Diversity of Lactobacillus sakei strains investigated by phenotypic and genotypic methods

A Lactobacillus sakei strain named FLEC01 was isolated from human feces and characterized genotypically. Comparison of the genetic features of this strain with those of both the meat-borne L. sakei strain 23K and another human isolate, LTH5590, showed that they belong to different but closely related clusters. The three L. sakei strains did not persist and only transited through the gastrointestinal tracts (GITs) of conventional C3H/HeN mice. In contrast, they all colonized the GITs of axenic mice and rapidly reached a population of 10 9 CFU/g of feces, which remained stable until day 51. Five days after mice were fed, a first subpopulation, characterized by small colonies, appeared and reached 50% of the total L. sakei population in mice. Fifteen to 21 days after feeding, a second subpopulation, characterized by rough colonies, appeared. It coexisted with the two other populations until day 51, and its cell shapes were also affected, suggesting a dysfunction of the cell division or cell wall. No clear difference between the behaviors of the meat-borne strain and the two human isolates in both conventional and axenic mice was observed, suggesting that L. sakei is a food-borne bacterium rather than a commensal one and that its presence in human feces originates from diet. Previous observations of Escherichia coli strains suggest that the mouse GIT environment could induce mutations to increase their survival and colonization capacities. Here, we observed similar mutations concerning a food-grade grampositive bacterium for the first time.Although initially characterized from rice wine (28), the lactic acid bacterium species Lactobacillus sakei is considered the main representative flora of meat products, representing the major population of many fermented meat products and of raw meat stored under vacuum-packaged conditions (10,12,13). L. sakei is naturally present in many fish and meat products that are traditionally processed without the use of starter cultures (33). In addition, when small-scale facilities producing traditional dry fermented sausage were searched, L. sakei was detected only in the meat matrix, suggesting that meat is contaminated by this species mainly during the early processing steps (certainly by hide or feces of the animals) and not later on or by contact with the environment or materials within the facilities (2).L. sakei shows high degrees of phenotypic and genomic diversity (11-13) that may explain the difficult detection and misidentification of it in the past. For instance, although the human gut microbiota has been intensively investigated by different microbial and molecular methods for many years, the presence of L. sakei in the feces of healthy humans was reported only recently (16,17,26,39). The presence of the meat-borne species L. sakei in human feces, similar to that of several other lactobacilli, could be correlated to human diet, including raw and fermented meat (or fish), for millennia (37). Considering its relatively high concentration in human feces (10 6 per g) that was p...

supporting

confidence: 66%

Behavior of the Meat-Borne Bacterium Lactobacillus sakei during Its Transit through the Gastrointestinal Tracts of Axenic and Conventional Mice

Chiaramonte

Blugeon²,

Chaillou

et al. 2009

“…AFLP provides a high-throughput method for high-resolution genomic fingerprinting (20) that has been employed frequently for the classification of strains of various species, including lactobacilli (2,33). AFLP classification of 65 L. rhamnosus strains enabled the distinction of 11 genotypic groups at a similarity cutoff of 70%, with all L. casei strains constituting an outgroup, since they belong to a closely related but genetically distinct species.…”

Section: Resultsmentioning

confidence: 99%

Correlation of Lactobacillus rhamnosus Genotypes and Carbohydrate Utilization Signatures Determined by Phenotype Profiling

Ceapă

Lambert

Limpt

et al. 2015

e Lactobacillus rhamnosus is a bacterial species commonly colonizing the gastrointestinal (GI) tract of humans and also frequently used in food products. While some strains have been studied extensively, physiological variability among isolates of the species found in healthy humans or their diet is largely unexplored. The aim of this study was to characterize the diversity of carbohydrate utilization capabilities of human isolates and food-derived strains of L. rhamnosus in relation to their niche of isolation and genotype. We investigated the genotypic and phenotypic diversity of 25 out of 65 L. rhamnosus strains from various niches, mainly human feces and fermented dairy products. Genetic fingerprinting of the strains by amplified fragment length polymorphism (AFLP) identified 11 distinct subgroups at 70% similarity and suggested niche enrichment within particular genetic clades. High-resolution carbohydrate utilization profiling (OmniLog) identified 14 carbon sources that could be used by all of the strains tested for growth, while the utilization of 58 carbon sources differed significantly between strains, enabling the stratification of L. rhamnosus strains into three metabolic clusters that partially correlate with the genotypic clades but appear uncorrelated with the strain's origin of isolation. Draft genome sequences of 8 strains were generated and employed in a gene-trait matching (GTM) analysis together with the publicly available genomes of L. rhamnosus GG (ATCC 53103) and HN001 for several carbohydrates that were distinct for the different metabolic clusters: L-rhamnose, cellobiose, L-sorbose, and ␣-methyl-Dglucoside. From the analysis, candidate genes were identified that correlate with L-sorbose and ␣-methyl-D-glucoside utilization, and the proposed function of these genes could be confirmed by heterologous expression in a strain lacking the genes. This study expands our insight into the phenotypic and genotypic diversity of the species L. rhamnosus and explores the relationships between specific carbohydrate utilization capacities and genotype and/or niche adaptation of this species.

“…No differences in the degradation capacity of these substrates among the various strains tested were found. However, a wide phenotypic and genotypic heterogeneity regarding structural and metabolic properties within strains of L. sakei has been reported before (3,24). Inosine consumption resulted in the excretion of hypoxanthine in the fermentation medium, whereas for adenosine conversion, adenine and not hypoxanthine was released into the medium.…”

Section: Discussionmentioning

confidence: 99%

The Pentose Moiety of Adenosine and Inosine Is an Important Energy Source for the Fermented-Meat Starter Culture Lactobacillus sakei CTC 494

Rimaux

Vrancken

Vuylsteke

et al. 2011

The genome sequence of Lactobacillus sakei 23K has revealed that the species L. sakei harbors several genes involved in the catabolism of energy sources other than glucose in meat, such as glycerol, arginine, and nucleosides. In this study, a screening of 15 L. sakei strains revealed that arginine, inosine, and adenosine could be used as energy sources by all strains. However, no glycerol catabolism occurred in any of the L. sakei strains tested. A detailed kinetic analysis of inosine and adenosine catabolism in the presence of arginine by L. sakei CTC 494, a fermentedmeat starter culture, was performed. It showed that nucleoside catabolism occurred as a mixed-acid fermentation in a pH range (pH 5.0 to 6.5) relevant for sausage fermentation. This resulted in the production of a mixture of acetic acid, formic acid, and ethanol from ribose, while the nucleobase (hypoxanthine and adenine in the case of fermentations with inosine and adenosine, respectively) was excreted into the medium stoichiometrically. This indicates that adenosine deaminase activity did not take place. The ratios of the different fermentation end products did not vary with environmental pH, except for the fermentation with inosine at pH 5.0, where lactic acid was produced too. In all cases, no other carbon-containing metabolites were found; carbon dioxide was derived only from arginine catabolism. Arginine was cometabolized in all cases and resulted in the production of both citrulline and ornithine. Based on these results, a pathway for inosine and adenosine catabolism in L. sakei CTC 494 was presented, whereby both nucleosides are directly converted into their nucleobase and ribose, the latter entering the heterolactate pathway. The present study revealed that the pentose moiety (ribose) of the nucleosides inosine and adenosine is an effective fermentable substrate for L. sakei. Thus, the ability to use these energy sources offers a competitive advantage for this species in a meat environment.Lactobacillus sakei is the most prevalent lactic acid bacterium (LAB) species encountered in spontaneously fermented sausages, which demonstrates its competitiveness in and adaptation to the meat environment (7,11,22,31,32). Accordingly, its use as a starter culture for meat fermentation is widespread (22). Meat is a rich substrate, allowing the growth of several bacteria, not only LAB but also spoilage and pathogenic bacteria (2, 21). However, the variety of carbohydrates in fresh meat is relatively restricted and their amounts are limited, with glucose being the main fermentable carbohydrate present in a concentration below 0.1 mg g Ϫ1 (1, 2, 17). To effectively cope with this environment, the flexible use of all available energy sources and nutrients in meat is of importance.The genome sequence of L. sakei 23K has revealed that the species L. sakei evolved through its adaptation to the meat environment (2). For example, L. sakei lacks most genes encoding the biosynthesis of amino acids. Meat, which provides an environment rich in amino acids because o...