Ent. mundtii CRL35 proteome (**Tables 1**, **2**).

### Differential Protein Expression of Ent. mundtii CRL35 Growing Alone Or in Co-culture

When proteomes from Ent. mundtii CRL35 grown in coculture were compared with Ent. mundtii growing alone in the sarcoplasmic model system, a significant protein over expression in co-culture was obtained at both analyzed times (T6 and T30). The identified proteins were successfully assigned to different functional categories. Specifically, at the first 6 h, 4 proteins related to carbohydrate (spot #1, enolase) (25%), amino acid metabolism (carbamoyl phosphate synthase large subunit, methionine ABC transporter substrate-binding Protein) (50%) and cell division (cell division protein FtsZ) (25%), resulted significantly over expressed by Ent. mundtii in co-culture (**Table 1**, **Figure 5**). On the other hand, after 30 h of growth in co-culture, 16 identified proteins resulted over expressed with a significant difference between 2.1 and 4.7 fold change. These proteins were involved in carbohydrate metabolism (phosphoglycerate kinase, fructose-bisphosphatealdolase, 6-phosphofructokinase, fructose-bisphosphatealdolase and enolase) (31.25%), energy production and conversion (pyruvate dehydrogenase E1 subunit alpha, L-lactate dehydrogenase, 2-oxoisovalerate dehydrogenase subunit beta) (18.75%), transcription (DNA-directed RNA polymerase subunit alpha) (6.25%), cell division (Cell division protein DivIVA oxidoreductase) (6.25%), cell wall biosynthesis (choloylglycine hydrolase) (6.25%), amino acid metabolism (hypothetical protein AK89\_04275) (6.25%), folding and protein processing (molecular chaperone DnaK) (6.25%), ribosomal structure (30S ribosomal protein S1) (6.25%) and stress (stress response regulator Gls24) (6.25%) (**Table 1**; **Figure 5**). These results indicate that the proteome of Ent. mundtii was affected by the presence of E. coli at 6 and 30 h although in a different way. In fact, a higher number of proteins were over produced at 30 h when the LAB achieved the stationary growth phase and the pathogen began its death cycle (**Table 1**; **Figure 3C**). Also, by performing a hypergeometric distribution, the probabilities of obtaining a certain COG category in our sample in relation with the ones encoded by the whole cell were analyzed. Whilst it was more probable to find one protein related with the metabolism and transport of carbohydrates (Figure S1A) we find five of them in our study (**Table 1**). This means that this category might be enriched by the obtained proteins. This also occurs with energy conversion and production, folding and protein processing and cell wall biosynthesis categories. For transcription, stress, ribosomal structure, and amino acid metabolism we found one of each as expected by the hypergeometric distribution. However, for the oxidoreductase (spot # 15) that includes three COG categories, was more probable to find two, and we found only one. This could imply an impoverishment of these categories (Figure S1A and **Table 1**).

### Differential Protein Expression in Ent. mundtii CRL35 Growing in Co-culture at 6 and 30 h

Regarding the differential protein expression when Ent. mundtii grew in co-culture at T6 vs. T30, a total of 21 proteins were successfully identified. Twenty spots

presented higher abundances at T6 than at T30 and one, the phosphoglucomutase/phosphomannomutase, was under expressed at T6. The overexpressed proteins participate of different functional categories, namely carbohydrate metabolism (phosphoglycerate mutase 1 family, glucose-6-phosphate isomerase, phosphoglycerate kinase, phosphopyruvate hydratase, phosphogluconate dehydrogenase, type I glyceraldehyde-3 phosphate dehydrogenase, transketolase) (40%), amino acid metabolism (glutamine synthetase, aminopeptidase, dipeptidase PepV and peptidase M13) (15%); energy production and

eMolecular Mass (Da).

f Calculated isoelectric point.

gRelative Fold change: Normalized Volumes of protein spot in

co-culture/Normalized

 Volumes of protein spots in individual growth.

 Score is

eMolecular Mass (Da).

f Calculated isoelectric point.

gRelative Fold change: Normalized Volumes of protein spot in

co-culture/Normalized

 Volumes of protein spots in individual growth.

−10\*Log(P),

 where P is the probability that the observed match is a random event. Protein scores larger than either 81 are considered significant (P < 0.05).

conversion (ATP synthase subunit alpha, 2-oxoisovalerate dehydrogenase and F0F1 ATP synthase subunit beta) (20%), transcription (DNA-directed RNA polymerase subunit alpha) (5%), nucleotide metabolism (adenylosuccinate synthase) (5%), translation (leucine–tRNA ligase) (5%), stress (general stress protein) (5%), cell wall biosynthesis (choloylglycine hydrolase) (5%) (**Table 2**, **Figure 5**). Moreover, according to the hypergeometric distribution, the categories related with carbohydrate metabolism, energy production and conversion, amino acid transport and metabolism, nucleotide transport and metabolism are enriched. While the categories of translation, transcription, cell wall biogenesis, and of unknown function were obtained as expected (Figure S1B).

In the three proteome comparisons, higher number of differentially expressed proteins were related with carbohydrate, energy production, and amino acid metabolism (**Figure 5**).

### Functional Analysis and Interaction of Proteins

The interactions among the over expressed proteins from Ent. mundtii were obtained using the STRING v10.05 database. This analysis contributes to understand microbial performance in the meat-based medium. Two protein-protein interaction networks were constructed: (i) containing proteins over expressed by Ent. mundtii in co-culture with respect to their individual growth at T30 (**Figure 6A**). A network for T6 was not built as only four proteins at this time were over expressed and (ii) containing proteins over expressed at T6 with respect to T30 when Ent. mundtii was grown in co-culture with E. coli (**Figure 6B**). As shown in **Figure 6A**, 4 out of the 15 proteins over expressed at T30 with respect to the single culture at T30 have no interactions in between each other. However 11 proteins were related showing interactions (32 edges). Four proteins are related with sugar carbohydrate metabolism and four of them with energy production and conversion, which shows very strong interactions in between each other. This also support the fact that carbohydrates metabolism (G) through glycolysis is enriched at T30 in the presence of E. coli.

The second network (**Figure 6B**) corresponds to the overexpressed proteins of Ent. mundtii CRL35 grown in coculture at T6 respect to their growth in co-culture at T30. Three out of 20 proteins were not included in the network as no interactions were found in the STRING database. In this network sixty eight interactions were obtained among the proteins overexpressed by Ent. mundtii, the thickness of lines between the nodes indicating the degree of interaction. As mentioned before, the overexpression of these 20 proteins in co-culture at T6 respect to T30 could be explained by the logarithmic growth phase of Ent. mundtii CRL35 at 6 h. Moreover, as it can be observed in **Figure 6B**, the main interactions found in the network are related with carbohydrate metabolism (9 proteins) in particular these involved in glycolysis and pentose phosphate pathway. This network support the activation and interaction of proteins related with carbohydrate metabolism in the exponential growth phase which is in agreement with the hypergeometric distribution test.

# DISCUSSION

### Performance of LAB and *E. coli* NCTC 12900 in a Meat-Based Medium. Physiological Results

The interaction and antagonistic activities of LAB with different pathogenic microorganisms were the focus of a number of studies. For instance, Atassi and Servin (2010) investigated the killing activity of Lactobacillus strains against Salmonella enterica serovar Typhimurium in co-cultures; Angmo et al. (2016) evaluated different LAB strains as biocontrol agents against Yersinia enterocolitica using agar spot tests as screening method. Thereafter, the growth of Y. enterocolitica in mixed cultures co-inoculated with two selected Lactobacillus strains was investigated. Also Yang et al. (2017) studied mixed cultures of bifidobacteria with Listeria monocytogenes to detect the changes in their growth pattern after mutual interaction by applying a proteomic approach. The present study is the first where the LAB inhibitory potential against E. coli O157:H7 is evaluated in co-cultures using physiological and proteomic approaches. The first objective of this study was to evaluate the inhibitory potential of three lactic acid bacteria strains toward E. coli O157:H7 NCTC12900. By a simple and rapid method such as the well-diffusion assay, a variable LAB inhibitory activity was evidenced. Then, the kinetic of growth in a meat model system in the presence of E. coli, was analyzed. The three LAB strains affected negatively the growth of EHEC in the meat environment after 8 h, evidencing a higher inhibitory potential of L. plantarum CRL681 and Ent. mundtii CRL35, which produced a significant decrease of E. coli counts at 96 h. It seems therefore that LAB trigged E. coli death, due to mechanisms other than acid effect or bacteriocin activity. In fact during the spot on lawn assay, no inhibitory halos were detected by supernatants containing acids or bacteriocins, they not triggering E. coli inhibition. Contrarily, Angmo et al. (2016) concluded that low pH and production of lactic acid were the main factors for inhibition of growth of Y. enterocolitica (Angmo et al., 2016). Moreover, even if all LAB strains were able to acidify the sarcoplasmic medium, they presented different inhibitory activity toward E. coli. According to this, EHEC has three powerful systems of resistance to acid stress; these including an acid-induced oxidative system, an acidinduced arginine-dependent system and a glutamate-dependent system (Bearson et al., 2009). These three systems of resistance to acids have different requirements, allowing an overlapping between them, ensuring that at least one of them will always be active to protect the cell in an acid environment (Bearson et al., 2009). These properties of EHEC contribute to low infectious doses by allowing small numbers of microorganisms to pass through the gastric acidity barrier. Therefore, its acid resistance ability is an important virulence factor and explains the absence of growth inhibition halos by culture supernatants observed herein. As regards to E. coli inhibition by bacteriocin action, it is known that these peptides do not act on Gram negative microorganisms unless they are combined with a treatment to damage the cell wall to allow bacteriocin entrance into the cell (Castellano et al., 2011). Therefore, our results, showing absence of inhibition due to Ent. mundtii CRL35 and L. curvatus CRL705 bacteriocins, suggest that EHEC antagonistic action involves other mechanisms such as competition for nutrients, quorum sensing, or a close cell-cell relationship where the bioprotective culture must preserve its vitality to cope with EHEC. In a similar work, Rios-Covian et al. (2015) reported a delayed growth of Bacillus fragilis by the presence of Bifidobacterim longun in coculture during the first 14 h. They observed an improved growth of bifidobacteria compared to the corresponding mono-culture. Our results showed that E. coli affects slightly to moderately the maximal cell densities achieved by LAB. Ent. mundtii CRL35 showed an earlier exponential phase and the stabilization of the stationary phase with a slight viability decrease after 40 h compared to its growth in mono-culture. Yang et al. (2017) studying the co-incubation of Bifidobacterium bifidum with Listeria monocytogenes, have also reported the earlier entrance into the logarithmic growth phase suggesting a mutual growth promoting effect during the co-cultivation.

### Differential Protein Expression Analyses Ent. mundtii CRL35 in Co-culture vs. Ent. mundtii in Mono-Culture

In this work, 2DE was employed to analyze differential expression induced by the interaction between Ent. mundtii CRL35 and E. coli NCTC12900, focusing on the LAB proteome. This approach allowed us to investigate the molecular basis of this interaction and the relation with the physiological changes undergoing during co-cultivation in the meat-based medium. Slight proteome variations were observed in Ent. mundtii during the first hours of co-culture with E. coli with respect to its growth as mono-culture at 6 h. One ABC transporter for methionine and the carbamoyl phosphate synthase large unit are among the over expressed proteins. They are related to amino acid biosynthesis and metabolism. One enzyme related to glycolysis, the enolase (spot #9), and one related to cell division (protein FtsZ, spot #14) resulted over expressed at 6 h and at 30 h indicating that LAB activated glycolysis and cell division to cope the presence of E. coli. It should be mentioned that enolase is also known as a moonlighting protein. These are proteins that display additional functions other than their major described biochemical catalytic activity. In general, these cytoplasmic/cell surface moonlighting proteins can be important in infection, virulence, or immune responses (Jeffery, 2015). For example enolase is also associated with epithelial cell binding (Castaldo et al., 2009). In fact, Peng et al. (2014) reported enolase as one of the actin-binding proteins in Enterococcus faecalis. It could therefore be suggested that Ent. mundtii can up-regulate enolase during co culturing with E. coli as an additional strategy to compete with E. coli for actin binding during adhesion to meat. On the other hand, Ent. mundtii proteome was much more affected in co-culture at 30 h than when it grew alone at the same time. In fact 16 proteins resulted over expressed, including some spots also upregulated at 6 h (enolase and cell division protein FtsZ). Proteinprotein network showed interaction in 12 of these proteins, mainly related with carbohydrate metabolism. The 31.25% of differentially over expressed proteins were involved in glycolysis. The 18.75% of proteins synthesized in higher amounts were related to other pathways, also involved in energy production and conversion, thus indicating that co-culturing with E. coli exerted more effective activation of these pathways at 30 h of co-culturing than during the first hours of the growth. In addition, physiological results indicate that at 30 h, E. coli is dying in co-culture, suggesting that E. coli viability decrease resulted convenient for Ent. mundtii which persisted in the stationary phase. Ent. mundtii during co-culture, resulted even more stable at stationary phase than when it grew alone. This fact is consistent with the over-expression of many proteins from sugar metabolism, energy production, transcription, cell division, and amino acid metabolism indicating an active metabolism of Ent. mundtii which allowed its persistence in the meat-based medium. It should also be highlighted the up regulation of proteins related to folding/processing and stress such as the chaperone DnaK and the stress response regulator Gls24 that could contribute to the satisfactory resistance of Ent. mundtii to stressful conditions dominating the microbial environment at 30 h in conjunction with a low pH (close to 4.0). There are some studies demonstrating the interaction of certain microorganisms during its growth in mixed cultures. Yang et al. (2017) proposed that the growth of Bifidobacterium bifidum WBBI03 and Listeria monocytogenes together promotes the growth of each other, resulting in earlier entry into the logarithmic phase, and the expression of proteins mostly tended

to be up regulated at the translational and transcriptional level. While Rios-Covián et al. (2016) reported the stimulation of the growth of Bifidobacterium longum in co-culture while retarding the growth of Bacteroides fragilis, with concomitant changes in the production of some proteins and metabolites of both bacteria. In the present work a different interaction seems to occur between Ent. mundtii CRL35 and E. coli O157:H7. In fact, a positive effect of E. coli on the fitness of the LAB could occur, while the latter triggered the pathogen death after 8 h of co-culture.

### Ent. mundtii CRL35 in Co-culture: T6 vs. T30

When comparing protein expression of co-cultures along the time (CC T6 vs. CC T30), 20 proteins resulted over expressed during the first hours (6 h). This is in relation to the exponential growth phase going through the microorganism, in which the general metabolism is activated as reported by other (Cohen et al., 2006; Koistinen et al., 2007). Those results are also supported by the observed protein network, where proteins related to carbohydrate metabolism presented stronger interactions. Among up regulated proteins, 7 spots were identified as belonging to carbohydrate metabolism and 1 to sugar transport. Five enzymes were related to glycolysis (spots #22, 23, 24, 25 and 27) and two involved in the pentose phosphate pathway (spots #26 and 28) (**Figure 7**). The over expression of phosphoenolpyruvate–protein phosphotransferase, involved in carbohydrate transport, could facilitate glucose entrance into the cell, as a consequence of a more efficient competition with the pathogen for sugar uptake, thus contributing to cope with E. coli presence which is also in the logarithmic growth state. Only one glycolytic enzyme, the phosphoglucomutase (spot #21) was under expressed at 6 h. This could be related to the up regulation of the PTS phosphoenolpyruvate-protein phosphotransferase (PTS system) involved in glucose transport by the generating glucose-6P which enters directly into the Embden-Meyerhorff-Parnas pathway, explaining the under expression of phosphoglucomutase which produces glucose-6P from glucose-1P coming from other pathways such as glycogen hydrolysis (Bonacina, 2017) (**Figure 7**). Glycogen metabolism would be less active than glucose during the first hours. In fact glucose is added to the meat-based medium which could be chosen firstly as primary energy source. On the other hand, two enzymes related to the pentose phosphate/phospohoketolase (PKP) pathway resulted over expressed in Ent. mundtii at 6 h, the 6-P gluconate dehydrogenase and transketolase, indicative also of the active metabolism of ribose, one of the sugars present in meat (Chaillou et al., 2005) (**Figure 7**). Concurring with our findings, Koistinen et al. (2007) reported that proteins preferentially expressed by L. plantarum in the early exponential phase were related to sugar consumption and biomass increase. Four enzymes related to amino acid metabolism were also up regulated by Ent. mundtii in co-culture at 6 h. Among them glutamine synthetase, a key enzyme of nitrogen metabolism that catalyzes the incorporation of ammonium into glutamate and is related to arginine biosynthesis, alanine, glutamate, and aspartate metabolism among other (Magasanik and Rothstein, 1980). In addition, some other peptidases and aminopeptidases resulted up regulated also at 6 h, indicating an active peptidolytic metabolism during the first hours of co-culturing. Also the adenylosuccinate synthase resulted 2.7-fold over expressed at 6 h with respect to T30 by Ent. mundtii in co-culture, this enzyme plays an important role in the de novo pathway of purine nucleotide biosynthesis, it catalyzing the first committed step in the biosynthesis of AMP from IMP, also indicating a more active metabolism of this LAB during the first hours of co-culture. Also, proteins involved in transcription and translation such as DNA-directed RNA polymerase subunit alpha, leucine-tRNA ligase resulted up regulated by this LAB strain during the first hours of co-culturing. In accordance, Yang et al. (2017), reported the up regulation of enzymes related to transcription and translation when mixed cultures of Bifidobaterium bifidum and Listeria monocytogenes were evaluated.

One general stress protein presented also higher amounts at T6, as well as, the choloylglycine hydrolase involved in lipid metabolism and cell wall biosynthesis. This pattern can be related with the exponential growth and the consequent active cellular division. Two ATP synthases (alpha and beta subunits) were found increased in this condition. They are the prime producers of ATP, using the proton gradient generated by oxidative phosphorylation. Finally, the 2-oxoisovalerate dehydrogenase beta subunit was 1.9 fold over expressed at 6 h during coculture. This enzyme belongs to the oxidoreductase family, being also implicated in Phe, Tyr, Trp, Leu, Ile, Val, Asp, and Asn degradation. The branched-chain alpha-ketodehydrogenase complex catalyzes the overall conversion of alpha-keto acids to acyl-CoA and CO2. Summarizing, differential protein expression by Ent. mundtii in co-culture at two different growth phases correlated with the observed physiological behavior. The up regulation of many enzymes implicated in sugar and nitrogen metabolism, transcription, translation and energy production was in relation with the metabolism and the physiological state crossing LAB during the first hours of growth. A direct consequence of this active global metabolism contributed to the competition with E. coli at this specific moment when the pathogen was going through the exponential phase, while at 30 h, Ent. mundtii CRL35 reaching the stationary phase did not have to face a strong competition with E. coli since it had already entered into its death phase.

According with the obtained results, on can postulate the positive effect of E. coli on the fitness of the LAB, while a negative impact exerted the LAB on the pathogen by triggering its death after 8 h of co-culture. On the other hand, differential overexpression of Ent. mundtii proteins was higher in co-culture with E. coli than when it grew alone at 30 h. Concomitantly, physiological results indicated that at 30 h, E. coli was dying in co-culture, suggesting that the decrease of E. coli viability resulted convenient for Ent. mundtii which persisted in the stationary phase. This fact is consistent with the over-expression of many proteins from sugar metabolism, energy production, transcription, cell division and amino acid metabolism. This fact indicating the active Ent. mundtii metabolism allowed its persistence over the pathogen in the meat medium. It should also be highlighted the up regulation of proteins related to

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asterisks are presented the proteins overexpressed in co-culture at 30 h with respect to 6 h.

folding/processing and stress such as the chaperone DnaK and the stress response regulator Gls24 that could contribute to the satisfactory resistance of Ent. mundtii to stressful conditions at 30 h, such as the low pH. When comparing protein expression of Ent. mundtii in co-culture along the time (CC T6 vs CC T30), the higher number of proteins over expressed during the first 6 h, was in relation with the metabolism and the physiological state crossing LAB during the first hours of co-culture. A direct consequence of this active global metabolism contributed to the competition with E. coli which was going through the exponential phase. Whereas at 30 h, Ent. mundtii was reaching the stationary phase and E. coli had already entered into its death phase. Indeed the LAB did not have to face a strong competition, accordingly, less differential protein expression was achieved at 30 h. Finally, mechanisms involved in this interaction, such as competition for nutrients, quorum sensing, or a close cell-cell relationship are suggested. The detailed study of these mechanisms is focus of ongoing investigations.

### CONCLUSION

Current results have demonstrated the true inhibitory potential of LAB against a pathogen of great concern such as E. coli O157:H7. Such inhibition was not due to acid or bacteriocin production but instead to a more complex relationship during the microbial interaction. The proteomic results herein presented supported physiological observations, demonstrating significant differences in protein expression in LAB (i) due to the presence of the pathogen and (ii) according to the growth phase analyzed. Even when more studies have to be performed in fresh meat to confirm in vitro observations, these results lay the foundations of the molecular basis for understanding the interaction between Ent. mundtii CRL35 and E. coli O157:H7 NCTC12900, as well as on the strategies of competition applied by both microorganisms. This work finally opens new perspectives for the application

### REFERENCES

of this bioprotective LAB to control E. coli O157:H7 in meat products.

### AUTHOR CONTRIBUTIONS

AO carried out the experiments, analyzed the results and wrote the paper. SF conceived the idea of the project, coordinated the study, analyzed and discussed the results and wrote the paper. MS coordinated the study, discussed the results and wrote the paper. LT performed the in silico analysis of the identified proteins (STRING, COGNITOR) and the analysis of the hypergeometric distribution. JR and AA contributed in mass spectrometric analysis (MALDI-TOF/TOF) and discussed the paper. GV contributed to the discussion of the paper. All authors read and approved the final manuscript.

### FUNDING

This study was funded by FONCyT (PICT06 0813; PICT2011 0175) and CONICET (PIP0406; PIP0530).

### ACKNOWLEDGMENTS

The authors thank Elena Bru for her statistical assistance. The FONCyT and CONICET organizations for the funding of this research carried out within the framework of AO doctoral thesis are fully acknowledged. We also thank CONICET for the doctoral scholarship of AO to support the completion of her doctoral degree.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.01083/full#supplementary-material

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Orihuel, Terán, Renaut, Vignolo, De Almeida, Saavedra and Fadda. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Edited by: Fliss Ismail, Laval University, Canada

### Reviewed by:

Luis Augusto Nero, Universidade Federal de Viçosa, Brazil Young Min Kwon, University of Arkansas, USA

### \*Correspondence:

Céline Delbès<EMAIL_ADDRESS>
### †Present address:

Sabine Nidelet, Institut National de la Recherche Agronomique, UMR 1062, Centre de Biologie pour la Gestion des Populations, Montferrier-sur-Lez, France Pierre Delpech, Department of Health and Life Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow, UK

‡These authors have contributed equally to this work.

### Specialty section:

This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

Received: 28 September 2016 Accepted: 21 February 2017 Published: 08 March 2017

### Citation:

Delpech P, Rifa E, Ball G, Nidelet S, Dubois E, Gagne G, Montel M-C, Delbès C and Bornes S (2017) New Insights into the Anti-pathogenic Potential of Lactococcus garvieae against Staphylococcus aureus Based on RNA Sequencing Profiling. Front. Microbiol. 8:359. doi: 10.3389/fmicb.2017.00359

# New Insights into the Anti-pathogenic Potential of Lactococcus garvieae against Staphylococcus aureus Based on RNA Sequencing Profiling

Pierre Delpech1† , Etienne Rifa1 , Graham Ball2 , Sabine Nidelet3† , Emeric Dubois3 , Geneviève Gagne1 , Marie-Christine Montel1 , Céline Delbès1 \* ‡ and Stéphanie Bornes1‡

1 Université Clermont Auvergne, INRA, UMRF, Aurillac, France, 2 John van Geest Cancer Research Centre, School of Science and Technology, Nottingham Trent University, Nottingham, UK, 3 Montpellier GenomiX, Institut de Génomique Fonctionnelle, Montpellier, France

The bio-preservation potential of Lactococcus garvieae lies in its capacity to inhibit the growth of staphylococci, especially Staphylococcus aureus, in dairy products and in vitro. In vitro, inhibition is modulated by the level of aeration, owing to hydrogen peroxide (H2O2) production by L. garvieae under aeration. The S. aureus response to this inhibition has already been studied. However, the molecular mechanisms of L. garvieae underlying the antagonism against S. aureus have never been explored. This study provides evidence of the presence of another extracellular inhibition effector in vitro. This effector was neither a protein, nor a lipid, nor a polysaccharide, nor related to an L-threonine deficiency. To better understand the H2O2-related inhibition mechanism at the transcriptome level and to identify other mechanisms potentially involved, we used RNA sequencing to determine the transcriptome response of L. garvieae to different aeration levels and to the presence or absence of S. aureus. The L. garvieae transcriptome differed radically between different aeration levels mainly in biological processes related to fundamental functions and nutritional adaptation. The transcriptomic response of L. garvieae to aeration level differed according to the presence or absence of S. aureus. The higher concentration of H2O2 with high aeration was not associated with a higher expression of L. garvieae H2O2-synthesis genes (pox, sodA, and spxA1) but rather with a repression of L. garvieae H2O2-degradation genes (trxB1, ahpC, ahpF, and gpx). We showed that L. garvieae displayed an original, previously undiscovered, H2O2 production regulation mechanism among bacteria. In addition to the key factor H2O2, the involvement of another extracellular effector in the antagonism against S. aureus was shown. Future studies should explore the relation between H2O2-metabolism, H2O2-producing LAB and the pathogen they inhibit. The nature of the other extracellular effector should also be determined.

Keywords: Lactococcus garvieae, Staphylococcus aureus, hydrogen peroxide, transcriptome, growth inhibition, anti-pathogenic interactions

# INTRODUCTION

fmicb-08-00359 March 6, 2017 Time: 16:45 # 2

Interest in Lactic Acid Bacteria (LAB) as bio-preservation agents against foodborne pathogens has been growing for the last 15 years (Ito et al., 2003; Batdorj et al., 2007; Charlier et al., 2009; Adesokan et al., 2010). Considering their beneficial properties, the dairy industry could find it useful to employ Lactococcus garvieae strains as starter or adjunct cultures, provided the strains are safe (Fernández et al., 2010). Aquatic strains of L. garvieae are frequently referenced as fish pathogens (Eldar and Ghittino, 1999; Vendrell et al., 2006) but other strains are regarded as opportunistic pathogens for humans (Aguado-Urda et al., 2011; Ortiz et al., 2014).  L. garvieae is of interest for bio-preservation owing to its ability to inhibit the growth of staphylococci, particularly Staphylococcus aureus, as has been observed in milk, in cheese and in vitro (Alomar et al., 2008a,b; Delbes-Paus et al., 2010; Delpech et al., 2015). The transcriptome response of S. aureus to this inhibition has already been explored (Delpech et al., 2015). It is associated with a repression of the stress response (especially H2O2 response) and of cell division genes and with modulation of the expression of virulence genes (particularly agrA, hld, and enterotoxin-encoding genes) genes. However, the molecular mechanisms of L. garvieae underlying the antagonism against S. aureus have never been explored.

Since L. garvieae produces low amounts of acetic and lactic acid in milk (Alomar et al., 2008a,b; Nouaille et al., 2009; Sip et al., 2009; Delbes-Paus et al., 2010; Delpech et al., 2015; Morandi et al., 2015), its antagonism against S. aureus is not associated with acidification. It is also unlikely to be associated with nutritional competition (Alomar et al., 2008a). With high aeration, S. aureus inhibition is mainly associated with hydrogen peroxide (H2O2) production by L. garvieae, as already observed in vitro (Delbes-Paus et al., 2010; Delpech et al., 2015). With low aeration, the inhibition is weaker and H2O2 is not detected. The addition of catalase (an H2O2-degrading enzyme) partly suppressed the inhibition (Delbes-Paus et al., 2010). Delbes-Paus et al. (2010) therefore suggested that at least one other molecule from L. garvieae must be involved in the residual inhibition of S. aureus in the absence of H2O2. The role and nature of this molecule have not yet been determined.

In Gram-positive bacteria, the aeration level and the presence of H2O2 generally strongly affect the expression of H2O2 metabolism genes. The expression of major H2O2 degradation genes (ahpC, ahpF, gshR, gpo, trxA, and trxB) and their related proteins is strongly induced in L. lactis (Pedersen et al., 2008) under aeration and in Bacillus subtilis in the presence of H2O2 (Mostertz et al., 2004). As regards the major H2O2 synthesis genes, sodA is generally regulated in the same way as H2O2 degradation genes while the pyruvate oxidase (pox) gene is not affected by these parameters. Biological functions of L. lactis related to aerobiosis, i.e., O2 response, menaquinone metabolism and stress response, are variably affected by the presence of S. aureus (Nouaille et al., 2009). However, little is known about the H2O2-metabolism of L. garvieae as an anti-pathogenic process.

In this study, we aimed to characterize the antagonism of L. garvieae against S. aureus in vitro in greater depth. Firstly, we investigated the presence of potential antagonist molecules in H2O2-free L. garvieae supernatants and their impact on S. aureus growth. Secondly, the transcriptome of L. garvieae with different aeration levels and in different biotic environments (absence or presence of S. aureus) was determined through RNA sequencing, an accurate and efficient method for revealing bacterial transcriptome profiles (Pinto et al., 2011). The resulting data led us to a better understanding of L. garvieae hydrogen peroxide metabolism and to some initial hypotheses on the nature of a new inhibition effector.

# MATERIALS AND METHODS

### Strains and Culture Conditions

Lactococcus garvieae N201 and S. aureus SA15, isolated from raw milk, were obtained from the INRA UR545 collection (Alomar et al., 2008a). Both strains were aerobically grown in Brain-Heart Infusion broth ("BHI", Biokar Diagnostic, Pantin, France) for 20 h, at 30◦C for L. garvieae and at 37◦C for S. aureus. They were then inoculated separately or in co-culture at 106 cells.mL−1 for S. aureus and 107 cells.mL−1 for L. garvieae into BHI buffered at pH = 7 with phosphate buffer KH2PO4, 3H2O/K2HPO4 at 0.1 mol.L−1 (KH2PO4, 3H2O, Riedel-de-Haen, Honeywell GmbH, Seelze, Germany; K2HPO4, Merck KGaA, Darmstadt, Germany) previously equilibrated at 30◦C. Pure cultures and co-cultures of both strains were performed with either a high or a low aeration depending on the experiment. Low aeration cultures were set in static, fully filled and sealed, 50-mL Nunc EZ Flip conical centrifuge tubes (Sigma–Aldrich, St. Louis, MO, USA). High aeration was obtained by a mechanical shaking at 150 rpm on 50-mL cultures in 250-mL Erlenmeyers. All cultures were incubated at 30◦C for 24 h in an Infors HT Minitron (Infors AG, Bottmingen, Switzerland). The cultivable cell counts were determined after plating for each sampling time as described by Delpech et al. (2015).

### Impact of H2O2-Free Co-culture Supernatants on S. aureus Growth

The potential presence of other antistaphylococcal molecules in L. garvieae N201 and S. aureus SA15 co-culture supernatant and their impact on S. aureus planktonic growth were investigated. After adding catalase at 400 U.mL−1 , a pure culture of S. aureus SA15, a co-culture of S. aureus SA15 and L. garvieae N201 and plain BHI were incubated with low aeration as described above. After 6 h of incubation, 40 mL of each tube were centrifuged at 9,600 × g for 10 min at 4◦C and supernatants stored at 4◦C until utilization. Four milliliters of the remaining S. aureus culture were centrifuged at 7,500 × g for 10 min. The cell pellet was resuspended at a concentration of ∼106 cells.mL−1 in 20 mL of

fresh 2X-concentrated BHI buffered at pH = 7. The supernatants were filtered through a cellulose acetate membrane (pore size 0.45 µm; GVS S.p.A., Zola Predosa, Italy). They were either treated with proteinase K (AMRESCO LLC, Solon, OH, USA, ref: 0706-100MG) and pronase E (Merck KGaA, ref: 537088), i.e., with two proteases, or treated with lipase (Sigma–Aldrich, ref: L3126-100G), or treated with α-amylase (Sigma–Aldrich, ref: A3176-500KU), or not treated at all. Each enzyme was added at 0.2 mg.mL−1 from stock solutions at 10 mg.mL−1 prepared in 200 mM of phosphate buffer at pH = 7. Treatment with proteases consisted of a first incubation of the supernatants with proteinase K at 50◦C for 2 h 15 and a second incubation with pronase E at 37◦C for 2 h 15. Treatments with lipase and α-amylase consisted of an incubation of the supernatants with the enzyme at 37◦C for 5 h. After each incubation step, the enzyme was inactivated by heating (95◦C during 10 min) and all supernatants were filtered again through a cellulose acetate membrane (pore size 0.45 µm; GVS S.p.A.). Prepared supernatants and S. aureus culture in 2X-BHI were then distributed at 1:1 volume ratio (total volume = 1.5 mL) in a CytoOne 24-well cell plate covered with a lid (ref: CC7672-7524; STARLAB, Hambourg, Germany). The cell plate was incubated for 25 h in a SAFAS Xenius XC spectrophotometer (SAFAS Monaco, Monaco) and thermostated at 30◦C with a Julabo CryoStat (JULABO Gmbh, Seelbach, Germany). S. aureus growth in each well was determined by measuring the OD600 every 15 min for 1 h and then every 30 min for 24 h. Before each OD600 measurement, the cell plate was shaken at 5 Hz for 20 s with an orbital diameter of 6 mm. The whole experimental design was repeated three times. To identify sample means that were significantly different from each other, statistical analyses were performed on values at 9, 12, 15, 18, 21, and 24 h, using Statistica software (StatSoft) with a single-factor analysis of variance (ANOVA) followed by a Newman–Keuls post hoc test.

# Sample Preparation for RNA Analysis

Pure cultures and co-cultures of L. garvieae N201 and S. aureus SA15 were grown with high or low aeration, as specified above. After 3, 6, 9, and 24 h of incubation, 40 mL of each culture were centrifuged at 9,600 × g for 10 min at 4◦C. Hydrogen peroxide concentrations and pH values were determined on the supernatants by enzymatic reaction and spectrophotometry as described by Delbes-Paus et al. (2010). The cell pellets were immediately frozen in an ethanol bath and stored at −80◦C. Extraction of total RNA from the frozen cell pellets was performed as described by Delpech et al. (2015). For each sample of total RNA obtained with one culture condition, a first part of the aliquot was used for RNA sequencing after rRNA depletion and a second part of the aliquot was used for RT-qPCR analyses. The whole experimental design was repeated three times.

# Determination of L. garvieae Transcriptome Changes by RNA Sequencing

Ribosomal RNAs were removed from the total RNA (2 × 5 µg of RNA by sample) using a RiboZero Magnetic Kit for Gram Positive Bacteria (Illumina Inc., San Diego, CA, USA) according to the manufacturer's instructions. The quality and concentration of RNA in each sample were assessed using a RNA 6000 pico kit (Agilent Technologies, Santa Clara, CA, USA). The following steps were performed by the MGX Platform (Montpellier GenomiX, CNRS, Montpellier, France) using Illumina kits and devices (Illumina Inc.): construction of the mRNA library using a TruSeq Stranded mRNA Sample Preparation Kit; cluster generation with the cBot system using a Cluster Generation Kit; hybridization of the sequencing primer on the flow-cell; 50-bp single-read sequencing using a HiSeq 2000 device with SBS technology; informatic pretreatments, i.e., image analysis with the HiSeq Control Software and Real-Time Analysis component, base-calling with the RTA software and demultiplexing with CASAVA (Illumina). The RNAseq data are available from NCBI GEO datasets under the accession number GSE74030.

The quality scores across all bases of all reads and the N (non-attributed bases) content across all bases were determined for each condition with the FastQC software from the Babraham Institute1 . Both analyses showed good quality values (data not shown). The reads were next aligned simultaneously on reference genomes, i.e., L. garvieae N201 (unpublished, GOLD project ID = Gp0034836 and NCBI BioProject ID = 184287) and S. aureus MW2 (RefSeq number = NC\_003923.1), using the BWA package (Li and Durbin, 2009) with a seed of 32 bases and a maximum of two mismatches tolerated on the seed. Using the Samtools suite (Li et al., 2009) we excluded from further analyses those reads with low alignment quality scores (MAPQ index < 20). Reads which mapped on multiple sites (between 0.3 and 3.2% depending on the sample) and reads which did not map on any site considering the stringency applied (between 8.7 and 15.5% depending on the sample) were excluded from further analysis (see Supplementary Table 1). Reads overlapping genes were counted with the HTSeq Count software in Union mode (Anders et al., 2015). Differentially expressed genes were identified using the Bioconductor R2 packages EdgeR, DESeq and DESeq2 (Anders and Huber, 2010; Robinson et al., 2010; Love et al., 2014). Genes with less than 15 reads (from the three biological replicates of two compared samples) were filtered and thus removed from the analysis. Data were normalized using the Relative Log Expression (RLE) normalization factor for EdgeR and the DESeq normalization factor for DESeq and DESeq2. Gene expression changes with adjusted p-value of less than 0.05 (by the FDR method from Benjamini–Hochberg) were declared differentially expressed. Differentially expressed genes highlighted by at least one of the three packages were considered for further investigation.

Genes were sorted into two lists: one of genes differentially expressed in both pure culture and co-culture and one of genes differentially expressed only in pure culture or only in co-culture. These lists were separately subjected to a

1http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ 2http://www.R-project.org/

Blast2Go analysis (Conesa et al., 2005). Blast2Go analysis consisted first of a BlastX3 on the nr database with 20 hits and a maximum E-value of 1.0E −15. The resulting data were enriched with an InterPro scan analysis (Zdobnov and Apweiler, 2001). Blast hits of each sequence were then mapped with Gene Ontology terms (Gene Ontology Consortium, 2008) annotated with a maximum E-value of 1.0E −6 , a cut-off of 55 and a GO weight of 5. The number of genes involved in each Biological Process GO category was calculated from combined graphs with no filter and a score alpha of 0.6. Since more than 450 different biological processes were identified for each category of genes, we excluded biological processes involving less than nine genes. When several biological processes involved the same genes and were associated with comparable functions, we considered only the one most relevant to our scientific hypotheses.

For a deeper analysis of S. aureus direct effect on L. garvieae gene expression regardless of aeration level, data mining was undertaken using the regression functions in Microsoft excel. Expression values were systematically plotted for each pair of samples (co-culture versus pure culture), a standard residual value was determined from the regression analysis for each gene across multiple pair wise comparisons and the mean value was determined. This mean standard residual value was used to rank the genes and identify the strongest gene expression differences. A standard residual cut-off of 5 was used as the threshold for significance to account for false discovery using a Bonferroni correction and approximating the standard residual to an equivalent p-value for the number of comparisons.

### Determination of Gene Expression by RT-qPCR

Total RNA was retro-transcribed using a High Capacity cDNA Reverse Transcription kit (Invitrogen, Life Technologies, Carlsbad, CA, USA) following the supplier's instructions. Ct of genes of interest (see Supplementary Table 2) were determined by RT-qPCR as described by Delpech et al. (2015). All primers were designed using PrimerExpress R software (Applied Biosystems R , Life Technologies). By comparison with the Ct of the tufB reference gene stable in our conditions (data not shown), expression of a gene of interest ("goi") was calculated using the formula introduced by Pfaffl (2001).

The influence of two experimental factors was studied: the presence of S. aureus (with high or low aeration, gene expression in co-culture divided by gene expression in pure culture) and aeration level (in presence or absence of S. aureus, gene expression in shaken condition divided by gene expression in static condition). Statistical analyses were performed using Statistica software (StatSoft, Inc., StatSoft France, Maisons-Alfort, France) by single-factor ANOVA followed by a Newman–Keuls post hoc test.

### RESULTS

### Effect of Enzyme-Treated Co-culture Supernatants on S. aureus Planktonic Growth

Since the inhibition of S. aureus may not be related solely to hydrogen peroxide, we looked for another possible antistaphylococcal molecule produced by L. garvieae in the culture supernatants. We monitored S. aureus growth (OD600) over 24 h in either non-inoculated BHI or in supernatants prepared from H2O2-free cultures (presence of catalase) with low aeration (either pure culture of S. aureus or co-culture of L. garvieae and S. aureus). In order to avoid any nutritional competition, BHI at a final concentration of 1X was added to each culture.

Growth of S. aureus in supernatant from S. aureus pure culture was comparable to that in plain BHI (data not shown). During the stationary phase, the OD600 measured in S. aureus cultures in supernatant from co-culture was lower than that in supernatant from S. aureus pure culture (see Data Sheet 1). This inhibition was still observed when these supernatants were treated with proteases, lipase or α-amylase, the main macromolecule-degrading enzymes. pH values remained between 6.9 and 7.1 in all cultures (data not shown).

### Determination of Aeration and S. aureus Effects on L. garvieae Transcriptome by RNA Sequencing

To identify the best conditions for studying the transcriptome of L. garvieae N201 with respect to its capacity to inhibit S. aureus SA15, we followed the growth of the two bacteria in pure cultures and co-cultures in BHI for 24 h under high or low aeration conditions (**Table 1**).

The growth of L. garvieae was not affected by S. aureus. The growth of S. aureus was inhibited by L. garvieae at both aeration levels. With high aeration, maximal inhibition was observed from 9 to 24 h (difference with pure culture of 4 and 4.5 log CFU/ml, respectively, at 9 and 24 h). At 9 h, H2O2 concentration reached a peak concomitant with the lowest S. aureus concentration. With low aeration, inhibition was weaker than with high aeration and was observed later, at 24 h. Concomitantly, H2O2 was not detected in these cultures. pH values remained between 6.9 and 7.1 in all cultures (data not shown).

Considering these data, we analyzed the L. garvieae transcriptome in pure culture and in co-culture with S. aureus after 9 h of incubation with high and the low aerations. RNA sequencing generated a number of reads per sample, ranging from 12,365,133 to 15,670,131 depending on the sample after the initial quality filter (see Supplementary Table 1). Between 83.1 and 91.0% of these reads mapped correctly onto the reference genomes. Considering the direct effect of S. aureus on L. garvieae gene expression, analysis of the mapped reads using the Bioconductor R packages failed to identify expression differences (data not shown). Additional regression analyses highlighted 39 genes differentially

3http://blast.ncbi.nlm.nih.gov/Blast.cgi

TABLE 1 | Cell counts and H2O2 concentration over 24 h in cultures of Lactococcus garvieae N201 and Staphylococcus aureus SA15 with high or low aeration levels.

In the same column, letters (a, b, and c) indicate homogeneous statistical groupings (p-value < 0.05 by Newman–Keuls method). ND, non-detectable values (below the spectrometry detection limit). NT, not tested.

expressed in presence of S. aureus, of which 4 genes under low aeration and 35 genes under high aeration conditions (see Supplementary Table 3). The expression of ∼18% of L. garvieae genes, i.e., 358 genes, differed between the two aeration levels (see Supplementary Table 4). Among these 358 genes, the expression of 181 genes differed regardless of the presence or absence of S. aureus (i.e., similarly in pure culture and in co-culture). The expression of 177 L. garvieae genes responded differently to different aeration levels, depending on the presence or absence of S. aureus: 88 gene expressions differed only in pure culture and 89 gene expressions differed only in co-culture.

### Effect of Aeration Level on L. garvieae Biological Processes

After RNA sequencing data treatments, 22 biological processes (named according to Gene Ontology termes) related to L. garvieae genes differentially expressed depending on aeration level in both pure culture and co-culture were identified using Blast2Go (**Figure 1**). Changes in gene expression are shown in Supplementary Table 4.

Most of the biological processes affected were related to fundamental growth functions (**Figures 1A,B**) and nutrition (**Figure 1C**). With high aeration, two genes (ilvA, LCGN\_1922) related to "threonine metabolism" were repressed while two genes (LCGN\_1919, LCGN\_1920) were over-expressed.

Fourteen genes related to "transport" processes were differently expressed depending on aeration level both in pure culture and co-culture, including genes involved in the transport of metals (lead, cadmium, zinc, copper and/or mercury) and vitamins (riboflavin and folate).

Genes and GO biological processes related to O2 and H2O2 metabolism were also affected. "Oxidation-reduction process" and "response to stress" biological processes involved more repressed genes (17 and 8, respectively) than overexpressed genes (6 and 1, respectively). Three genes related to H2O2 metabolism (ahpF, pox, and spxA1) and one gene related to O2 consumption (lox) were repressed with the high aeration level. Several stress response genes (hrcA, groES, groEL, dnaK, dnaJ, grpE, clpB, and five genes belonging to the universal stress protein family) were repressed. Genes related to peroxide resistance (ohrA and ohrR) were strongly overexpressed under high aeration conditions. Electron Transport Chain (ETC) genes (cydB, menH, and ubiE) were overexpressed.

### Modulation by S. aureus of L. garvieae Response to Aeration

We identified 27 Gene Ontology biological processes related to genes differentially expressed depending on aeration level either in pure culture or in co-culture (**Figure 2**).

Changes in gene expressions are shown in Supplementary Table 4. Most of the biological processes affected were related to fundamental growth functions (**Figures 2A,B**) or nutrition (**Figure 2C**). Under high aeration conditions, four lysine metabolism genes (LCGN\_0575, LCGN\_0576, LCGN\_0577, LCGN\_0578) and two threonine metabolism genes (LCGN\_0576 and LCGN\_0577) were over-expressed exclusively in pure culture while four galactose metabolism genes were overexpressed exclusively in co-culture (LCGN\_1809, LCGN\_1810, LCGN\_1811, and LCGN\_1812). Eight genes involved in pyruvate and carbohydrate metabolism and the citrate cycle (glmS, glmM, galE, PTS-Man-EIIC and EIID pdhA, pdhB, DLAT) were repressed by S. aureus. With low aeration, the enolase encoding gene was repressed by S. aureus.

Four biological processes related to transport functions were affected (**Figure 2D**): "transport," "transmembrane transport," "organic substance transport" and "ion transport." In pure culture, genes involved in the transport of cobalt/zinc/cadmium (LCGN\_1867) and an unspecified monosaccharide (LCGN\_0332) were over-expressed with high aeration while a gene involved in copper transport (LCGN\_1427) was repressed. Two genes of the fur regulon (ferrous iron transport) were over-expressed with high aeration, one of them in pure culture (feoA) and one in co-culture (feoB).

With low aeration, the H2O2 synthesis gene sodA was up-regulated by S. aureus. The H2O2 degradation gene ahpC was repressed by high aeration in co-culture but not in pure culture. In pure culture, two genes related to O2 consumption, noxE and LCGN\_0208, were repressed and six genes related to ETC, cydA, cydC, menB, menC, menD, and LCGN\_0364, were over-expressed.

### Impact of Aeration on the Expression of L. garvieae Genes Potentially Involved in the Antagonism against S. aureus

To complete the RNA-seq data obtained only at 9 h, we determined the expression of ten genes from the cultures previously used for RNA-seq analyses, at 6, 9, and 24 h by RT-qPCR. These genes, potentially involved in the antagonism mechanisms, were involved in O2 consumption (noxE and lox), H2O2 synthesis (pox and sodA), H2O2 degradation (ahpC, ahpF, gpx, and trxB1) and resistance to other peroxides (ohrA and ohrR).

The presence of S. aureus induced only slight changes in their expression (data not shown). With high aeration, ahpC and pox were over-expressed 2.4-fold and 2.0-fold in the presence of S. aureus at 6 h. With low aeration, S. aureus induced a 3.1-fold over-expression of trxB1 at 9 h and a 3.3-fold repression of gpx at 24 h.

Conversely, RT-qPCR results showed major differences in H2O2-metabolism gene expression between the two aeration levels (**Table 2**). The expression of ohrA was strongly induced under high aeration conditions in both pure culture and co-culture at 9 h. While RNA sequencing identified trxA2 as up-regulated with high aeration in both pure culture and co-culture at 9 h (Supplementary Table 4), RT-qPCR identified other H2O2-degradation genes (ahpC, ahpF, gpx, and trxB1) as repressed at 6, 9, or 24 h. The lactate mono-oxygenase gene lox seemed slightly repressed with high aeration but this modification was significant only at 9 h in co-culture. In pure culture with high aeration, the expression of noxE reached a peak at 6 h when it was 11.0 times higher than with low aeration. With low aeration, noxE expression gradually increased over time. No significant difference in the expression of H2O2-synthesis genes (pox and sodA) according to aeration level was observed.

### DISCUSSION

This study aimed to improve understanding of the mechanisms underlying the antagonism of L. garvieae against S. aureus, especially as regards H2O2-related pathways.

The high aeration level and the resulting high H2O2 concentration were associated with stronger inhibition of S. aureus, confirming previous observations under the same conditions (Alomar et al., 2008a; Delbes-Paus et al., 2010; Delpech et al., 2015). It was already known that this inhibition is associated with a modulation of the expression of S. aureus virulence genes and a repression of the H2O2 response, stress response and cell division genes of S. aureus by L. garvieae and with high aeration (Delpech et al., 2015). However, the transcriptome adaptation of L. garvieae to prevailing aeration conditions during this interaction had not been explored until

this study. It is known that difference in aeration is associated with drastic modifications of the L. lactis transcriptome (Pedersen et al., 2008; Dijkstra et al., 2014) and consequent metabolic adaptations (Jensen et al., 2001; Nordkvist et al., 2003; Pedersen et al., 2008; Dijkstra et al., 2014; Larsen et al., 2016a,b). In accordance with these findings, we found that different aeration levels were associated with significant differences in the L. garvieae transcriptome in biological processes related to fundamental growth functions, nutritional and metabolic adaptations and transport functions. Moreover, L. garvieae genes involved in fundamental growth functions were slightly repressed by S. aureus under both aeration levels suggesting an impact of S. aureus on L. garvieae metabolism. While L. garvieae is a catalase-negative bacterium, the main S. aureus enzyme involved in H2O2 dismutation is the O2-forming catalase KatA (Cosgrove et al., 2007). The S. aureus catalase may affect the transcriptomic response of L. garvieae to different aeration conditions by modulating O2 and H2O2 concentrations. It is known that the katA gene of S. aureus SA15 is repressed by L. garvieae in high aeration conditions but not in low aeration conditions (Delpech

a Indicated values, at 6, 9, or 24 h in pure culture or in co-culture, corresponds to the ratios of gene expressions with high aeration to gene expressions with low aeration. Only ratios that are significant according to the Newman–Keuls test are shown (∗p-value < 0.1, ∗∗p-value < 0.05). NT, not tested.

et al., 2015). This may explain why the presence of S. aureus (via its catalase production) modulated the L. garvieae transcriptome response under different aeration conditions. Indeed, with low aeration, the L. garvieae H2O2 synthesis gene sodA expression was slightly higher in presence of S. aureus. With high aeration, the main enzymes involved in O2 consumption of L. lactis (NADH-oxidase NoxE and ETC enzymes (Tachon et al., 2010), were repressed only in L. garvieae pure culture. The expression of the L. garvieae noxE reached a peak at 6 h (beginning of exponential growth) and then decreased until 24 h. This suggests that L. garvieae NoxE is the main enzyme responsible for O2 consumption at the beginning of exponential growth with high aeration, as observed for L. lactis NoxE (Lopez de Felipe and Hugenholtz, 2001; Tachon et al., 2010). The induction of most of the L. garvieae ETC genes (cydA, cydB, cydC, menB, menC, menD, menH, and LCGN\_0364) under high aeration conditions at 9 h (beginning of stationary phase) suggested that ETC may be involved in O2 consumption by L. garvieae during the stationary phase, as already shown with L. lactis (Tachon et al., 2009, 2010).

Previous studies have showed that hydrogen peroxide production by L. garvieae depends on aeration level and plays a key role in S. aureus inhibition. As regards S. aureus, Delpech et al. (2015) suggested that the stronger inhibition with a high aeration level may be caused by the higher concentration of H2O2 associated with the L. garvieae-induced repression of S. aureus genes involved in the H2O2 response (katA and sodA at 6 and 9 h) and cell division (mraZ, mraW and potentially the dcw cluster). As regards L. garvieae, our RNAsequencing and RT-qPCR analyses showed an overexpression of the H2O2-degradation genes ahpC, ahpF, gpx, and trxB1 under low aeration conditions compared to high aeration, while the expression of H2O2 synthesis genes pox and sodA remained stable. The expression of the main H2O2 degradation genes of Gram-positive bacteria is generally induced more under aeration and in the presence of H2O2 (Mostertz et al., 2004; Pedersen et al., 2008). Also, H2O2 metabolism may be very different in L. garvieae compared to other Gram-positive bacteria. Although the difference in H2O2 concentration between the two aeration levels was probably primarily conditioned by the availability of O2, our transcriptome results suggest that it was also associated with a control of H2O2 degradation by L. garvieae rather than with a control of H2O2 synthesis. Since the AhpCF peroxyredoxin system was repressed, the OhrAR system was probably essential for the resistance of L. garvieae to ROS (other than H2O2) under the high aeration conditions. The widespread organic hydroperoxide detoxifying system ohrAR, known to be over-expressed under high O2 conditions (Mongkolsuk et al., 1998; Fuangthong et al., 2001; Chuchue et al., 2006; Oh et al., 2007; Atichartpongkul et al., 2010; da Silva Neto et al., 2012; Clair et al., 2013), was consistently induced in L. garvieae at the high aeration level.

The fact that S. aureus population levels were lower in the stationary phase in H2O2-free supernatant from a coculture of S. aureus and L. garvieae revealed the presence of a new molecule involved in this inhibition. This effector is extracellular, is produced by L. garvieae during its exponential growth phase and can reduce the population level of S. aureus during its stationary growth phase. In view of the results of our enzymatic treatments on supernatants, the inhibitory gap during the stationary phase was probably caused neither by hydrogen peroxide, nor by a protein, nor by a lipid, nor by a polysaccharide. The only putative bacteriocin identified in the L. garvieae N201 genome was homologous to garvieaecin Q (GarQ, data not shown), a class IId bacteriocin (Tosukhowong et al., 2012). Class IId bacteriocins are generally sensitive to protease treatments as stringent as the one we used in this study (Kuo et al., 2013; Song et al., 2014), suggesting that garvieacin Q is unlikely to be the effector we are seeking. This effector may instead be related to several genes identified by RNA seq as being regulated by aeration level, such as genes involved in metal homeostasis (e.g., siderophores (Hannauer et al., 2015), chemical and ionic equilibrium (Doyle et al., 1975; Chudobova et al., 2015), transport of vitamin-related compounds (Schlievert et al., 2013) and export of unknown proteins. For example, the differential expression of ferrous ion transport encoding genes has already been observed in L. lactis under oxidative stress in milk (Larsen et al., 2016b). It may also be related to signaling molecules (stress, quorum sensing). It is known that L. garvieae can modify the expression of several S. aureus genes involved in environment-sensing systems like the agr system, CodY or two-component systems SaeRS and SrrAB (Delpech et al., 2015).

RNA-seq revealed variations in the expression of the codY gene (involved in nutritional adaptation (Guédon et al., 2001; Ercan et al., 2015), and of several nutritional-related metabolisms (lysine, threonine, mannitol, aspartate, ribose, fructose, and galactose). This suggests that L. garvieae adapts its nutritional behavior to the prevailing aeration level and the presence or absence of S. aureus. In a rich medium like BHI, there should be little nutritional competition. It is known that L. garvieae can consume all the L-threonine in micro-filtered milk in less than 3 h (Alomar et al., 2008b) and that S. aureus growth could be inhibited by L-threonine depletion (Pohl et al., 2009). However, we showed that the antagonism of L. garvieae against S. aureus was not associated with nutritional competition for L-threonine in micro-filtered milk (see Supplementary Table 5).

### CONCLUSION

RNA sequencing analyses revealed a L. garvieae transcriptome adaptation to aeration level. This adaptation differed depending on the presence or absence of S. aureus. Our findings show that the control of autogenic H2O2 levels by L. garvieae was probably carried out by H2O2 degradation genes rather than H2O2 synthesis genes. Our study also leads us to suggest that an unidentified effector was involved in the inhibition of S. aureus in the stationary phase. The potential inhibitory role of metals, siderophores and signal molecules (e.g., stress signal, quorum sensing) generated by L. garvieae should be investigated. In order to promote the use of H2O2-producing bacteria as bio-preservation agents, future studies should explore the relation between H2O2-metabolism, H2O2-producing LAB and the pathogen they inhibit.

### AUTHOR CONTRIBUTIONS

fmicb-08-00359 March 6, 2017 Time: 16:45 # 9

PD carried out all the experiments, excluding preparation of cDNA libraries and RNA sequencing, and drafted the manuscript helped by CD and SB. ER, ED, and GB analyzed RNA sequencing data. SN performed the RNA sequencing (preparation of libraries and the sequencing itself). GG, M-CM, CD and SB conceived the study. All authors read and approved the final manuscript.

### FUNDING

PD received a Ph.D. fellowship from the FEDER (Fond Européen de Développement Economique et Régional) and the Conseil Régional of the Auvergne region in the framework of the Pharmabiotic Research Institute (PRI) cluster.

### REFERENCES

### ACKNOWLEDGMENTS

The authors would like to thank Adrien Nivoliez for his help in determining the quality of our RNAs in the Probionov laboratory, Magali Cordaillat-Simmons, David Tropel, Laurent Rios, Monique Zagorec, Yves Le Loir, Pierre Renault, Hélène Falentin, and Christophe Chassard for their helpful advice and Maryline Bornes and Harriet Coleman for English proofreading.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2017.00359/full#supplementary-material

and protective genes against organic hydroperoxide stress in Agrobacterium tumefaciens. J. Bacteriol. 188, 842–851. doi: 10.1128/JB.188.3.842-851.2006

reduction in the electron transport chain of Lactococcus lactis. Microbiology 155, 2941–2948. doi: 10.1099/mic.0.029678-0

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Delpech, Rifa, Ball, Nidelet, Dubois, Gagne, Montel, Delbès and Bornes. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Pentocin MQ1: A Novel, Broad-Spectrum, Pore-Forming Bacteriocin From Lactobacillus pentosus CS2 With Quorum Sensing Regulatory Mechanism and Biopreservative Potential

Samson B. Wayah and Koshy Philip\*

Microbiology Division, Faculty of Science, Institute of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia

Micrococcus luteus, Listeria monocytogenes, and Bacillus cereus are major food-borne pathogenic and spoilage bacteria. Emergence of antibiotic resistance and consumer demand for foods containing less of chemical preservatives led to a search for natural antimicrobials. A study aimed at characterizing, investigating the mechanism of action and regulation of biosynthesis and evaluating the biopreservative potential of pentocin from Lactobacillus pentosus CS2 was conducted. Pentocin MQ1 is a novel bacteriocin isolated from L. pentosus CS2 of coconut shake origin. The purification strategy involved adsorption-desorption of bacteriocin followed by RP-HPLC. It has a molecular weight of 2110.672 Da as determined by MALDI-TOF mass spectrometry and a molar extinction value of 298.82 M−1 cm−1 . Pentocin MQ1 is not plasmid-borne and its biosynthesis is regulated by a quorum sensing mechanism. It has a broad spectrum of antibacterial activity, exhibited high chemical, thermal and pH stability but proved sensitive to proteolytic enzymes. It is potent against M. luteus, B. cereus, and L. monocytogenes at micromolar concentrations. It is quick-acting and exhibited a bactericidal mode of action against its targets. Target killing was mediated by pore formation. We report for the first time membrane permeabilization as a mechanism of action of the pentocin from the study against Gram-positive bacteria. Pentocin MQ1 is a cell wall-associated bacteriocin. Application of pentocin MQ1 improved the microbiological quality and extended the shelf life of fresh banana. This is the first report on the biopreservation of banana using bacteriocin. These findings place pentocin MQ1 as a potential biopreservative for further evaluation in food and medical applications.

Keywords: bacteriocin, cell-wall associated bacteriocin, quorum sensing, Lactobacillus pentosus, bactericidal, pore formation, broad-spectrum bacteriocin, biopreservation

# INTRODUCTION

Consumer's requisition for food products containing less chemical preservatives (Barbosa et al., 2017) and emergence of antibiotic resistance among pathogenic and food spoilage bacteria prompted the search for novel antimicrobials (Berendonk et al., 2015; Jia et al., 2017). Bacteriocins are an attractive class of natural antimicrobials with potential for future use as a synergistic

### Edited by:

Aldo Corsetti, Università di Teramo, Italy

### Reviewed by:

Daniela Fiocco, University of Foggia, Italy Fohad Mabood Husain, King Saud University, Saudi Arabia

> \*Correspondence: Koshy Philip<EMAIL_ADDRESS>
### Specialty section:

This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

Received: 08 December 2017 Accepted: 12 March 2018 Published: 27 March 2018

### Citation:

Wayah SB and Philip K (2018) Pentocin MQ1: A Novel, Broad-Spectrum, Pore-Forming Bacteriocin From Lactobacillus pentosus CS2 With Quorum Sensing Regulatory Mechanism and Biopreservative Potential. Front. Microbiol. 9:564. doi: 10.3389/fmicb.2018.00564 combination with antibiotics or complete replacement of antibiotics (Behrens et al., 2017; Collins et al., 2017) and currently used chemical preservatives (Kaškoniene et al., 2017; Wiernasz et al., 2017). This is due to their ability to inhibit some drugresistant pathogens (Mathur et al., 2017). These interesting antimicrobial peptides are of bacterial origin and are ribosomally synthesized (Langa et al., 2017). They have narrow or broad spectrum of inhibitory activity (Hanchi et al., 2017).

Bacteriocins are a highly diverse group of antimicrobial peptides. They have variations in molecular weight, inhibitory spectrum, mode of action, mechanism of biosynthesis and externalization, and self-protection mechanism (Salazar et al., 2017). They are part of the inherent defense system of bacteria and play other roles such as niche colonization, direct killing of competing strains and signaling (cross-talk and quorum sensing) within bacterial communities (Dobson et al., 2012; Inglis et al., 2013; Yang et al., 2014). Bacteriocins are commonly classified into two groups namely class I (undergo posttranslational modification) and class II (unmodified). In less popular classification schemes class III (high molecular weight and heat-sensitive bacteriocins) (Alvarez-Sieiro et al., 2016) and class IV (bacteriocins with carbohydrate or lipid moieties) (Kaškoniene et al., 2017) were introduced. Bacteriocins are produced by lactic acid bacteria (LAB) and non-lactic acid bacteria (Mechoud et al., 2017). LAB bacteriocins are given more attention because they are generally recognized as safe (GRAS) facilitating their use in situ and ex situ in preservation of foods (Bali et al., 2016; Castro et al., 2017; Hu et al., 2017). Moreover, they are inactivated by gut proteases, heat-stable, active at various pH, potent even at nanomolar concentration and their biosynthetic gene cluster is often plasmid-borne, facilitating the use genetic engineering approaches in improving production (Cotter et al., 2013; Lakshminarayanan et al., 2013; Messaoudi et al., 2013; Woraprayote et al., 2016).

Biopreservation involves the use of microorganisms or their products and other natural bio-products to enhance safety and extend shelf life of food. This is achieved either by killing or reduction of the load of food spoilage microorganisms (Johnson et al., 2017; Saraoui et al., 2017). The concept of biopreservation of food has recently intensified due to growing consumer inclination toward foods containing biopreservatives or less synthetic chemical preservatives, fear of side effects of currently used chemical preservatives, demand for fresh-tasting and less processed food (Kashani et al., 2012; Woraprayote et al., 2016; Barbosa et al., 2017). Fruits and vegetables are one the major reservoirs of minerals, vitamins, and fiber and are consumed worldwide. Fresh fruits such as banana have short shelf life due to their high moisture content (Joardder et al., 2014). Preservation of banana is a huge task especially if required fresh. Moreover, eating them fresh exposes consumers to food-borne pathogens (Berger et al., 2010; Tian et al., 2012).

It has been suggested that live cultures of LAB can be used to prevent the growth of food spoilage and pathogenic bacteria on the surfaces of fruits and vegetables (Trias et al., 2008). In situ biopreservation of foods has not been quite successful due to two main reasons. Firstly, difficulty in getting the LAB strain adapted and established to the new environment. Secondly, difficulty in the production of an effective concentration of bacteriocin required to control spoilage and food-borne pathogens. However, a few breakthroughs have been reported. Leuconostoc mesenteroides was effective at inhibiting the growth of Listeria monocytogenes on fresh apples and lettuce (Trias et al., 2008). The Safety and shelf life of minimally processed lettuce and apples were improved by the inoculation of two strains of Lactobacillus plantarum (Siroli et al., 2015). A bacteriocinogenic strain of Lactococcus lactis inhibited the growth of yeasts and L. monocytogenes in minimally processed apples. Furthermore, extension of shelf life was observed (Siroli et al., 2016). Growth of L. monocytogenes and Salmonella on minimally processed pear was controlled by the inoculation of Lactobacillus rhamnosus GG (Iglesias et al., 2017). Addition of L. rhamnosus GG to fresh pear was an effective strategy in controlling the growth of L. monocytogenes (Iglesias et al., 2018).

Bacteriocin was first discovered in early 1925 when antagonistic activity was observed among strains of Escherichia coli (Ghazaryan et al., 2014). The discovery of colicin as the first bacteriocin was closely followed by that of nisin (1928), the first LAB bacteriocin (Shin et al., 2016). Despite the long history of LAB bacteriocins only nisin and pediocin PA-1/AcH have gained approval for preservation of selected foods (Saraniya and Jeevaratnam, 2014; Barbosa et al., 2017). The potential of bacteriocins in the biopreservation of fresh fruits or minimally processed fruits has been highly underexploited. Combined application of nisin-EDTA and chlorine was effective at reducing the surface microbial load of whole melon (Ukuku and Fett, 2002). Application of nisin, hydrogen peroxide, citric acid and sodium lactate effectively reduced the transfer of pathogens from the surface of melons to freshly cut pieces (Ukuku et al., 2005). Load of pathogens on the surfaces of minimally processed mangoes was controlled by packaging in nisin films (Barbosa et al., 2013). Enterocin AS-48 was effective at controlling contamination of raw fruits by L. monocytogenes (Molinos et al., 2008). Enterocin 416K1 inhibited the growth of L. monocytogenes on apples and grapes (Anacarso et al., 2011). The potential of preserving minimally processed papaya by applying alginate coatings containing pediocin has been demonstrated (Narsaiah et al., 2015). Biopreservation of fresh banana using bacteriocin has not been investigated.

Although Lactobacillus pentosus has been isolated from various sources, bacteriocinogenic strains are rare (Liu et al., 2008). A bacteriocin-producing strain of L. pentosus with probiotic potential has been reported (Aarti et al., 2016). Bacteriocins of L. pentosus origin have not been adequately studied. Pentocins have been poorly characterized and their regulatory mechanisms have not been sufficiently investigated. Their modes of action are unknown. Moreover biopreservation of fresh banana using L. pentosus-derived bacteriocins (commonly called pentocins) has not been studied. In this

**Abbreviations:** CFS, Cell-free supernatant; FU, Fluorescence unit; HIC, Hydrophobic interaction chromatography; LAB, Lactic acid bacteria; MALDI-TOF, Matrix-assisted laser desorption ionization time-of-flight; MIC, Minimum inhibitory concentration; MRS, De Man, Rogosa and Sharpe; RP-HPLC, Reversedphase high performance liquid chromatography; SDS, Sodium dodecyl sulfate.

study a novel bacteriocin (pentocin MQ1) from L. pentosus CS2 of coconut shake origin was purified to homogeneity and characterized. Its regulatory mechanism and mode of action was investigated. Finally, its ability to preserve fresh banana was studied in-vivo.

# MATERIALS AND METHODS

# Bacterial Strains and Culture Media

Streptococcus pyogenes, Enterococci, Bacillus cereus, Micrococcus luteus, and L. lactis were obtained from American Type Culture Collection (ATCC). L. monocytogenes NCTC 10890 was obtained from National Collection of Type Culture (NCTC). Staphylococcus aureus RF122, Streptococcus mutans GEJ11, Pseudomonas aeruginosa PA7, Corynebacterium spp. GH17, E. coli UT181, L. plantarum K25, and L. pentosus CS2 were taken from the culture collection of Microbial Biotechnology Laboratory, Division of Microbiology, Institute of Biological Science, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia. L. plantarum K25 and L. pentosus CS2 were maintained on MRS agar (Merck, Darmstadt, Germany). S. pyogenes ATCC 12344 and S. mutans GEJ11 were maintained on Todd-Hewitt agar (Difco, Le Pont de Claix, France). M. luteus ATCC 10240, B. cereus ATCC 14579, S. aureus RF122, P. aeruginosa PA7, Corynebacterium spp. GH17 and E. coli UT181 were maintained on Mueller-Hinton agar (Merck, Darmstadt, Germany). Enterococcus faecium ATCC BAA-2127 and E. faecium ATCC 349 were maintained on Tryptic soy agar (Merck, Darmstadt, Germany) while other enterococcal strains and L. monocytogenes NCTC 10890 were maintained on Brainheart infusion agar (Merck, Darmstadt, Germany). L. lactis ATCC 11454 was maintained on M17 agar (Merck, Darmstadt, Germany) supplemented with 5% glucose (Merck, Darmstadt, Germany).

# Isolation and Screening of LAB for Bacteriocin Production

Indigenously sourced coconut shake was inoculated into freshly prepared De man Rogose and Sharpe (MRS) broth and incubated at 37◦C for 24 h. The culture was serially diluted in peptone water and LAB was isolated by growing on MRS agar plate (Merck Germany) at 37◦C. MRS broth was inoculated with single colonies from a 24 h old MRS agar LAB culture and incubated aerobically at 37◦C for 24 h. Screening of LAB for bacteriocin production was carried out using well diffusion assay in which cell-free supernatant (CFS) was tested for inhibitory activity against M. luteus ATCC 10240, L. monocytogenes NCTC 10890, B. cereus ATCC 14579, and S. aureus RF122. MRS agar used for well diffusion assay was supplemented with 0.1% CaCO3 (Friedemann Schmidt Chemical, Germany) to neutralize acidity due to organic acids.

Molecular identification of LAB was conducted by amplifying 16S rRNA gene via PCR using the universal primers 27F [5′ -AGAGTTTGATC(A/C)TGGCTCAG-3′ ] and 1492R [5′ - ACGG(C/T)TACCTTGTTACGACTT-3′ ]. The 16S rRNA gene was sequenced and similarity search was performed using NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

# Purification, Determination of Molecular Weight, and Molar Extinction Coefficient

Bacteriocin was purified using adsorption-desorption approach followed by reversed-phase high performance liquid chromatography (RP-HPLC). A 24-h old culture of L. pentosus CS2 was subcultured in freshly prepared MRS broth in a bioreactor (Sartorius Stedim, Germany). The Bioreactor was set up (agitation at 150 rpm, temperature at 37◦C) and run for 20 h after which the culture was collected and the pH was adjusted to 5.8 and allowed for 1 h. The culture was centrifuged (9,000 × g, for 20 min at 4◦C) and the cell pellet re-suspended in 95% methanol (Merck, Darmstadt, Germany) with pH-value adjusted to 2. The cell suspension was stirred overnight at 4◦C and subsequently centrifuged (9,000 × g for 30 min at 4◦C) to obtain the supernatant which was filtered using 0.22µm sterilized cellulose membrane (Millipore). The clear supernatant was evaporated to dryness at 40◦C using a water bath and the crude bacteriocin was reconstituted in ultrapure water. Inhibitory activity was tested using well diffusion assay and crude bacteriocin was subjected to RP-HPLC containing SemiPrep RP-18e 100-10 mm column. The mobile phase consisted of two solvents: A (95% Mili-Q water Millipore, USA) and 5% acetonitrile (Merck, Germany) and B (100% acetonitrile). Elution was done using a biphasic gradient of 20–80% acetonitrile at a flow rate of 1 ml/min over 65 min. Fractions were collected and evaporated using a vacuum evaporator. Antibacterial activity of HPLC fractions were was tested. Molecular weight of the bacteriocin was determined by subjecting the active HPLC fraction to MALDI-TOF mass spectrometry. To ascertain the molar extinction coefficient, 2-fold dilutions of the bacteriocin were prepared and bacteriocin concentration was expressed in molar units. Absorbance at 280 nm was measured and a standard curve was generated from which the molar extinction coefficient was determined.

# Antibacterial Spectrum

This experiment was done to ascertain the inhibitory spectrum of pentocin MQ1. Bacteriocin producer was grown in MRS broth for 20 h and CFS (400 AU/ml) was used in well diffusion assay to test antibacterial activity against selected targets. All agar plates were supplemented with 0.1% CaCO3 (Friedemann Schmidt Chemical, Germany) to neutralize acidity.

# Bacteriocin-Cell Wall Association Assay

An investigation was done to assess the association of pentocin MQ1 with the cell wall of its producer. Overnight broth culture of bacteriocin producer was centrifuged (9,000 × g for 20 min) at 4◦C. Antibacterial activity of CFS was tested using well diffusion assay. The cell pellet was re-suspended in 95% methanol (Merck, Darmstadt, Germany) adjusted to pH = 2 and stirred overnight at 4◦C on a magnetic stirrer. The cell suspension was centrifuged (9,000 × g for 30 min at 4◦C) and the supernatant was filtered using a Millipore filter (0.22µm). This was followed by evaporation of methanol on a water bath at 40◦C. The cell extract was reconstituted in ultrapure water and antibacterial activity was tested.

### Bacteriocin Stability Test

In order to ascertain the stability of pentocin MQ1, bacteriocin preparation was exposed to different temperatures: 40, 60, and 80◦C for 40 min; 100 and 121◦C for 15 min. Samples were cooled to room temperature before testing antibacterial activity. Stability of bacteriocin to different enzymes (Sigma-Aldrich, St. Louis, USA) namely: proteinase K, lysozyme, pepsin, lyticase, catalase, trypsin, α-chymotrypsin, protease, proteinase, and hyaluronidase was tested. This was achieved by adding different enzyme preparations to a final enzyme concentration of 1 mg/ml and incubating for 1 h at 37◦C. Inhibitory activity was tested afterwards. Bacteriocin was adjusted to various pH (2, 3, 5, 8, and 10) and incubated for 2 h at room temperature. Antibacterial activity was tested. Stability of bacteriocin upon exposure to different chemicals viz: 1% (v/v) Tween 80, 1% (v/v) Tween 20, 1% (w/v) sodium dodecyl sulfate (SDS) (Fisher scientific, New Jersey, USA) and 1% (v/v) triton X-100 was investigated. These chemicals were added to the bacteriocin and incubated for 2 h at room temperature after which antibacterial activity was tested.

### Plasmid Isolation

To investigate if plasmids harbor the bacteriocin structural gene or not, plasmid isolation was carried out. This was done using easy pure R plasmid miniprep kit (TransGen Biotech, Beijing) according to manufacturer's instruction.

### Regulatory Mechanism

This experiment was done to understand the regulatory system of pentocin MQ1 production. The bacteriocin was semi-purified using ammonium sulfate precipitation. This was achieved by the following procedure. An overnight MRS broth culture of the bacteriocin-producing phenotype of L. pentosus CS2 was centrifuged (9,000 × g, for 20 min at 4◦C). The supernatant was collected and filtered using 0.22µm sterilized cellulose membrane (Millipore) to obtain CFS. The CFS was subjected to 80% ammonium sulfate precipitation after which it was centrifuged at the same condition to obtain the precipitate. The resulting active ammonium sulfate precipitate (semi-purified bacteriocin) was dissolved in minimum ultrapure water. Another semi-purified form of the bacteriocin was obtained through hydrophobic interaction chromatography (HIC) by the following method. Active CFS from an overnight culture of the bacteriocinproducing phenotype of L. pentosus CS2 was subjected to HIC. Acetonitrile (Merck, Darmstadt, Germany) gradient (20, 40, 60, and 80% v/v) was used for elution of the bacteriocin adsorbed onto the surfaces of amberlite XAD-16 particles (Sigma-Aldrich, St. Louis, USA) packed in a glass column. Fractions were evaporated and antibacterial activity was determined using well diffusion assay. The active fraction from HIC was used in the next experiment. In order to investigate the regulatory mechanism of pentocin MQ1 production a bacteriocin-negative (bac−) phenotype was generated using the following procedure. Ten milliliters (10 ml) of fresh MRS broth was inoculated with colonies from an overnight culture of the bacteriocin-producing phenotype of L. pentosus CS2 and incubated at 37◦C for 20 h. Cell pellet was collected by centrifugation at 2,000 rpm for 5 min. It was re-suspended in saline solution (0.85%) and washed three times at the same condition to produce a bac− phenotype of L. pentosus CS2. Thereafter, 100 µl of this bacterial suspension was added to 900 µl of fresh MRS broth in 2 ml Eppendorf tube. This was followed by the addition of 50 µl of 0.21µM pentocin MQ1, active ammonium sulfate precipitate (8 AU/ml), and the active fraction from HIC (8 AU/ml). These tubes were marked as "induced" while tubes that do not contain the bacteriocin were marked as "control." All tubes were incubated at 37◦C for 20 h after which 50 µl of 0.21µM pentocin MQ1was added to the control tube. CFS from all tubes was tested for antibacterial activity. Induction of pentocin MQ1 production was said to occur if CFS from an induced tube produced inhibition zone while the control tube did not.

### Minimum Inhibitory Concentration

Minimum inhibitory concentration (MIC) was determined by employing the broth microdilution assay as described by Mota-Meira et al. (2000) with little modifications. Two-fold dilutions of bacteriocin were prepared in adequate media and 10 µl for pentocin MQ1 was added to 96-well microtiter plate. Overnight culture of indicator bacteria was diluted (1 × 108 CFU/ml) and added to 150 µl of each bacteriocin preparation. Wells containing indicator without pentocin MQ1 were used as positive control while wells containing only the media were used as blank. Incubation was done at 37◦C and optical density at 600 nm was monitored with a multiskan GO microplate reader (Multiskan GO, Thermo Scientific) over a period of 24 h. MIC was defined as the bacteriocin preparation which caused growth reduction by more than 90% compared with the positive control.

# Mode of Action

### Time-Killing

This assay was done to investigate the mode and speed of action of pentocin MQ1. Indicators were grown for 10 h and centrifuged (2,000 rpm for 5 min) to collect cell pellet. Each cell pellet was resuspended in ice-cold 5 mM sodium phosphate buffer (pH 7.2) and washed twice. The cell suspension was mixed at a ratio of 1:1 with the bacteriocin preparation (5 X MIC) and incubated at 37◦C. Control consisted of bacterial suspension without the addition of bacteriocin. Experiments were done in triplicates. Growth was monitored over a period of 120 min.

### Membrane Permeabilization

Pore formation assay was done to understand the mechanism of action of pentocinMQ1. M. luteus was grown in Mueller-Hinton broth until OD600nm = 0.45 after which 5µM SYTOX green dye (Invitrogen, USA) was added. Ninety microliters of stained bacteria was added to MicroAmp Fast Optical 96-well reaction plate (Applied Biosystems, Life Technologies, USA). After a stable line base was attained, 10 µl of pentocin MQ1 (5 X MIC) was added to the stained bacteria. Sodium phosphate buffer (5 mM) and nisin (Sigma-Aldrich, USA) were added to stained bacteria in different wells to serve as negative and positive controls, respectively. Experiments were done in triplicates. Fluorescence as a result of binding of SYTOX green to leaked intracellular DNA was monitored using Real-Time PCR (Applied Biosystems, USA).

### Biopreservation of Banana

To investigate the biopreservative potential of pentocin MQ1, a bacteriocin preparation (66.4µM) was topically applied to mature, fresh banana samples. This was achieved by soaking sterile cotton swab in the bacteriocin preparation and gently rubbing the surfaces of banana samples with it. Some pentocin MQ1-treated banana samples were kept at ambient condition while others were refrigerated. Control samples consisted of non-pentocin MQ1-treated banana samples kept at ambient condition and others refrigerated. Samples were monitored for morphological changes. At the onset of deterioration of control samples, sterile cotton swabs were used to collect surface microflora of both control and pentocin MQ1-treated banana samples and bacterial count (CFU/ml) was measured. The experiment was allowed to proceed until the onset of deterioration of bacteriocin-treated banana samples. Shelf-life was measured. Experiments were done in triplicates.

# RESULTS

# Isolation and Screening of LAB for Bacteriocin Production

Sixteen different strains of LAB were isolated and identified based on 99% sequence homology. The 16S rRNA gene sequences of all isolated LAB strains were deposited in the NCBI database. L. pentosus CS2 with accession number MG976651 exhibited the strongest antibacterial activity and broadest antibacterial spectrum. The remaining 15 LAB strains and their accession numbers are Lactobacillus fermentum HFCS1(MG966463), Weissella cibaria BavoCS3 (MG976765), Weissella confusa PBCS4 (MG976757), W. confusa CS5 (MG976685), W. cibaria CS6 (MG976766), W. confusa CS7 (MG976686), Lactobacillus nagelii CS8SB (MG976652), W. confusa CS9 (MG980308), L. fermentum CS10 (MH032758), L. fermentum CS11 (MG966465), L. fermentum CS12 (MG966459), L. fermentum CS13 (MG966468), L. fermentum CS14 (MG980307), W. cibaria CS15 (MG982483), and L. fermentum CS16 (MG980073).

### Purification, Determination of Molecular Weight, and Molar Extinction Coefficient

Purification of bacteriocin by a combination adsorptiondesorption method and RP-HPLC proved successful. Bacteriocin was obtained at a retention time of 31–33 min (**Figure 1**). The purified bacteriocin resulting from RP-HPLC had a purification yield of 1.6% (**Table 1**). MALDI-TOF mass spectrometry revealed that the molecular weight is 2110.672 Da (**Figure 2**). A molar extinction coefficient of 298.82 M−1 cm−1 was obtained.

### Antibacterial Spectrum

Pentocin MQ1 displayed strong inhibitory activity toward L. monocytogenes NCTC 10890, M. luteus ATCC 10240 and B. cereus ATCC 14579. It was also inhibitory albeit to a less extent toward S. pyogenes ATCC 12344, S. aureus RF122, P. aeruginosa PA7, E. faecium ATCC 19434, E. faecium ATCC 27270, E. faecium ATCC 27273, E. faecium ATCC BAA-2318, E. faecium ATCC BAA-2127, E. faecium ATCC 6569, E. faecium ATCC 25307, and E. faecium ATCC 349 but was not active against S. mutans GEJ11, L. lactis ATCC 11454 and Corynebacterium spp. GH17 (**Table 2**).

# Bacteriocin-Cell Wall Association Assay

This experiment was designed to know whether pentocin MQ1 abounds in the supernatant or cell wall of the producer. Of the total activity of 6.9 × 104 AU, 66.67% (4.6 × 104 AU) was detected in the cell extract while 33.33% (2.4 × 104 AU) was found in the CFS (**Table 3**).This shows the cell-wall adhering characteristic of pentocin MQ1.

### Bacteriocin Stability Test

Stability of pentocin MQ1 under different conditions of heat, enzyme and pH are shown in **Table 4**. Its stability when exposed to different chemicals (1% Tween 80, Tween 20, SDS and triton X-100) are not shown in **Table 4** because it retained 100% residual activity. Residual activities of 99.82, 97.99, 91.32, 90.78, and 83.11% were obtained after heating at 40, 60, 80, 100, and 121◦C revealing its high thermal stability. Proteinase K, pepsin, and proteinase significantly reduced its activity (**Table 4**). There was a complete loss of activity when it was treated with trypsin, αchymotrypsin, and protease. Pentocin MQ1 retained its activity after exposure to lyticase, catalase, and hyaluronidase (**Table 4**). pH variation had effect on its activity. It had higher activity in the pH range of 2–5 than at pH-value of 8. There was no activity at pH-value of 10 (**Table 4**).

# Plasmid Isolation

This experiment was done to ascertain if genes encoding pentocin MQ1 production are plasmid-borne. After agarose gel electrophoresis, clear bands were observed for the 1 kb molecular ladder but no band was seen for L. pentosus CS2. This indicates the absence of plasmids in L. pentosus CS2 (Supplementary Figure 1).

### Regulatory Mechanism

This assay was done to investigate the regulatory mechanism of pentocin MQ1 production by L. pentosus CS2. A bacteriocinnegative (bac−) phenotype of L. pentosus CS2 was produced by washing off pentocin MQ1 from the cell wall of the producer. Addition of inducible concentrations (quorum) of active ammonium sulfate precipitate, active HIC fraction and pure pentocin MQ1 to the bac− phenotype of L. pentosus CS2 restored pentocin MQ1 production. This was made evident by the presence of inhibition zones for the induced tubes but the absence of zone of inhibition for the non-induced (control) tubes (Supplementary Figure 2).

### Minimum Inhibitory Concentration

Pentocin MQ1 exhibited strong inhibitory effect against L. monocytogenes NCTC 10890, M. luteus ATCC 10240, and B. cereus ATCC 14579. MIC-value for M. luteus and L. monocytogenes and B. cereus were 1.66, 1.66, and 3.32µM, respectively.

FIGURE 1 | Reversed-phase HPLC chromatogram of crude cell extract from Lactobacillus pentosus CS2. Bacteriocin released into MRS broth was adsorbed onto the cell wall of the producer by increasing the pH of the medium to 5.8 followed by desorption from the cell wall by lowering the pH to 2. Total crude bacteriocin obtained was subjected to RP-HPLC. The vertical lines indicate the retention time.

TABLE 1 | Purification of bacteriocin using adsorption-desorption followed by RP-HPLC.

### Mode of Action Time-Killing

Pentocin MQ1 caused a decline in the Log10 CFU/ml of L. monocytogenes and B. cereus (**Figure 3**). After 120 min the Log10 viable cell count for L. monocytogenes had decreased from 10.27 to 1.80 (82.47% reduction) while that of B. cereus had decreased from 9.27 to 3.10 (66.56% reduction).

### Membrane Permeabilization

Treatment of M. luteus with pentocin MQ1 caused an increase in fluorescence intensity over the course of the study indicating pore formation. Similar observation was made for nisin although higher fluorescence intensity was observed. Fluorescence intensity of the untreated bacterial cells remained stable (**Figure 4**).

### Biopreservation of Banana

Total surface bacterial count and LAB count of 4.00 × 107 CFU/ml and 2.10 × 103 CFU/ml (0.005% of total bacterial count), 3.70 × 105 CFU/ml and 9.40 × 102 CFU/ml (0.254% of total bacterial count), 2.14 × 104 CFU/ml and 1.76 × 103 CFU/ml (8.22% of total bacterial count), 7.30 × 102 CFU/ml and 3.20 × 102 CFU/ml (43.84% of total bacterial

count) were obtained for nonbacteriocin-treated sample stored at ambient condition, nonbacteriocin-treated sample stored at refrigeration condition, pentocin MQ1-treated sample stored at ambient condition and pentocin MQ1-treated sample stored at refrigeration condition, respectively (**Table 5**). The shelf life of nonbacteriocin-treated sample stored at ambient condition, nonbacteriocin-treated sample stored at refrigeration condition, pentocin MQ1-treated sample stored at ambient condition and pentocin MQ1-treated sample stored at refrigeration condition are 3, 5, 7, and 11 days, respectively. Total surface bacterial count and shelf had a Pearson correlation coefficient (r) value TABLE 2 | Antibacterial Spectrum of pentocin MQ1.

+ + ++ Inhibition zone >20mm, + + + Inhibition zone 15–20 mm, ++ Inhibition zone <15mm, – No inhibition.

TABLE 3 | Pentocin MQ1 recovered from the cell-free supernatant and cell extract of Lactobacillus pentosus CS2.

of −0.779 indicating a strong inverse relationship between the two parameters. An r-value of 0.863 was obtained for Pearson correlation analysis between percentage of LAB and shelf life of banana suggesting a strong direct relationship between the two parameters. Changes in organoleptic characteristics of nonbacteriocin-treated banana occurred much earlier than in bacteriocin-treated samples (**Figure 5**).These results show that treatment of banana with pentocin MQ1 extended its shelf. The microbiological quality and shelf life of pentocin MQ1-treated banana stored at refrigeration condition was better than that of pentocin MQ1-treated banana stored at ambient condition.

### DISCUSSION

Bacteriocin-producing LAB confer various beneficial effects (such as improvement of quality and shelf life extension) on dairy products (Sultan et al., 2017). As such, the presence of L. pentosus CS2 in coconut shake suggests it has bioprotective role. Although L. pentosus CS2 has been isolated from vagina (Okkers TABLE 4 | Stability tests for pentocin MQ1.

et al., 1999), fermented Xuan-Wei ham (Zhang et al., 2009) and fermented shrimp (Watthanasakphuban et al., 2016), this is the first report of its isolation from coconut shake considered as a dairy product. Purification of pentocin MQ1 by sequential use of adsorption-desorption method and RP-HPLC proved successful. At low pH bacteriocins are released into the culture medium but when pH is increased to around 5.8–6.0 they become adsorbed onto the producer cells. This phenomenon was observed in this study due to the fact that no activity was detected in the CFS after the adsorption process. Adsorption-desorption approach has been used previously in the purification of some bacteriocins (Siying et al., 2000). Adsorption-desorption method has some advantages over traditional approaches such ammonium sulfate precipitation. These include reduced time of processing, purer crude bacteriocin and cheap running cost (Mu-xu and Zhi-jiang, 2009; Jia-qi et al., 2011). Based on the retention time of pentocin MQ1 which corresponds to high concentration of acetonitrile (high hydrophobicity), it can be deduced that it contains slightly more hydrophobic amino acid residues than polar or hydrophilic ones.

MALDI-TOF mass spectrometry revealed that the molecular weight of pentocin MQ1 is 2110.672 Da. There are only a few reports on purification of bacteriocin from L. pentosus. To date pentocins that have been successfully purified to homogeneity

and molecular weight accurately determined are pentocin TV35b (3929.63 Da) (Okkers et al., 1999), pentocin 31-1 (5592.225 Da) (Zhang et al., 2009), and bacteriocin K2N7 (2.017 kDa) (Watthanasakphuban et al., 2016). The molecular weight of pentocin MQ1 does not match with any of the reported pentocins. Hence, it is a novel pentocin. Molar extinction coefficient is an important biophysical parameter that can facilitate the quantitation and future industrial application of pentocin MQ1.

Pentocin MQ1 displayed a broad spectrum of antibacterial activity. This attribute has been observed in cerein 7 (Oscáriz et al., 1999), enterocin P (Cintas et al., 1997), and enterocin LR/6 (Kumar and Srivastava, 2010). It was reported that pentocin TV35b is not inhibitory toward B. cereus (Okkers et al., 1999). Liu et al. (2008) also reported that pentocin 31-1 is a broad spectrum bacteriocin with inhibitory activity against L. monocytogenes, B. cereus, S. aureus and E. coli. Watthanasakphuban et al. (2016) reported that bacteriocin K2N7 has a narrow spectrum of antibacterial activity and was not inhibitory against L. monocytogenes, B. cereus, S. aureus, E. coli, and E. faecium. Pentocin MQ1 is different from pentocin TV35b, pentocin 31-1 and bacteriocin K2N7 in that in addition to the aforementioned bacterial targets it is also inhibitory against M. luteus, S. pyogenes, P. aeruginosa, and E. faecium. It also showed inhibitory activity against closely related species L. plantarum K25. Bacteriocins from many LAB strains have been found to inhibit the growth of both closely related and distantly-related bacterial strains (Müller et al., 2009). Broad spectrum of antibacterial activity is one of the important criteria for selection of bacteriocin for use in the biopreservation of foods (Johnson et al., 2017; Kaškoniene et al., 2017). The broad antibacterial spectrum of pentocin MQ1 well positions it as a good candidate for preservation of various types of foods.

Investigating the association of pentocin MQ1 with the cell wall of the producer is important because it can reveal whether the bacteriocin abound in the supernatant or on the cell wall. In this study more activity was detected on the cell wall than

TABLE 5 | Effect of bacteriocin application on surface bacterial count and shelf-life of banana.

P, pentocin MQ1; C, negative control; A, ambient condition; R, refrigerated.

the supernatant indicating the cell wall-binding characteristic of pentocin MQ1. This finding shows that for a better recovery of pentocin MQ1 produced by L. pentosus CS2, an adsorptiondesorption approach facilitated by pH modifications should be employed. In this study, an adsorption-desorption approach suitable for purification of pentocin MQ1 was demonstrated. Association of bacteriocin with the cell wall of the producer is thought to enhance niche competition. Cell-wall associated bacteriocins have also been described in Lactobacillus crispatus, Streptococcus salivarius, and Streptococcus bovis HC5 (Tahara and Kanatani, 1997; Mantovani et al., 2002; Barbour and Philip, 2014).

Pentocin MQ1 was highly stable to all chemical treatments investigated. This is evidenced by its retention of 100% residual antibacterial activity. It exhibited high thermal and pH stability. Higher activity was detected in the acidic pH range (2– 5) while moderately alkaline pH (pH 8) caused a drastic reduction in activity. Lack of activity at pH 10 (high alkaline pH) indicates severe denaturing of pentocin MQ1. Pentocin MQ1 is a proteinaceous biomolecule due to its susceptibility to proteinases. Retention of high antibacterial activity after exposure to lyticase, catalase and hyaluronidase provides more evidence on its proteinaceous nature. Pentocin TV35b was active in the pH range of 1–10 and after heating at 60– 100◦C (Okkers et al., 1999). Pentocin 31-1 was active at pH 2–10 and at 60–121◦C but sensitive to SDS (Liu et al., 2008). Bacteriocin K2N7 retained activity at pH 2–12 but unlike pentocin MQ1 it was inactive at 121◦C (Watthanasakphuban et al., 2016). The combined attributes of chemical, pH and thermal stability of pentocin MQ1 favors its future application in food systems subjected to harsh processing conditions (Hemu et al., 2016; Yi et al., 2016). Its sensitivity to proteases is a desirable characteristic in that its chances of inhibiting beneficial components of the gut microbiota is reduced thereby, enhancing its safety (Zacharof and Lovitt, 2012; Hemu et al., 2016). Moreover, degradation of bacteriocin by proteases reduces the time of interaction between fragments of a given bacteriocin and its target thereby decreasing the possibility of resistance development (Perez et al., 2014). Its application in the treatment of gut infection would require encapsulation in nanoparticles or bioengineering to make it resistant to protease of the gut (Zhang L. et al., 2010; Arthur et al., 2014; Cavera et al., 2015).

nonbacteriocin-treated stored at refrigeration condition (C) pentocin MQ1-treated stored at ambient condition (D) pentocin MQ1-treated stored at refrigeration condition.

Genetic element harboring L. pentosus-derived bacteriocin has not been reported. Genes encoding bacteriocin production have been detected on plasmids and chromosomes (Garcia et al., 2010). Absence of plasmids in L. pentosus CS2 suggests that genes encoding pentocin MQ1 production are chromosomeborne. It is thought that chromosome-encoded bacteriocin genes are more stable than plasmid-encoded bacteriocin genes because plasmids, being small and mobile genetic elements can be lost by leaking out of bacterial cells (Sengupta and Austin, 2011). Hence, bacteriocin-producing LAB strains harboring chromosome-borne bacteriocin genes have an edge over those with plasmid-borne bacteriocin genes. Thus, L. pentosus CS2 is genetically stable. Chromosome-borne bacteriocins include enterocin A (Aymerich et al., 1996) and ABP-118 (Flynn et al., 2002) and acidocins LF221 (Majhenic et al., 2003 ˇ ).

Pentocin MQ1 production was restored when active ammonium sulfate precipitate, active HIC fraction and pure pentocin MQ 1 were added separately to the bac− phenotype of L. pentosus CS2. This shows that pentocin MQ1 production is auto-inducible suggesting its regulation by a two-component quorum sensing mechanism involving an inducing peptide (the bacteriocin), a histidine protein kinase (HPK) and a response regulator (RR). Pentocin MQ1 acts as the inducing peptide. Upon attaining an inducible concentration (quorum) the bacteriocin binds to HPK and activates it. Activated HPK phosphorylates RR which in turn binds to the promoter of bacteriocin genes and upregulates bacteriocin production (Chanos and Mygind, 2016). Nisin production is regulated by a similar mechanism (Dobson et al., 2012). Pentocin 31-1 production is also controlled by quorum sensing (Zhang et al., 2012). Regulation of bacteriocin production via quorum sensing mechanism is commonly found among class II bacteriocins. (Straume et al., 2007; Di Cagno et al., 2010, 2011).

Pentocin MQ1 was strongly inhibitory against L. monocytogenes NCTC 10890, M. luteus ATCC 10240, and B. cereus ATCC14579 at micromolar concentrations. High activity at low concentration is a desirable property of natural biopreservatives (Bali et al., 2016). MIC-values for nisin A against Micrococcus spp. and Bacillus spp. (Mota-Meira et al., 2000) are lower than that of pentocin MQ1. However, the broad antibacterial spectrum of pentocin MQ1 suggests wide food and medical applications. Pentocin MQ1 exhibits a bactericidal mode of action against L. monocytogenes and B. cereus. After 120 min the viable cell count for L. monocytogenes and B. cereus had been reduced significantly. This shows the quick-acting characteristic of pentocin MQ1 against these pathogens. Pentocin 31-1 was also shown to exert a bactericidal effect against L. monocytogenes (Liu et al., 2008). Pentocin TV35b had a bactericidal activity against Listeria innocua (Okkers et al., 1999).

The two main modes of action of LAB bacteriocins against Gram-positive bacteria are pore formation and inhibition of cell wall synthesis (Cotter et al., 2013). Inhibition of cell wall biosynthesis of M. luteus by salivaricin B did not cause an increase in fluorescence within 30 min. But within the same time frame nisin, a known pore former caused an increase in fluorescence intensity (Barbour et al., 2016).  Since increase in fluorescence intensity was observed for the pentocin MQ1 treated M. luteus within 30 min it can be concluded that it was due to membrane permeabilization. Pentocin MQ1 caused membrane permeabilization of M. luteus leading to leakage of intracellular DNA and consequently death of the bacteria. This is the first report on membrane permeabilization as a mechanism of action of L. pentosus-derived bacteriocins. It is thought that pore formation also led to loss of other valuable intracellular molecules such as ATP contributing to the rapid death of the bacterial target. Pentocin MQ1 was quick-acting against its target. It is thought that resistance to a quick-acting antimicrobial agent is less likely to occur compared to a slow-acting one. Pore formation has been reported for several LAB bacteriocins (Perez et al., 2014; Snyder and Worobo, 2014).

Banana is one of the most consumed fruit in the tropics and subtropics (Huang et al., 2014). It is a good source of antioxidants, carbohydrates, calcium, and potassium (Mohapatra et al., 2011). As a perishable and climacteric crop it has a short shelf life. Preserving fresh banana is quite challenging (Mohapatra et al., 2010). Various chemical and physical approaches are employed in the preservation of banana (Zaman et al., 2007; Kudachikar et al., 2011; Mohapatra et al., 2011). However, consumer inclination toward foods containing biopreservatives and less chemical preservatives triggered the search for natural products that can be used for biopreservation (Barbosa et al., 2017). In a recent study, combined application of phenylurea and gibberellins was effective at extending the shelf life of banana (Huang et al., 2014). Although the bioprotective capabilities of several bacteriocins have been reported (Galvez et al., 2008; Abriouel et al., 2010; Bhatia et al., 2016), no report has been made for banana. Only one report has been made on biopreservation of food using pentocin. In that study, the potential of pentocin 31-1 for preserving pork meat was demonstrated (Zhang J. et al., 2010). Topical application of pentocin MQ1 extended the shelf of banana in this study. Shelf life extension was due to decrease in total bacterial count

and increase in the percentage of LAB compared to the other microflora (**Table 5**). It can be deduced that pentocin MQ1 decreased the population of pathogenic and spoilage bacteria on the surface of banana. Moreover, it had a positive effect on the population dynamics of the surface microflora such that decrease in spoilage bacteria enhanced the growth of beneficial LAB strains leading to shelf life extension. These results reveal the biopreservative potential of pentocin MQ1. Furthermore, bacteriocin treatment and refrigeration had a synergistic effect on the microbiological quality of banana resulting in extension of shelf life. These findings pave the way for future ex situ application of pentocin MQ1 in the biopreservation of banana.

In conclusion, this is the first report on the presence of bacteriocinogenic strain of L. pentosus in coconut shake. L. pentosus CS2 produces a novel bacteriocin (pentocin MQ1) with a broad spectrum of antibacterial activity, high chemical, thermal and pH stability but sensitive to proteolytic enzymes. It is cell-wall associated and possesses a bactericidal mode of action. Pentocin MQ1 acted against its target through pore formation. Genes encoding pentocin MQ1 production are not plasmid-borne.  Its ability to preserve fresh banana was

### REFERENCES

demonstrated in this study. The characteristics of pentocin MQ1 show its potential for the preservation of food.

### AUTHOR CONTRIBUTIONS

KP designed, supervised execution of the experiments, and wrote the manuscript; KP also edited the manuscript; SW designed the experiments, performed it, and wrote the manuscript.