Fig 1 - uploaded by Mari Luz Mohedano
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Physical map of pRCR12. The plasmid carries the following: the mrfp gene encoding the mCherry protein under the control of the Px promoter of S. pneumoniae and the cat gene, which confers chloramphenicol resistance, and it contains the replicon of plasmid pSH71 from L. lactis
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A critical feature of probiotic microorganisms is their ability to colonize the intestine of the host. Although the microbial potential to adhere to the human gut lumen has been investigated in in vitro models, there is still much to discover about their in vivo behaviour. Zebrafish is a vertebrate model that is being widely used to investigate var...
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... pRCR12 plasmid ( Fig. 1) constructed in this work is based on the promoter-probe pRCR plasmid (Mohedano et al. 2015). The pRCR plasmid carries the replication origin from the lactococcal plasmid pSH71, the cat gene responsible for chloramphenicol resistance and a synthetic mrfp gene codon optimized for LAB (García-Cayuela et al. 2012), which encodes the monomeric mCherry fluorescent protein. pRCR12 (Fig. 1) was constructed by insertion of the strong P x pneumococcal promoter of the malXCD operon (Nieto et al. 1998) into the pRCR plasmid upstream of mrfp (GenBank ID: KP182347) generating the transcriptional fusion P x -mrfp in pRCR12. To this end, a 0.5 kb amplicon (GenBank ID: KP182346) was generated by use of chromosomal DNA of S. pneumoniae JNR7/87 (Bricker and Camilli 1999) as template and ForP x (5 - GGAAGATCTCTTTTCAAGCATAATTGCAAGCG-3 ′ ) and RevP x (5 ′ -CTAGTCTAGACCTCCTAAAGAATAGCAAGT TTTATTG-3 ′ ) primers. After digestion with Bgl II and Xba I, the amplicon was introduced into the multicloning site of pRCR, previously digested with the same restriction enzymes. The ligation mixture was used to transform competent Escherichia coli DH5 α cells, and transformants, carrying the pRCR12 plasmid, were selected by chloramphenicol resistance and detected on Luria broth agar (Oxoid) by the red colour of the colonies. The presence of the transcriptional fusion in pRCR12 was confirmed by DNA sequencing with ForP and RevmRFP ...
Context 2
... pRCR12 plasmid ( Fig. 1) constructed in this work is based on the promoter-probe pRCR plasmid (Mohedano et al. 2015). The pRCR plasmid carries the replication origin from the lactococcal plasmid pSH71, the cat gene responsible for chloramphenicol resistance and a synthetic mrfp gene codon optimized for LAB (García-Cayuela et al. 2012), which encodes the monomeric mCherry fluorescent protein. pRCR12 (Fig. 1) was constructed by insertion of the strong P x pneumococcal promoter of the malXCD operon (Nieto et al. 1998) into the pRCR plasmid upstream of mrfp (GenBank ID: KP182347) generating the transcriptional fusion P x -mrfp in pRCR12. To this end, a 0.5 kb amplicon (GenBank ID: KP182346) was generated by use of chromosomal DNA of S. pneumoniae JNR7/87 (Bricker and Camilli 1999) as template and ForP x (5 - GGAAGATCTCTTTTCAAGCATAATTGCAAGCG-3 ′ ) and RevP x (5 ′ -CTAGTCTAGACCTCCTAAAGAATAGCAAGT TTTATTG-3 ′ ) primers. After digestion with Bgl II and Xba I, the amplicon was introduced into the multicloning site of pRCR, previously digested with the same restriction enzymes. The ligation mixture was used to transform competent Escherichia coli DH5 α cells, and transformants, carrying the pRCR12 plasmid, were selected by chloramphenicol resistance and detected on Luria broth agar (Oxoid) by the red colour of the colonies. The presence of the transcriptional fusion in pRCR12 was confirmed by DNA sequencing with ForP and RevmRFP ...
Citations
... In fact, probiotic colonization of the host gastrointestinal tract (GIT) is an issue that deserves additional attention. While some of the potential aquaculture probiotics were reported as delivering their positive effect while being transient colonizers [20,21], colonization is a recommended property of probiotics that can help stimulate the host's immune system to protect against pathogens. Moreover, different functional ecological niches (organs and tissues) of the aquaculture animals can be subject to the effect of the probiotics. ...
The use of microorganisms as beneficial crops for human and animal health has been studied for decades, and these microorganisms have been in practical use for quite some time. Nowadays, in addition to well-known examples of beneficial properties of lactic acid bacteria, bifidobacteria, selected Bacillus spp., and yeasts, there are several other bacteria considered next-generation probiotics that have been proposed to improve host health. Aquaculture is a rapidly growing area that provides sustainable proteins for consumption by humans and other animals. Thus, there is a need to develop new technologies for the production practices associated with cleaner and environment-friendly approaches. It is a well-known fact that proper selection of the optimal probiotics for use in aquaculture is an essential step to ensure effectiveness and safety. In this critical review, we discuss the evaluation of host-specific probiotics in aquaculture, challenges in using probiotics in aquaculture, methods to improve the survival of probiotics under different environmental conditions, technological approach to improving storage, and delivery along with possible negative consequences of using probiotics in aquaculture. A critical analysis of the identified challenges for the use of beneficial microbes in aquaculture will help in sustainable aquafarming, leading to improved agricultural practices with a clear aim to increase protein production.
... Apart from related genera, constitutive promoters from phylogenetically distant bacteria have been also tested in L. plantarum strains. The constitutive promoter P X , isolated from the Gram-positive bacteria Streptococcus pneumoniae, was able to drive high expression of the red fluorescent (mRFP5) protein in probiotic L. plantarum 90 and B2 strains (Russo et al., 2015). The strong fluorescent protein expression allowed monitoring of bacterial colonization in the intestinal tract of zebrafish larvae. ...
Lactobacilli are ubiquitous in nature and symbiotically provide health benefits for countless organisms including humans, animals and plants. They are vital for the fermented food industry and are being extensively explored for healthcare applications. For all these reasons, there is considerable interest in enhancing and controlling their capabilities through the engineering of genetic modules and circuits. One of the most robust and reliable microbial chassis for these synthetic biology applications is the widely used Lactiplantibacillus plantarum species. However, the genetic toolkit needed to advance its applicability remains poorly equipped. This mini‐review highlights the genetic parts that have been discovered to achieve food‐grade recombinant protein production and speculates on lessons learned from these studies for L. plantarum engineering. Furthermore, strategies to identify, create and optimize genetic parts for real‐time regulation of gene expression and enhancement of biosafety are also suggested.
... The gnotobiotic zebrafish were prepared following the protocol of Russo et al. [33] with slight modifications. The embryo wash buffer was replaced with the E3 medium, and the embryos were incubated overnight at 28.5 ℃ with antibiotic solution and phenyl-thio-urea (PTU). ...
... For the in vivo studies, the probiotic B. subtilis was tagged with pDsRed-monomer. A similar study was conducted by Russo et al. [33], who used mCherry protein. The fluorescent signals in exposed fish and further confirmation by re-isolating the RFP-tagged probiotic bacterium, and amplifying pDsRed-monomer using pDsRed-specific primers proved the robustness of the technique for in vivo validation. ...
The indiscriminate use of antibiotics in aquaculture has led to the emergence of resistance; hence, eco-friendly, host-specific alternatives to mitigate bacterial infections have become imminent. In this study, bacteria that could possibly serve as probiotics were isolated and evaluated for their efficacy with in vitro experiments and in vivo zebrafish gut model. One isolate from each of the 23 rohu fish (Labeo rohita) was shortlisted after preliminary screening of several isolates and tested for their ability to inhibit two important warm water bacterial fish pathogens, Aeromonas hydrophila, and Edwardsiella tarda. An isolate (RODK28110C3) that showed broad-spectrum inhibitory activity against a battery of different isolates of the two fish pathogens included in this study and maintained in our repository was selected for further characterization. The culture was identified phenotypically as Bacillus subtilis and confirmed by 16S rDNA sequencing. The isolate was able to hydrolyze fish feed constituents that include starch, protein, and cellulose. Further in vitro tests ensured that the potential isolate with probiotic attributes could tolerate different gut conditions, which included a range of pH, salinity, and varying concentrations of bile salt. Exposure of 4 days post fertilization zebrafish embryos to the RFP-tagged isolate confirmed the colonization of B. subtilis in the gut of the zebrafish embryo, which is an important attribute of a probiotic. The isolate was able to inhibit both A. hydrophila and E. tarda in gnotobiotic zebrafish embryo in triplicate. The study demonstrates the probiotic characteristics of the B. subtilis isolated from L. rohita and its ability to inhibit A. hydrophila and E. tarda using in vitro conditions and in the zebrafish gut and could serve as an effective alternative to antibiotics in aquaculture.
... Altogether, there are interesting applications for vitamin-overproducing strains as their antioxidant and anti-inflammatory properties can improve host health and the vitamin is protected from the harsh environment of the GI tract as it is secreted directly into the large intestine [31,33, 35,36]. ...
... Zebrafish embryos were obtained from wild-type adult zebrafish (Danio rerio, Hamilton 1822), bred and maintained in the AZTI Zebrafish Facility (REGA number ES489010006105; Derio, Spain) as previously described (Russo et al., 2015) and following standard conditions (Sullivan and Kim, 2008). All experimental procedures were approved by the Regional Animal-Welfare Body. ...
Some lactic acid bacteria (LAB) strains isolated from alcoholic beverages are able to produce exopolysaccharides (EPS). The present work focuses on the physico-chemical characterization of the heteropolysaccharides (HePS) produced by Liquorilactobacillus sicerae CUPV261T (formerly known as Lactobacillus sicerae) and Secundilactobacillus collinoides CUPV237 (formerly known as Lactobacillus collinoides) strains isolated from cider. Genome sequencing and assembly enabled the identification of at least four putative HePS gene clusters in each strain, which correlated with the ability of both strains to secrete EPS. The crude EPS preparation from CUPV261T contained glucose, galactose and rhamnose, and that of CUPV237 was composed of glucose, galactose and N-acetylglucosamine. Both EPS were mixtures of HePS of different composition, with two major soluble components of average molecular weights (Mw) in the range of 106 and 104 g.mol-1. These HePS were resistant to gastric stress conditions in an in vitro model, and they significantly reduced zebrafish larvae mortality in an in vivo model of inflammatory bowel disease.
... The in vivo colonization ability of three probiotic lactic acid bacteria [viz., Lactobacillus plantarum Lp90, L. plantarum B2, and Lactobacillus fermentum PBCC11.5] was assessed in the gnotobiotic zebrafish larvae as the model system (Russo et al. 2015). These bacterial species were transformed with plasmid pRCR12, which contained the mCherry protein. ...
The microbiome is a complex yet defined set of microbial species in a given habitat. The mining of the human microbiome is pursued in several research projects globally. The microbiota of other vertebrate animals also needs to be studied. Fish represent a diverse group of vertebrates and are the primary source of animal protein. The fish gut microbiota plays a crucial role in the host’s metabolism and can be manipulated to maximize production. Similar to humans, the fish microbiota is studied using culture-dependent and culture-independent techniques, including next-generation sequencing, bioinformatics analysis, and omics. Various fish organs have been examined for their dominant and distinctive microbiota species, including the gills, skin, gut, kidney, liver, and spleen. Under various environmental, dietary, and other physiological situations, the gut microbiota is explored. Gnotobiotic zebrafish represent a valuable model for studying microbe–microbe and microbe–host interactions. Microbe–microbe interactions can be modulated by pre-, pro, and synbiotics, influencing the development of the fish immune system, which will eventually reduce the need for antibiotics. This chapter highlights the necessity of research to advance knowledge of fish health, immune system, metabolism, probiotic research, etc., and to extract novel biomolecules with functional features for creating new medications and pharmaceuticals.
... For silage, adding lactic acid bacteria could reduce pH; increase lactic acid concentration and inhibit the production of mycotoxins [32]; improve the growth performance of calves [33]; and inhibit the attachment of Escherichia coli, Klebsiella ent of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa [34]. However, a problem that arises and needs to be solved is whether lactic acid bacteria can rapidly colonize after addition, and whether lactic acid bacteria can be induced to play a better supportive role [35]. In this study, E. avium EA2, S. lutetiensis SL2 and SL3, and S. equinus SE1 had faster growth rates, which means that these strains may rapidly proliferate to become the dominant floras and competitively inhibit the growth of pathogenic bacteria [36]. ...
Lactic acid bacteria are some of the dominant bacteria in the rumen, and they have a high ability for lactic acid production. The present study aimed to screen and evaluate the performance of culturable rumen bacteria from Chinese Holstein dairy cows as a potential probiotic or inoculant for silage production, in order to isolate ruminal lactic acid bacteria and evaluate their potential as probiotics. Three strains of Enterococcus avium (E. avium, EA1-3); three strains of Streptococcus lutetiensis (S. lutetiensis, SL1-3); and six strains of Streptococcus equinus (S. equinus, SE1-6) were successfully identified from the rumen fluid using modified De Man Rogosa sharp medium supplemented with 0.325% lactic acid. E. avium, S. lutetiensis and S. equinus are clustered in the phylogenetic tree. All the 12 Gram-positive strains reached the plateau growth phase in 6–10 h, with an OD600 at about 1.8. Both gas and acid accumulation reached plateaus at about 10–12 h in all strains, and S. equinus showed the strongest capacity. The highest lactic acid accumulation was detected in S. equinus broth (up to 219.77 μmol/L). The growth of all isolates was inhibited at pH 4.0, and EA2, SL1, SL2, SL3 and SE2 were tolerant to 0.1%, 0.2% and 0.3% bile salt. In addition, the supernatants of the strains had inhibitory effects on Escherichia coli and Staphylococcus aureus. Specifically, the S. equinus strains exhibited the strongest inhibition of the pathogens. In conclusion, these 12 strains had good potential as silage inoculants or probiotics for edible animals, especially S. equinus.
... In this sense, a study using gnotobiotic zebrafish demonstrated the importance of the presence of specific adhesion factors not only for the establishment of probiotic bacteria in the intestine, but also for their protective effect against pathogen infection (Rendueles et al. 2012). Other Lactobacillus species such as L. plantarum, L. brevis, L. rhamnosus, and L. casei were also able to adhere to and colonize the zebrafish gut (Zhou et al. 2012;Rieu et al. 2014;Russo et al. 2015). ...
Studies about probiotics isolated from Patagonian fish are scarce. Three autochthonous lactic acid bacteria (LAB) were previously isolated from Patagonian fish: Carnobacterium sp. T4, Lactococcus lactis TW34, and Lactobacillus pentosus H16. Zebrafish has been extensively used as an experimental vertebrate model; however, this model has recently been used to assess the intestinal bacterial colonization. In this study, we researched the in vitro probiotic properties of the Patagonian LABs T4, TW34, and H16, and their in vivo capability to colonize the gastrointestinal tract of zebrafish. Such strains were tolerant to trout bile and acid pH values, which is an essential property for their survival in the fish gut. H16 was the most resistant strain to low pH values. It specifically adhered to mucus, unlike T4 and TW34 that showed nonspecific adhesion. Zebrafish were fed daily with commercial food supplemented with T4, TW34, or H16 at a final concentration of 10⁷ CFU g⁻¹ of dry food, while the control group was fed only with commercial food. The presence of T4, TW34, and H16 was detected in the zebrafish intestines of the treated groups. After the treatments, changes in the zebrafish intestinal microbiota were recorded. The counts of vibrios and enterobacteria decreased, while the LAB count increased. This study showed that autochthonous LAB strains isolated from Patagonian fish were able to colonize the intestine of the zebrafish and modify the gut microbiota balance towards health-promoting bacteria. These findings propose them as interesting probiotic candidates for use in aquaculture purposes.
... L. plantarum B2 (CECT 8328, Valencia, Spain), B 2 overproducing strain was characterized for its probiotic potential in vitro and in vivo and used in the production of B 2 -enriched foods. 13,17,18 In continuation of our previous report on B 2 bioenriched soymilk fermented with B 2 overproducing L. plantarum B2 (CECT 8328, Valencia, Spain), 13,19 herein, we established the B 2 deficient mice model to evaluate the reversion effects of normal soymilk, commercial B 2 , and B 2 bioenriched soymilk on B 2 deficiency biomarkers (phenotypic symptoms and EGRAC), serum oxidative stress markers (SOD, CAT, MDA, T-AOC), and inflammation symptoms (tissue structure of the liver and colon), which were confirmed at mRNA and protein levels. Further, changes in mice intestinal microflora (diversity and variation analysis of gut microbiota and cecal short-chain fatty acid content) responding to oxidative stress and inflammation were also evaluated. ...
... Other papers have reported that Pediococcus acidilactici (Bactocell ® , Lallemand Inc., Montreal, QC, Canada) [5], Bacillus paralicheniformis FA6 [6], Lactiplantibacillus plantarum G1 [7], Lacticaseibacillus casei ATCC 393 [8], Latilactobacillus sakei CLFP 202 [9], Lactococcus lactis CLFP 100 [9] and Leuconostoc mesenteroides CLFP 196 [9] can also colonize the GI tract of goldfish, grass carp, shabout fish and rainbow trout. However, some papers have shown that probiotic strains, including Lactobacillus, in the GI tract rapidly decreases following the withdrawal of supplementation [10][11][12][13][14][15][16], indicating their transient nature. Meanwhile, Ringø et al. [17] raised the following question: "Are probiotics permanently colonizing the GI tract?". ...
Lactic acid bacteria (LAB) are commonly applied to fish as a means of growth promotion and disease prevention. However, evidence regarding whether LAB colonize the gastrointestinal (GI) tract of fish remains sparse and controversial. Here, we investigated whether Lacticaseibacillus casei ATCC 393 (Lc) can colonize the GI tract of crucian carp. Sterile feed irradiated with 60Co was used to eliminate the influence of microbes, and 100% rearing water was renewed at 5-day intervals to reduce the fecal–oral circulation of microbes. The experiment lasted 47 days and was divided into three stages: the baseline period (21 days), the administration period (7 days: day −6 to 0) and the post-administration period (day 1 to 19). Control groups were fed a sterile basal diet during the whole experimental period, whereas treatment groups were fed with a mixed diet containing Lc (1 × 107 cfu/g) and spore of Geobacillus stearothermophilus (Gs, 1 × 107 cfu/g) during the administration period and a sterile basal diet during the baseline and post-administration periods. An improved and highly sensitive selective culture method (SCM) was employed in combination with a transit marker (a Gs spore) to monitor the elimination of Lc in the GI tract. The results showed that Lc (
