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Analyses of the probiotic property and stress resistance-related genes of Lactococcus lactis subsp. lactis NCDO 2118 through comparative genomics and in vitro assays - BEST PROBIOTICS FOR IBS

Analyses of the probiotic property and stress resistance-related genes of Lactococcus lactis subsp. lactis NCDO 2118 through comparative genomics and in vitro assays

Citation: Oliveira LC, Saraiva TDL, Silva WM, Pereira UP, Campos BC, Benevides LJ, et al. (2017) Analyses of the probiotic property and stress resistance-related genes of Lactococcus lactis subsp. lactis NCDO 2118 through comparative genomics and in vitro assays. PLoS ONE 12(4):
e0175116.

https://doi.org/10.1371/journal.pone.0175116

Editor: Luca Cocolin, University of Torino, ITALY

Received: November 14, 2016; Accepted: March 21, 2017; Published: April 6, 2017

Copyright: © 2017 Oliveira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: Support was provided by: National Counsel of Technological and Scientific Development [http://cnpq.br/]; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior [https://www.capes.gov.br/]; Fundação de Amparo à Pesquisa de Minas Gerais [http://www.fapemig.br/en/].

Competing interests: The authors have declared that no competing interests exist.

Introduction

The genus Lactococcus is part of the lactic acid bacteria (LAB), one of the most biotechnologically important groups of bacteria, which is composed of Lactococcus, Streptococcus, Lactobacillus, Weissella and others [1]. LAB species share in common the ability to convert sugar (mainly glucose) into lactic acid through specific metabolic pathways. Additionally, these species are facultative anaerobic, catalase negative and non-motile. Moreover, there is a close phylogenetic relationship between the bacteria of this group [2].

Many LAB species are biotechnologically important due to their safety aspects, achieved because they have been used for years in the preservation and maintenance of food [3]. Previous studies highlight the importance of genome sequencing in the discovery of new features related to LAB: genes coding for proteolytic enzymes (which participate in cheese maturation) in Lactobacillus helveticus [4], identification of citrate catabolic pathways in Lactobacillus casei [5], and genes responsible for decarboxylation of alpha-keto acid branched chain in Lactococcus lactis [6; 7].

Genome sequencing studies have also helped in the elucidation of probiotic effects exerted by LAB. For instance, in Lactobacillus reuteri, genome analyses have focused on the capacity to adapt to nutrient availability and environmental conditions of the GI tract, the adhesion mechanisms, the production of antimicrobial compounds, and the mechanisms of immunomodulation, such as the synthesis of pro-inflammatory extracellular polymeric substances (EPS compounds) [8]. Moreover, Lactobacillus rhamnosus and L. casei strains isolated from marketed probiotic products were compared with the well-studied L. rhamnosus GG and L. casei BL23, mainly focusing on pilus gene clusters and metabolic pathways analyses [9]. Interestingly, a new adhesion-associated protein, cwaA, was identified through genome sequencing and comparative genomics analyses of Lactobacillus plantarum NL42. The expression of cwaA in L. lactis has significantly increased its autoaggregation, hydrophobicity and exclusion ability, where the mutant strain was able to inhibit the adhesion of Staphylococcus aureus and Escherichia coli to HT-29 cells [10]. Another study illustrated the mechanisms by which Lactobacillus species from the intestinal niche have adapted to the gastrointestinal tract (GIT) by acquiring traits, such as stress tolerance, carbohydrate absorption, adhesion to epithelial cells and mucus [11].

Additionally, many species of this group are important for their probiotic effects, such as the genus Lactobacillus, which is used in the production of the fermented milk Yakult [12], and Bifidobacteria, widely known for their beneficial effects on the host intestinal tract [13]. However, although several works highlight the probiotic effects of LAB, most focus on Lactobacillus and Bifidobacterium species [14], whereas few studies report the beneficial effects of L. lactis strains. For instance, Lactococcus lactis subsp. cremoris FC has an important anti-inflammatory activity [15]. The probiotic properties of L. lactis subsp. cremoris IBB477 have attracted attention due to their adhesion mechanisms and survival in the intestinal environment [16; 17]. Additionally, it was recently demonstrated, through the evaluation of three L. lactis strains in vitro, that Lactococcus lactis subsp. lactis NCDO 2118 has anti-inflammatory and immunomodulatory activity that can alleviate colitis symptoms [18]. This strain was described as a gamma-aminobutyric acid (GABA) producer [19]. It has been extensively used for heterologous expression [20], and its probiotic effect is associated with exported proteins [18].

Here, we use comparative genomics and in silico analyses to provide insights into the probiotic nature of L. lactis NCDO 2118. The criteria for screening LAB strains before their use as probiotics include assessing functional features, such as the ability to resist environmental conditions found in the digestive tract (low gastric pH and bile salts) and the ability to antagonize or competitively exclude pathogens, which is achieved by secreting antimicrobial substances or competing for nutrients and epithelial adhesion sites. LAB produce different antimicrobial components, such as organic acids, hydrogen peroxide, carbon peroxide, diacetyl, low molecular weight antimicrobial substances, bacteriocins and adhesion inhibitors. The adhesiveness of LAB may involve passive forces, electrostatic interactions, hydrophobic steric forces, lipoteichoic acids, and lectins [21]. The hydrophobic nature of the outermost surface of microorganisms facilitates the adhesion of bacteria to the host epithelium, thereby conferring competitive advantages during the colonization of the GIT [22]. The antimicrobial susceptibility of intestinal microorganisms is an important criterion for the selection of probiotic strains, mainly due to the potential transfer of those genes to pathogenic or commensal bacteria that inhabit the GIT [23]. In the following sections, we present comparative genomic analyses of L. lactis NCDO 2118 and other Lactococcus species and predict genes that putatively code for acid stress resistance proteins, bacteriocins, adhesins and exported proteins.

Results

General features, phylogenomics and synteny analyses

The general genomic features of all genomes used in this work are summarized in Table 1.

Briefly, Lactococcus garvieae strains have the highest G+C content, ~38.80%, whereas the lowest G+C contents, ~34.86%, were from L. lactis NCDO 2118 and L. lactis KF147, both isolated from vegetables. Additionally, the genome sizes of the Lactococcus species range from ~1.95 Mb to ~2.60 Mb, and the two L. garvieae strains have the smallest genomes.

In this work, the only species harboring plasmids were L. lactis NCDO 2118, L. lactis KF147, Lactococcus lactis subsp. lactis KLDS 40325 and Lactococcus lactis subsp. lactis CV56 strains, Lactococcus lactis subsp. cremoris A76, Lactococcus lactis subsp. cremoris SK11 and Lactococcus lactis subsp. cremoris UC5099 (L. cremoris UC5099) strains, where the latter harbored the greatest number of plasmids (Table 1).

From the heatmap created with Gegenees (Fig 1), it is possible to visualize a high similarity between the subspecies of Lactococcus, with nucleotide similarities ranging from 40% to 100%. Additionally, the species and subspecies clustered separately, creating 3 green blocks of strains at the chart, represented by L. lactis subsp. lactis and L. lactis subsp. cremoris, with similarities ranging from 91% to 100%, and L. garvieae, in which the two strains of this species were 100% similar to each other.

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Fig 1. 16S phylogenetic tree and genomic heatmap of Lactococcus genus.

The Streptococcus thermophilus LMD-9 (position 17) was added to root the tree. The species in comparison are distributed from 1 to 17 in the same order, both vertically and horizontally. The numbers in the heatmap show the percentage of similarity between the species, varying from yellow (low similarity) to green (high similarity), or from 40% to 100%, respectively. The heatmap and the phylogenetic tree were created with the software Gegenees and Mega (Neighbor-Joining method with 1000 bootstraps replicates), respectively.


https://doi.org/10.1371/journal.pone.0175116.g001

On the phylogenetic tree created using 16S, the species and subspecies also clustered together, forming two main clades corresponding to the best similarity among L. lactis subsp. lactis and L. lactis subsp. cremoris (Fig 1). Additionally, L. garvieae strains appeared in an outside node compared to L. lactis species and are the two most distinct and distant species of Lactococcus on the heatmap and phylogenetic tree. Briefly, on the heatmap, the degree of intraspecies similarity varies from 91% to 100%, whereas interspecies similarity varies from 40% to 65%.

From the genome synteny analysis (S1 Fig), all strains from L. lactis subsp. lactis presented a high degree of synteny, where the most conserved genome compared to L. lactis NCDO 2118 (chosen as reference genome) was L. lactis KF147. Additionally, we performed a comparison with the plasmids of L. lactis NCDO 2118 and L. lactis KF147 strains. However, we verified a high degree of similarity from the beginning to the end of each plasmid sequence, meaning that they possibly harbored the same plasmid (data not shown).

Metabolic pathways prediction

To identify conserved or non-conserved metabolic pathways, we used three different datasets, consisting of (1) the closely related L. lactis NCDO 2118, L. lactis KF147 and L. lactis IL1403, (2) all strains from L. lactis subsp. lactis and L. lactis subsp. cremoris (non-pathogenic dataset), and (3) all strains from this study (including L. garviae). The number of metabolic pathways harbored by each genome varies from 148 to 206, with a general mean of ~183 pathways. Both L. garvieae strains contained 148 metabolic pathways, L. lactis subsp. lactis showed an average of ~192 metabolic pathways, and L. lactis subsp. cremoris showed ~186 pathways.

The main differences were that the strain L. lactis NCDO 2118 contains more peptidoglycan biosynthesis pathways than L. lactis KF147 and L. lactis IL1403 strains. Other exclusive metabolic features of L. lactis NCDO 2118 in this context were complete anaerobic respiration pathways, fermentation of pyruvate to acetate, fermentation of fumarate, complete heterolactic fermentation, valine degradation, L-serine degradation, ammonia assimilation to glutamate, complete superpathway of acetate utilization and formation, protein degradation, initial pathway of sucrose degradation I, valine degradation, lysine degradation I and acyl-ACP thioesterase pathway (S1 Table).

Genome plasticity

We identified 5 prophages in L. lactis NCDO 2118, of which 2 were incomplete, and 3 were considered intact (Table 2). The three intact phages harbored important genes such as rusA, arsC1, arsC3, amtB, rpmE2, carA, pyrB, pyrP and pepT.

Additionally, we used BRIG to visualize the plasticity events from phage sequences (Fig 2). According to the BRIG analyses, phage 1 was incomplete in all species, except for the reference genome L. lactis NCDO 2118 and L. lactis KF147. Both phages 2 and 3, predicted as intact in the reference, were also present in L. lactis KF147, L. lactis IL1403, Lactococcus lactis subsp. cremoris NZ9000 and L. cremoris MG1363, whereas the former phage was also found in Lactococcus lactis subsp. cremoris KW2. Phage 4, also intact in the reference genome, was present in all other species. Phage 5, predicted as incomplete in the reference genome, was absent in L. lactis IO-1, L. cremoris KW2, L. cremoris UC5099 and partially present in both L. garvieae strains.

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Fig 2. Circular comparison of the Lactococcus genus using L. Lactis NCDO 2118 as a reference.

Each ring of the circle corresponds to a specific complete genome represented in the legend on the right. The similarity between species is represented by the intensity of the color. Darker colors represent higher similarities than bright ones. Deleted regions are represented by blank spaces inside the circles. (GEI = Genomic Island; MI = Metabolic Island; SI = Symbiotic Island; MSI = Miscellaneous Island, harboring both metabolic and symbiotic factors). Genomic islands and phage sequences were predicted with GIPSy and PHAST, respectively. The circular genomic comparisons were created with BRIG.


https://doi.org/10.1371/journal.pone.0175116.g002

In the GIPSy predictions, we identified 9 Genomic Islands (GEIs), 5 Metabolic Islands (MIs), 4 Symbiotic Islands (SIs) and 3 Miscellaneous Islands (MSIs), which were predicted as harboring both metabolic and symbiotic related factors. The GEIs are listed in S2 Table.

All SIs were only partially present in the other strains, except for SI4, which was absent from all L. garviae strains, L. lactis subsp. cremoris strains and L. lactis IL 1403 (Fig 2). Additionally, all MIs presented regions of deletions in the pathogenic species L. garviae. The most prominent GEIs were MI3, which was only present in the two strains isolated from plants (L. lactis NCDO 2118 and L. lactis KF147), and MSI 2, which presented the biggest region of deletion in all Lactococcus, except for L. lactis NCDO 2118 and L. lactis KF147.

Antibiotic resistance

LAB that are widely used as probiotics or in starter cultures have the potential to host antibiotic resistance genes, thereby presenting a risk of transferring such genes to many lactic acid bacteria and other pathogenic bacteria [23]. In the antibiogram assay, L. lactis NCDO 2118 was susceptible to ceftriaxone, erythromycin, tetracycline, ampicillin, penicillin and chloramphenicol and resistant to vancomycin, oxacillin and amikacin antibiotics (Table 3). Additionally, we tried to correlate the antibiogram profile with the genome content of L. lactis NCDO 2118, which presented 22 antibiotic resistance-related genes putatively coding for a VanZ family protein (NCDO2218_1094), penicillin-binding proteins (NCDO2118_0402, NCDO2118_0445, NCDO2118_0526, NCDO2118_0880 and NCDO2118_2216), and multidrug efflux pump proteins (Table 4). Additionally, no antibiotic resistance related gene presented deviation in its genomic signature.

Identification of genes involved in acid stress and bile salt resistance

We searched the genome sequence of L. lactis NCDO 2118 for genes previously shown to be differentially expressed on cells cultivated under low and optimum pH (5.1 and 6.5, respectively) in L. cremoris MG1363 [38] (Table 5). Additionally, we also searched for genes differentially regulated by bile exposure in Bifidobacterium animalis and Bifidobacterium longum NCIMB 8809 [39; 40] and/or identified on the total proteome and surfome of Lactobacillus rhamnosus GG using proteomics analyses (Table 5). Here, we identified some genes in L. lactis NCDO 2118 that were previously reported to be involved in the acid stress response, including genes coding for chaperones (dnaK) and stringent response. Additionally, DnaK and Enolase are plasminogen receptors involved in bile modulation during intestinal colonization.

Additionally, we assayed L. lactis NCDO 2118 to see how it responds to the challenges of acid pH and bile salt secretion in the gastrointestinal tract. When in contact with artificial gastric juice, 48% of the L. lactis NCDO 2118 was not inhibited and was able to grow after acid pH challenge, whereas the contact with bile salts inhibited 95% of the bacteria growth, showing a high sensibility, as a result of three independent experiments (S2 Fig).

Identification of genes coding for adhesins and adhesion-related proteins

Based on literature data, we predicted proteins involved in the adhesion mechanisms of L. lactis NCDO 2118, shown in Table 6. L. lactis NCDO 2118 harbors 19 genes putatively coding for adhesion-related proteins, such as the gene chiA (NCDO2118_2053) and the genes coding for the Chitin binding protein (CBP–NCDO2118_2054) and the laminin-binding protein (NCDO2118_1446).

To determine whether L. lactis NCDO 2118 exhibited adhesive ability, corroborating the in silico data, we performed microbial adhesion to solvents (MATS) experiments, which demonstrated a moderate cell surface hydrophobicity, as suggested by Nader-Macías et al., (2008) [41], with 52% association with xylene.

Bacteriocins and other competitive exclusion mechanisms

To predict putative bacteriocins, we used the software BAGEL [42]. In addition to identification, BAGEL also classifies the bacteriocins into three classes: (i) lanthionine-containing bacteriocins/lantibiotics, (ii) non-lanthionine-containing bacteriocins and (iii) bacteriolysins/non-bacteriocin lytic proteins [43].

In L. lactis NCDO 2118, BAGEL predicted one bacteriocin for each of the three classes (Fig 3): a lanthipeptide (class I), NCDO2118_1768 (putative Bacteriocin-lactococcin-A—class II) and a putative bacteriocin (class III), located between NCDO2118_2257 and NCDO2118_2258. The class III putative bacteriocin was not described in the L. lactis NCDO 2118 genome, possibly because the gene-finding methodology failed to predict it. The bacteriocin of class I is a lantibiotic Nisin coded by the nisZ gene (NCDO2118_1272), a natural variant of nisA [44]. Briefly, Nisin is commonly produced by strains of L. lactis, and the cluster of genes coding for the nisin biosynthesis proteins consists of 11 genes: nisABTCIP (biosynthesis and immunity), nisFEG (immunity) and the two-component regulatory system nisRK [45]. L. lactis NCDO 2118 harbors a nisBCIP operon (where nisP is a pseudogene), a nisRK two-component system and a nisFEG operon. Additionally, BAGEL has predicted the presence of another putative bacteriocin between NCDO2118_1258 and NCDO2118_1259 that is located close to the class I cluster of genes. However, the amino acid sequence predicted from this region only presents similarity to a hypothetical protein. Lactococcin A is a class IId, non-pediocin-like, single-peptide bacteriocin normally produced by strains of L. lactis. Four genes are responsible for the biosynthesis of lactococcin: the lactococcin-A coding gene, one immunity gene and the dedicated ABC transporter system along with its accessory protein. L. lactis NCDO 2118 harbors an immunity protein (NCDO2118_1767) and lactococcin-A (NCDO2118_1768). As for the class III prediction, the predicted putative bacteriocin is located upstream of two hypothetical proteins (NCDO2118_2258 and NCDO2118_2259); however, little is known about the organization of the gene cluster of class III bacteriocins [45], and the putative bacteriocin predicted by BAGEL only presents similarity to hypothetical proteins in GENBANK.

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Fig 3. Regions of bacteriocins predicted with BAGEL in L. lactis NCDO 2118.

BAGEL predicted three putative bacteriocins, one of each class. (A) Putative bacteriocin/Class I predicted on orf010 (pseudogene) and nisZ was found with manual curation. (B) Putative bacteriocin/Class II predicted on orf027 (pseudogene). (C) Putative bacteriocin/Sactipeptidase predicted on orf011 (this region was not previously characterized in the L. lactis subsp. lactis NCDO 2118 genome). All putative bacteriocins were also identified in Bactibase.


https://doi.org/10.1371/journal.pone.0175116.g003

Moreover, an additional bacteriocin-coding gene was harbored by GEI 9 (S2 Table), which was not predicted by BAGEL. Through blast analyses, we found a significant amino acid similarity, with identities varying from 76% to 98%, between this gene and a bacteriocin-coding gene from other L. lactis in the UNIPROT and NCBI BLAST databases. However, many of the genes were also described as hypothetical proteins. In addition, we also searched for other genes that could possibly play a role in the competitive exclusion of other bacteria. A Lyzozyme M1 and a Macrolide biosynthetic protein encoding genes were also included in S3 Table after manual curation in the L. lactis NCDO 2118 genome.

In the present study, a deferred agar spot assay was used for the initial determination of antagonistic activity via diffusible compound(s) produced by L. lactis NCDO 2118. To assay whether L. lactis NCDO 2118 could affect the growth of pathogenic bacteria, we used an approach to measure its antagonistic activity against the strains Salmonella enterica ATCC 14028, Escherichia coli ATCC 25723, Staphylococcus aureus 29213, Bacillus cereus ATCC 11778, Listeria monocytogenes ATCC 15313, Enterococcus faecalis ATCC 19433 and Pseudomonas aeruginosa ATCC 5853. L. lactis NCDO 2118 showed no effect on the growth of the abovementioned pathogenic strains.

In silico identification of putatively secreted proteins

Here, we strove to predict genes encoding secreted proteins from L. lactis NCDO 2118 that are absent from the genomes of the strains L. lactis IL1403 and L. cremoris MG1363, as the secreted proteins of L. lactis NCDO 2118 are possibly responsible for the immunomodulatory effects of this transient bacterium inside the host [18].

To predict the secreted proteins, we used the software SurfG+, which classifies the proteins using an identification approach based on the presence/absence of signal peptides, signal retention and transmembrane helix [46], which are correlated with the cell wall thickness of the bacteria. To determine the cell wall thickness, we made photomicrographs of L. lactis NCDO 2118 (Fig 4); the cell wall was measured more than 270 times, showing an average size of ~20 nm, and this value was used to determine the motifs. If none of the motifs were found in the protein sequence, SurfG+ characterized the protein as cytoplasmic (CYT) [47]. Using SurfG+, we predicted 94 secreted proteins in L. lactis NCDO 2118.

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Fig 4. Photomicrograph of L. lactis NCDO 2118.

The measurements of the membrane wall were performed with ImageJ software using images generated with electron microscopy with EM10A equipment (Zeiss). Top: magnification of 50,000 times; bottom: magnification of 100,000 times.


https://doi.org/10.1371/journal.pone.0175116.g004

From this data, the secreted proteins of L. lactis NCDO 2118 were compared to the proteins identified in L. lactis IL1403 using OrthoMCL [48]. In this comparison, 26 of the secreted proteins were exclusive from L. lactis NCDO 2118. Because the probiotic effect was searched using secreted proteins previously expressed in vitro, we searched for proteins that were expressed in L. lactis NCDO 2118 in vitro using proteomics analyses. Five proteins were both present in the 26 secreted proteins that were exclusive from L. lactis NCDO 2118 and in the 867 expressed proteins from proteomic analyses (Table 7). The complete lists of genes identified in proteomic analyses, in the prediction of subcellular location and the exclusive proteins of L. lactis NCDO 2118 are described in S4 Table.

Discussion

Genomic characterization of L. lactis NCDO 2118 and comparison with other species

The genomic lengths of the Lactococcus species analyzed here are highly variable (from ~1.95 Mb to ~2.60 Mb). However, the finding that L. garvieae strains have the smallest genomes compared to L. lactis strains is in agreement with the lifestyle of L. garvieae, isolated from diseased fish. Because pathogenic bacteria may scavenge compounds from the host for their own metabolism, they tend to lose genes involved in biosynthetic pathways, thus, presenting smaller genomes [49].

The high similarity at the subspecies level may be related with some specific characteristics already described in literature, such as the propensity of L. lactis subsp lactis to form longer chains. Besides, L. lactis subsp. lactis are able to produce GABA, ammonia from arginine, carbon dioxide and diacetyl formation from citrate as opposing to L. lactis subsp. cremoris subspecies. Additionally, analyses using southern hybridization, PFGE, 16 rRNA and housekeeping genes (atpA, rpoA, pheS, pepN, bcaT, pepX) showed two separate clusters formed by L. lactis subsp. lactis and L. lactis subsp. cremoris with a low degree of similarity between them [5052].

From the genome synteny analyses, we have found a high degree of synteny between L. lactis KF147 and L. lactis IL1403, which was already reported in a previous work [25]. However, there was no other genome sequence of any Lactococcus species correlated with plants available at the time the work was performed. Here, we found that the most conserved genome compared to L. lactis NCDO 2118 was L. lactis KF147. The material of fermented plant covers a highly variable niche according to some characteristics as: chemical composition and physical conditions. Thus, plant-related strains posses a great metabolic diversity that certainly extrapolates that from dairy strains [53].

Finally, although L. lactis NCDO 2118 shares several pathways in common with L. lactis KF147 and L. lactis IL1403, it presents several exclusive metabolic features that may be explored for future utilization in industry.

Evaluation of safety aspects in the use of L. lactis NCDO 2118 by genome plasticity and antibiotic resistance approaches

Plasmid-linked antibiotic resistance is not very common among LAB, but it does occur, and safety implications should be taken into consideration. Strains harboring resistance plasmids should not be used as human or animal probiotics. Checking the ability of a proposed probiotic strain to act as a donor for conjugative antibiotic resistance genes may be a sensible precaution in some instances [54].

To provide a better understanding of the putative plasticity of L. lactis NCDO 2118, we have predicted putative phage and genomic islands of this species. The presence of phage regions may contribute to the acquirement of antibiotic resistance, the ability to survive in a new environment, the improvement of adhesion ability, or even to turning the bacteria pathogenic [55]. Here, we found 5 phages; the 3 intact phages harbored important genes such as rusA, arsC1, arsC3, amtB, rpmE2, carA, pyrB, pyrP and pepT. The rusA gene is associated with the prophage sequences of several genera of bacteria, including Bacillus, Streptococcus, Staphylococcus, and Enterococcus, and it is also present in Lactococcus lactis phage r1t [56]. The arsC1 gene is related to arsenate resistance in Corynebacterium glutamicum [57]. arsC3 codes for a thioredoxin-dependent arsenate reductase of the Mycobacterium sp. A33 [58]. amtB is a gene of the ammonia transporter family, which is found in eubacteria, archaea, fungus, plants and animals, whereas in prokaryotes, its homologue is co-transcribed with a PII paralogue, GlnK, in response to nitrogen limitation [59]. The rpmE2 gene codes for a L31 ribosomal protein. The genes carA, pyrB and pyrP are organized as an operon in L. cremoris MG1363, where pyrP encodes a membrane-bound protein with high affinity to uracil permease and pyrimidines, and pyrB and carA encode pyrimidine biosynthetic enzymes [60]. Finally, the gene pepT encodes for a tripeptidase.

Additionally, we predicted 9 GEIs, 5 MIs, 4 SIs and 3 MSIs in the genome sequence of L. lactis NCDO 2118. Interestingly, all MIs present deletions in the pathogenic species L. garvieae, which is a common feature of pathogenic bacteria that adapted to scavenge nutrients from the host [61]. Additionally, MI3 is only present in the L. lactis NCDO 2118 and L. lactis KF147 and may be important for the adaptation of those strains to plants.

We have also assayed L. lactis NCDO 2118, aiming to characterize its antibiotic resistance profile. L. lactis NCDO 2118 is susceptible to most of the antibiotics assayed here. Although L. lactis NCDO 2118 presented resistance to oxacillin and susceptibility to penicillin, it only harbored genes coding for a VanZ family protein, which may be related to the vancomycin resistance, penicillin-binding proteins, and multidrug efflux pump proteins.

The efflux pumps are membrane transporter proteins responsible for the extrusion of relevant antibiotics, which are found in both Gram-positive and Gram-negative bacteria [62; 63]. Penicillin-binding proteins are transpeptidases or caboxypeptidases that harbor specific motifs that limit the active site serine penicillin-recognizing enzyme family, including class A and C β-lactamases [64]. Vancomycin is a glycopeptide antibiotic used in severe infections. Some species used in the food industry or found naturally in raw food material present an intrinsic resistance to vancomycin, including L. rhamnosus, L. casei, Lactobacillus plantarum, and Leuconostoc lactis [65].

Finally, although L. lactis NCDO 2118 does present genes putatively coding for antibiotic resistance-related proteins, none of those genes present anomalous G+C or codon usage deviation, nor are they harbored by the putative horizontally acquired regions predicted by GIPSy or PHAST. More interestingly, no Resistance Island was identified in L. lactis NCDO 2118, corroborating its safety aspects [66].

In vitro and in silico analyses of survival, exclusion mechanisms and probiotic properties of L. lactis NCDO 2118

Susceptibility of L. lactis NCDO 2118 to acid stress and bile salts.

Concerning the acid stress, lowering the intracellular pH reduces the transmembrane pH difference and the activity of acid-sensitive enzymes and damages proteins and DNA [67].The first mechanism used by L. lactis species to cope with acid stress is to maintain a low intracellular pH (pHi) by using membrane ATPase FoF1 [68; 69] and the generation of alkaline substances through the catabolism of amino acids (deamination, for example) [70; 71]. Bile salts, on the other hand, are surface-active, amphipathic molecules with a potent antimicrobial activity, and they act as detergents that disrupt biological membranes [67]. The percentage of resistance to bile salts also tends to vary among LAB and even between strains of the same species [72].

Here, we have identified 25 and 16 genes previously shown to be involved in acid stress and bile resistance in other species, respectively. In an in vitro assay, however, only 48% of L. lactis NCDO 2118 was able to grow after pH challenge, and 95% of bacteria was inhibited by bile salts. Other authors have already found that bacteria with an intestinal origin tend to be more resistant to stomach acids [73]. Therefore, this finding corroborates our results because L. lactis NCDO 2118 was isolated from frozen peas. Most of the genes found in L. cremoris MG1363 were also identified in L. lactis NCDO 2118. Additionally, a work using proteomics analyses identified some genes related to acid response and they are present in L. lactis NCDO 2118 genome (clpEP, ahpC, tig, hpr and luxS) [74] showing that other approaches may better elucidate the mechanism of survival to acid stress on this strain.

The high susceptibility of L. lactis NCDO 2118 to bile salts, on the other hand, must be further explored in vitro and in vivo using transcriptomics analyses to determine the expression rates of the described genes.

Competitive exclusion mechanisms of L. lactis NCDO 2118.

There are several mechanisms used by bacteria to competitively exclude other species, such as bacteriocin production, space competition through the use of adhesins or receptors that bind to specific surface features, predation and even rapid growth [75].

Adhesins are responsible for the recognition and colonization of host tissues through specific binding. This process may activate the innate host cells or the expression of new genes. Adhesins may be characterized as hair-like attachments named pili or fimbriae or in other cases, named non-pilus adhesin, related to the microbial cell surface [76].

In L. lactis NCDO 2118, we have identified the gene chiA (NCDO2118_2053) and the genes coding for the Chitin binding protein (CBP–NCDO2118_2054) and the laminin-binding protein (NCDO2118_1446), which are normally related to adhesion in other bacteria. Chitin is degraded by chitinases that belong to members of the glycoside hydrolase of family 18 [77]. One example of bacteria that produces chitinase is Serratia marcescens, one of the most efficient organisms in chitin degradation [78]. When E. coli was cloned with a chitin-binding protein of Serratia marcescens, there was a significant increase in its ability to adhere to human colon cells [77].

Chitin-binding encoding genes are broadly distributed in many microorganisms. The L. lactis IL1403 genome, for example, harbors chitinolytic machinery represented by one family 33 CBP (yucG; referred as LlCBP33A), one family 18 chitinase (chiA, referred as LlChi18A) and one family 20 N-acetylhexosaminidase [3; 79]. Another example of bacteria that present a high adhesion degree is Borrelia burgdorferi, which is able to bind to mammalian laminin, an important extracellular matrix (ECM) component [80]. A laminin-binding protein has also been identified in L. lactis NCDO 2118.

Additionally, we have found using MATS experiments that L. lactis NCDO 2118 presents a 52% of association to xylene, which supports the presence of genes coding for adhesion-related proteins in this strain. The hydrophobicity is directly related to the capacity of strains to adhere to surfaces. This capacity is determined by hydrophobic components present in the outer membrane of microorganisms, and it is known that hydrophobic interactions have an important role in the adhesion of bacteria to the epithelium. The application of MATS experiments facilitates a qualitative assessment of the polarity or non-polarity of the bacterial surface, which is important because it indicates the potential for probiotic adhesion to apolar surfaces in the intestinal and vaginal epithelia. However, this test is only a primary indicator of the adherence of microorganisms [81; 82].

The other bacterial competitive exclusion mechanism assayed here was the production of exclusion antimicrobial peptides, named bacteriocins. Bacteriocins produced by a bacterium may be activated against others, even ones from the same species, while the producer is immune to its own peptides [43]. This exclusion mechanism is very important for probioses, as it renders probiotic organisms able to compete with and kill pathogenic ones, promoting a health benefit to the host [2]. We have predicted one bacteriocin for each of the three classes in L. lactis NCDO 2118 (class I-III), which may be important for exclusion mechanisms of this bacteria. However, the lack of nisT and the pseudogenization of nisP on the class I gene cluster, the lack of ABC-transporters in the class II cluster and, also, the lack of information regarding the product of the putative bacteriocin in the class III cluster have to be further studied using in vitro analyses to elucidate whether those bacteriocins are produced and present antimicrobial activity or not.

We have also performed a deferred agar spot assay for the initial determination of antagonistic activity produced by L. lactis NCDO 2118. This test indicates the activity against various Gram-positive and -negative bacteria. This inhibitory effect may be due to H2O2, lactic acid, bacteriocins, antibiotic-like substances, or a combination of these compounds [83]. However, L. lactis NCDO 2118 showed no effect on the growth of the pathogenic strains assayed here.

Secreted proteins and immunomodulatory effects.

According to Luerce et al., (2014), the secreted proteins of L. lactis NCDO 2118 are possibly responsible for the immunomodulatory effects of this transient bacterium inside the host. In a comparison of the anti-inflammatory effects between L. lactis NCDO 2118, L. lactis IL1403 and L. cremoris MG1363 strains, only the L. lactis NCDO 2118 supernatant was able to decrease the IL-8 production (45%), showing its immunomodulatory ability against inflammation [18].

Here, we predicted 5 proteins that are present in the 26 secreted proteins exclusive from L. lactis NCDO 2118 and in the 867 expressed proteins from proteomic analyses and may thus be related to the probiotic effect of this strain (Table 7). From those 5 exclusive, secreted and expressed genes of L. lactis NCDO 2118, epsK and epsL are part of the operon epsABCDEFGHIJKLX, whereas there is an epsR gene located in another genomic region.

The EPSs are a type of biopolymer able to facilitate intense interactions of biofilm cells through adhesion, aggregation of bacterial cells, cohesion of biofilms, protective barriers, and cell component export [84]. Through microarray and electron microscopy analyses, Denou et al., 2008 found an eps cluster of genes exclusive from a probiotic Lactobacillus strain compared to a type strain and they have shown that deletion of this cluster from the probiotic strain results in lack of the fuzzy layer on the outside of the cell wall [85].

Altogether, the lack of further knowledge of the eps cluster of genes and the presence of three other genes coding hypothetical exclusive/secreted/expressed proteins highlight the need for additional studies to better elucidate the underlying mechanisms involved in the anti-inflammatory and immunomodulatory activities of this strain.

Conclusions

Although L. lactis NCDO 2118 presented a high similarity to the other L. lactis strains, it presents an SI that is commonly shared with L. lactis KF147, along with high genomic synteny conservation with this strain. Additionally, the antibiotic resistance of this strain to vancomycin, amikacin and oxacillin could be an obstacle for its use as a probiotic. However, the absence of resistance-related genes in regions acquired by HGT and the absence of RIs in the genome sequence corroborates its safety aspects and supports its use as a probiotic strain. Moreover, the high susceptibility of L. lactis NCDO 2118 to acid and bile salts stresses have to be further evaluated in a complete digestion simulation, using transcriptomics and proteomics analyses, to elucidate whether the identified genes are differentially expressed in those environmental conditions.

Interestingly, the adhesion of L. lactis NCDO 2118 to xylene and the putative production of three classes of bacteriocins are important indicators of the exclusion mechanisms used by this strain. However, the in vitro analyses have not shown any sign of an antagonistic effect against the assayed pathogenic bacteria. Future works could also take advantage of combined transcriptomics and proteomics analyses of L. lactis NCDO 2118 in vitro before and after intestinal passage to evaluate the expression of the identified genes. Additionally, the identification of the EPS cluster of genes putatively associated with the probiotic effect of L. lactis NCDO 2118 could be further explored in 16S metagenomics analyses of gut microbiota, after expression, purification and administration of EPS proteins. Finally, through the analyses of the safety, survival and probiotic aspects of L. lactis NCDO 2118, we highlight here the potential use of this strain as a target for the future development of probiotic foods.

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