PLoS ONE 12(6): e0176801. https://doi.org/10.1371/journal.pone.0176801 

In silico genomic insights into aspects of food safety and defense mechanisms of a potentially probiotic Lactobacillus pentosus MP-10 isolated from brines of naturally fermented Aloreña green table olives

Hikmate Abriouel1*, Beatriz Pérez Montoro1, María del Carmen Casado Muñoz1, Charles W. Knapp2, Antonio Gálvez1, Nabil Benomar1 

1Área de Microbiología, Departamento de Ciencias de la Salud, Facultad de Ciencias Experimentales, Universidad de Jaén, 23071-Jaén, Spain.

2Department of Civil and Environmental Engineering, University of Strathclyde, Glasgow, Scotland, G1 1XJ United Kingdom. 

*Corresponding author

E-mail: hikmate@ujaen.es

 

Abstract

Lactobacillus pentosus MP-10, isolated from brines of naturally fermented Aloreña green table olives, exhibited high probiotic potential. The genome sequence of L. pentosus MP-10 is currently considered the largest genome among lactobacilli, highlighting the microorganism’s ecological flexibility and adaptability. Here, we analyzed the complete genome sequence for the presence of acquired antibiotic resistance and virulence determinants to understand their defense mechanisms and explore its putative safety in food. The annotated genome sequence revealed evidence of diverse mobile genetic elements, such as prophages, transposases and transposons involved in their adaptation to brine-associated niches. In-silico analysis of L. pentosus MP-10 genome sequence identified a CRISPR (clustered regularly interspaced short palindromic repeats)/cas (CRISPR-associated protein genes) as an immune system against foreign genetic elements, which consisted of six arrays (4–12 repeats) and eleven predicted cas genes [CRISPR1 and CRISPR2 consisted of 3 (Type II-C) and 8 (Type I) genes] with high similarity to L. pentosus KCA1. Bioinformatic analyses revealed L. pentosus MP-10 to be absent of acquired antibiotic resistance genes, and most resistance genes were related to efflux mechanisms; no virulence determinants were found in the genome. This suggests that L. pentosus MP-10 could be considered safe and with high-adaptation potential, which could facilitate its application as a starter culture and probiotic in food preparations.

 

Supplement

Lactobacillus pentosus and L. plantarum are the predominant species isolated from naturally fermented table olives due to their capacity to resist low pH and high concentration of salt. Their increased importance as a potential probiotic from a non-dairy origin has been widely reported in the last decade. L. pentosus MP-10, isolated from Aloreña green table olives (1), exhibited in vitro attractive probiotic activities such as fermentation of several carbohydrates including lactose and different prebiotics, auto-aggregation, co-aggregation with pathogenic bacteria (Listeria innocua, Staphylococcus aureus, Escherichia coli, and Salmonella Enteritidis), antimicrobial activity and high potential adherence to 2-Caco and Hela cell lines (2). Genome sequencing and annotation provides useful tools to determine the safety and the functionality of probiotic bacteria. Thus, re-sequencing and re-annotation of L. pentosus MP-10 revealed that its genome was among the largest genome sequences in lactobacilli up to date (3), was composing of a chromosome at 3,698,214 bp, an estimated mol% G+C content of 46.32% and five plasmids ranging 29–46 kb (3, 4).

This study highlighted the defense mechanisms and the safety aspects of a potential probiotic L. pentosus MP-10 by in silico analysis of its genome sequence. We focused for the first time on the analysis of defense mechanisms present in both the chromosome and the five plasmids. Two CRISPR systems (CRISPR1 and CRISPR2) that represent an acquired and adaptive immune system that provides protection against mobile genetic elements (i.e., viruses, transposable elements and conjugative plasmids) were detected in L. pentosus MP-10 being distributed throughout the entire genome. To determine the CRISPR elements [CRISPR array and CRISPR associated protein responsible genes (cas genes)], we used the CRISPRs finder program. Thus, we could appreciate the high fitness of this bacterium resulting from its chromosomal plasticity, with nine arrays with spacer sequences chronologically acquired from phages and plasmids being detected. As such, we concluded that L. pentosus MP-10 possessed many defense mechanisms against several pathogens, which can be exploited in food systems and also as a probiotic in intestinal tracts. On the other hand, the presence of five temperate phage regions in L. pentosus MP-10 chromosome and plasmids as revealed by bioinformatic tool “PHAST” highlighted the high diversity and fitness of this bacterium.

Regarding mobile genetic elements, 29 transposases were found in L. pentosus MP-10, which were similar to lactobacilli such as L. plantarum; however, the number of transposases was higher than other Lactobacillus sp. This fact confirmed, once more, how the highly diversified of genome contribute to this bacterium’s adaptability to the changing environment and its increased fitness.

In this study, we also examined at molecular level the safety of L. pentosus MP-10 as a key criterium for probiotic bacteria selection. As such, in silico analysis of their annotated genome against the antibiotic resistance genes database (CARD) revealed the absence of acquired antibiotic resistance determinants. However, the Resistance Gene Identifier (RGI) tool available in CARD database revealed the presence of different antibiotic resistance genes (Fig. 1): alaS, mfd and ileS genes coding for aminocoumarin, fluoroquinolone and mupirocin resistance, respectively, along with mprF gene and several genes (lmrB, lmrD, emeA and arlR) encoding efflux pump proteins conferring multiple-antibiotic resistance (Fig. 1). When we analyzed the possibility of acquired resistance by horizontal gene transfer using ResFinder, no acquired antibiotic resistance determinants were detected for aminoglycoside, beta-lactam, colistin, fluoroquinolone, fosfomycin, fusidic acid, MLS-series (macrolide, lincosamide and streptogramin B), nitroimidazole, oxazolidinone, phenicol, rifampicin, sulphonamide, trimethoprim, tetracycline and glycopeptide.

We then investigated whether L. pentosus MP-10 had any virulence determinants by comparing the annotated genome sequence against a virulence gene database (PHAST). Results revealed 14 genes encoding prophage proteins, an alanine racemase and a DNA-binding ferritin-like protein, which were similar to L. plantarum WCFS1.

Based on the results. we consider the absence of virulence factors and acquired antibiotic resistance genes makes L. pentosus MP-10 safer for food production. Furthermore, its ability to adapt to broad ecological conditions and its increased fitness, L. pentosus MP-10 should be considered a promising candidate for probiotic applications besides its use as starter culture.

 

 

Figure 1. Heatmap analysis of antibiotic resistance in L. pentosus MP-10 showing the unique distribution profiles of antibiotic resistance genes [black: no sequence matching the protein; purple: precise match to known antimicrobial resistance (AMR) gene sequence].

 

References

(1) Abriouel H, Benomar N, Cobo A, Caballero N, Fernández Fuentes MÁ. Pérez-Pulido R, Gálvez A. (2012). Characterization of lactic acid bacteria from naturally-fermented Manzanilla Aloreña green table olives. Food Microbiol. 32: 308–316.

(2) Pérez Montoro B, Benomar N, Lavilla Lerma L, Castillo Gutiérrez S, Gálvez A and Abriouel H. (2016). Fermented Aloreña table olives as a source of potential probiotic Lactobacillus pentosus strains. Front. Microbiol. 7: 1583.

(3) Abriouel H, Pérez Montoro B, Casado Muñoz MC, Lavilla Lerma L, Hidalgo Pestaña M, Caballero Gómez N, Franz CMAP, Gálvez A, Benomar N. (2016). Complete genome sequence of a potentially probiotic Lactobacillus pentosus MP-10 isolated from fermented Aloreña table olives. Genome Announc. 4(5): e00854–16. pmid:27634988.

(4) Abriouel H, Pérez Montoro B, Casado Muñoz MdC, Knapp CW, Gálvez A, Benomar N. (2017). In silico genomic insights into aspects of food safety and defense mechanisms of a potentially probiotic Lactobacillus pentosus MP-10 isolated from brines of naturally fermented Aloreña green table olives. PLoS ONE 12(6): e0176801.