Reporter systems for<i>in vivo</i>tracking of lactic acid bacteria in animal model studies

Zyl, Winschau F. van; Deane, Shelly M.; Dicks, Leon M. T.

doi:10.1080/19490976.2015.1086058

Cited by 10 publications

(13 citation statements)

References 86 publications

(134 reference statements)

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“…In addition, the optical transparency of zebrafish larvae allows the easy detection of fluorescent-labeled bacteria for studies of host–microbe interactions ( Rieu et al, 2014 ; Oyarbide et al, 2015 ; Russo et al, 2015 ). Thus, the development of fluorescence reporter systems to label putative probiotic bacteria is useful for tracking them inside the animal model used ( van Zyl et al, 2015 ; Landete et al, 2016 ). Accordingly, we have developed the pRCR12 plasmid ( Russo et al, 2015 ) derived from the pRCR promoter probe vector ( Mohedano et al, 2015 ), which expresses constitutively the red fluorescent protein mCherry, it is useful for the fluorescent-labeling and detection of LAB, and it has been already validated in Lactobacillus acidophilus , Lactobacillus casei , Lactobacilus fermentum , Lactobacillus plantarum , Lactobacillus sakei , Lactococcus lactis , and Streptococcus pneumoniae ( Mohedano et al, 2015 ; Russo et al, 2015 ; Nácher-Vázquez et al, 2017 ; Pérez-Ramos et al, 2017b ).…”

Section: Introductionmentioning

confidence: 99%

β-Glucan-Producing Pediococcus parvulus 2.6: Test of Probiotic and Immunomodulatory Properties in Zebrafish Models

Pérez-Ramos

Mohedano

Pardo³

et al. 2018

Front. Microbiol.

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Lactic acid bacteria synthesize exopolysaccharides (EPS), which could benefit the host’s health as immunomodulators. Furthermore, EPS could protect bacteria against gastrointestinal stress, favoring gut colonization, thus protecting the host against pathogenic infections. Pediococcus parvulus 2.6, produces a 2-substituted (1,3)-β-D-glucan and, in this work, its probiotic properties as well as the immunomodulatory capability of its EPS have been investigated using Danio rerio (zebrafish). To this end and for a comparative analysis, P. parvulus 2.6 and its isogenic β-glucan-non-producing 2.6NR strain were fluorescently labeled by transfer of the pRCR12 plasmid, which encodes the mCherry protein. For the in vivo studies, there were used: (i) a gnotobiotic larvae zebrafish model for bacterial colonization, pathogen competition, and evaluation of the β-glucan immunomodulation capability and (ii) a transgenic (mpx:GFP) zebrafish model to determine the EPS influence in the recruitment of neutrophils under an induced inflammation. The results revealed a positive effect of the β-glucan on colonization of the zebrafish gut by P. parvulus, as well as in competition of the bacterium with the pathogen Vibrio anguillarum in this environment. The larvae treatment with the purified β-glucan resulted in a decrease of expression of genes encoding pro-inflammatory cytokines. Moreover, the β-glucan had an anti-inflammatory effect, when it was evaluated in an induced inflammation model of Tg(mpx:GFP) zebrafish. Therefore, P. parvulus 2.6 and its EPS showed positive health properties in in vivo fish models, supporting their potential usage in aquaculture.

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Section: Introductionmentioning

confidence: 99%

β-Glucan-Producing Pediococcus parvulus 2.6: Test of Probiotic and Immunomodulatory Properties in Zebrafish Models

Pérez-Ramos

Mohedano

Pardo³

et al. 2018

Front. Microbiol.

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“…A major advantage of using the BLI technique compared to conventional approaches is that it allows for drastic reductions in the number of animals to be sacrificed to establish the precise location of bacteria in mouse or rat models [ 20 ]. Moreover, more information is gathered over a shorter period per experiment compared to traditional pre-clinical animal trials [ 21 ]. Mouse models provide a complex whole body system for the non-invasive real-time monitoring of bioluminescent probiotic LAB through the GIT of the mammalian host [ 43 ].…”

Section: Discussionmentioning

confidence: 99%

“…Mouse models provide a complex whole body system for the non-invasive real-time monitoring of bioluminescent probiotic LAB through the GIT of the mammalian host [ 43 ]. The murine model is the predominant choice for the in vivo evaluation of probiotic properties and has been used to study the persistence and localization of potential probiotic LAB in several studies [ 21 , 24 , 27 , 33 , 43 ]. In the current study, an IVIS system and the BLI technique were used to study the colonization dynamics of the probiotic LAB strains L. plantarum 423 and E. mundtii ST4SA in the GIT of mice.…”

Section: Discussionmentioning

confidence: 99%

“…The best approach to study real-time interactions between probiotic bacteria and their mammalian host in the GIT is by labelling the cells with fluorescent or bioluminescent markers [ 20 – 26 ]. A selection of genes, encoding proteins that emit light at specific wavelengths, are available for cloning into plasmids or insertion into the genomes of recipient cells [ 21 , 22 ]. The most commonly used luciferase labelling systems used in in vivo and ex vivo tracking of bacteria are bacterial luxABCDE from Photorabdus luminescence [ 26 ], click beetle luciferase (CBluc) from Pyrophorus plagiophthalamus [ 23 ] and firefly luciferase (Ffluc) from Photinus pyralis [ 25 ].…”

Section: Introductionmentioning

confidence: 99%

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In vivo bioluminescence imaging of the spatial and temporal colonization of lactobacillus plantarum 423 and enterococcus mundtii ST4SA in the intestinal tract of mice

2018

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BackgroundLactic acid bacteria (LAB) are major inhabitants and part of the normal microflora of the gastrointestinal tract (GIT) of humans and animals. Despite substantial evidence supporting the beneficial properties of LAB, only a few studies have addressed the migration and colonization of probiotic bacteria in the GIT. The reason for this is mostly due to the limitations, or lack of, efficient reporter systems. Here we describe the development and application of a non-invasive in vivo bioluminescence reporter system to study, in real-time, the spatial and temporal persistence of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA in the intestinal tract of mice.ResultsThis study reports on the application of the firefly luciferase gene (ffluc) from Photinus pyralis to develop luciferase-expressing L. plantarum 423 and E. mundtii ST4SA, using a Lactococcus lactis NICE system on a high copy number plasmid (pNZ8048) and strong constitutive lactate dehydrogenase gene promoters (Pldh and STldh). The reporter system was used for in vivo and ex vivo monitoring of both probiotic LAB strains in the GIT of mice after single and multiple oral administrations. Enterococcus mundtii ST4SA reached the large intestine 45 min after gavage, while L. plantarum 423 reached the cecum/colon after 90 min. Both strains predominantly colonized the cecum and colon after five consecutive daily administrations. Enterococcus mundtii ST4SA persisted in faeces at higher numbers and for more days compared to L. plantarum 423.ConclusionsOur findings demonstrate the efficiency of a high-copy number vector, constitutive promoters and bioluminescence imaging to study the colonization and persistence of L. plantarum 423 and E. mundtii ST4SA in the murine GIT. The system allowed us to differentiate between intestinal transit times of the two strains in the digestive tract. This is the first report of bioluminescence imaging of a luciferase-expressing E. mundtii strain to study colonization dynamics in the murine model. The bioluminescence system developed in this study may be used to study the in vivo colonization dynamics of other probiotic LAB.Electronic supplementary materialThe online version of this article (10.1186/s12866-018-1315-4) contains supplementary material, which is available to authorized users.

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“…In the same way, the osmY gene showed the highest light emission in muscle tissue. Bioluminescence-based bacterial reporter genes are useful to identify and study in vitro and in vivo gene regulation ( Méndez and Guijarro, 2013 ; van Zyl et al, 2015 ). This result, together with that obtained using randomly selected clones cultivated in the presence of rainbow trout tissues, indicates that plate assays seem to be useful to determine Y. ruckeri differential gene expression in trout tissues.…”

Section: Discussionmentioning

confidence: 99%

Temperature-Dependent Gene Expression in Yersinia ruckeri: Tracking Specific Genes by Bioluminescence During in Vivo Colonization

2018

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Yersinia ruckeri is a bacterium causing fish infection processes at temperatures below the optimum for growth. A derivative Tn5 transposon was used to construct a library of Y. ruckeri mutants with transcriptional fusions between the interrupted genes and the promoterless luxCDABE and lacZY operons. In vitro analysis of β-galactosidase activity allowed the identification of 168 clones having higher expression at 18°C than at 28°C. Among the interrupted genes a SAM-dependent methyltransferase, a diguanylated cyclase, three genes involved in legionaminic acid synthesis and three transcriptional regulators were defined. In order to determine, via bioluminescence emission, the in vivo expression of some of these genes, two of the selected mutants were studied. In one of them, the acrR gene coding a repressor involved in regulation of the AcrAB-TolC expulsion pump was interrupted. This mutant was found to be highly resistant to compounds such as chloramphenicol, tetracycline, and ciprofloxacin. Although acrR mutation was not related to virulence in Y. ruckeri, this mutant was useful to analyze acrR expression in fish tissues in vivo. The other gene studied was osmY which is activated under osmotic stress and is involved in virulence. In this case, complemented mutant was used for experiments with fish. In vivo analysis of bioluminescence emission by these two strains showed higher values for acrR in gut, liver and adipose tissue, whereas osmY showed higher luminescence in gut and, at the end of the infection process, in muscle tissue. Similar results were obtained in ex vivo assays using rainbow trout tissues. The results indicated that this kind of approach was useful for the identification of genes related to virulence in Y. ruckeri and also for the in vivo and in vitro studies of each of the selected genes.

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Reporter systems forin vivotracking of lactic acid bacteria in animal model studies

Cited by 10 publications

References 86 publications

β-Glucan-Producing Pediococcus parvulus 2.6: Test of Probiotic and Immunomodulatory Properties in Zebrafish Models

β-Glucan-Producing Pediococcus parvulus 2.6: Test of Probiotic and Immunomodulatory Properties in Zebrafish Models

In vivo bioluminescence imaging of the spatial and temporal colonization of lactobacillus plantarum 423 and enterococcus mundtii ST4SA in the intestinal tract of mice

Temperature-Dependent Gene Expression in Yersinia ruckeri: Tracking Specific Genes by Bioluminescence During in Vivo Colonization

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