Use of green fluorescent protein to tag lactic acid bacterium strains under development as live vaccine vectors

The lactic acid bacteria (LAB) are safe microorganisms which are mainly used for the preparation of fermented foods and for probiotic applications. The potential of LAB as live vehicles for the production and delivery of therapeutic molecules such as antigens is also being actively investigated today. However, very little is known about the fate of live LAB when administered in vivo and about the interaction of these microorganisms with the nasal or gastrointestinal ecosystem. For future applications, it is essential to be able to discriminate the biotherapeutic strain from the endogenous microflora and to unravel the mechanisms underlying the postulated health-beneficial effect. We therefore started to investigate both aspects in a mouse model with two LAB species presently under development as live vaccine vectors, i.e., Lactococcus lactis and Lactobacillus plantarum. We have constructed different expression vectors carrying the gfp (green fluorescent protein [GFP]) gene from the jellyfish Aequoria victoria, and we found that this visible marker was best expressed when placed under the control of the inducible strong nisA promoter from L. lactis. Notably, a threshold amount of GFP was necessary to obtain a bright fluorescent phenotype. We further demonstrated that fluorescent L. plantarum NCIMB8826 can be enumerated and sorted by flow cytometry. Moreover, tagging of this strain with GFP allowed us to visualize its phagocytosis by macrophages in vitro and ex vivo and to trace it in the gastrointestinal tract of mice upon oral administration.     

Adaptation of the Nisin-Controlled Expression System in Lactobacillus plantarum: a Tool To Study In Vivo Biological Effects

The potential of lactic acid bacteria as live vehicles for the production and delivery of therapeutic molecules is being actively investigated today. For future applications it is essential to be able to establish dose-response curves for the targeted biological effect and thus to control the production of a heterologous biopeptide by a live lactobacillus. We therefore implemented in Lactobacillus plantarum NCIMB8826 the powerful nisin-controlled expression (NICE) system based on the autoregulatory properties of the bacteriocin nisin, which is produced by Lactococcus lactis. The original two-plasmid NICE system turned out to be poorly suited to L. plantarum. In order to obtain a stable and reproducible nisin dose-dependent synthesis of a reporter protein (β-glucuronidase) or a model antigen (the C subunit of the tetanus toxin, TTFC), the lactococcal nisRK regulatory genes were integrated into the chromosome of L. plantarum NCIMB8826. Moreover, recombinant L. plantarum producing increasing amounts of TTFC was used to establish a dose-response curve after subcutaneous administration to mice. The induced serum immunoglobulin G response was correlated with the dose of antigen delivered by the live lactobacilli.     

The clam Meretrix lamarckii (Bivalvia: Veneridae) is a rich repository of marine lactic acid bacterial strains

Lactic acid bacteria (LAB) were isolated from the intestinal tract of the wild clam Meretrix lamarckii caught from the coastal waters of Kashima, Ibaraki, Japan. As many as 415 isolates were obtained using the culture method, of which 70 were considered presumptive LAB strains based on phenotypic tests. Phylogenetic analysis of these presumptive isolates of LAB based on the sequence of the 16S rRNA gene demonstrated that the species belonged to several genera of Lactobacillus, Lactococcus and Pediococcus. Interestingly, however, the species composition was different between the samples in July and October 2010. Further analyses based on the fermentation profiles revealed that the LAB from the clam caught in July 2010 were identified to be Lactobacillus curvatus, Lactobacillus plantarum, Lactococcus lactis subsp. cremoris and Pediococcus pentosaceus, whereas those in October 2010 were identified to be Lactobacillus plantarum, Lactococcus lactis subsp. lactis and P. pentosaceus. The diversity of LAB in the intestinal tract of the clam suggests that the filter feeder bivalves such as M. lamarckii are a rich repository of marine isolates of LAB.     

Characterization,Identification and Application of Lactic Acid Bacteria Isolated from Forage Paddy Rice Silage

There has been growing interest to develop forage rice as a new feed resource for livestock. This study was to characterize the natural population of lactic acid bacteria (LAB) and select potentially excellent strains for paddy rice silage preparation in China. One hundred and twenty-six strains were isolated and screened from paddy rice silage prepared using a small-scale fermentation system, and ninety-nine of these isolates were considered to be LAB based on their Gram-positive and catalase-negative morphology and the production of most of their metabolic products as lactic acid. These isolates were divided into eight groups (A-H) on the basis of their morphological and biochemical characteristics. The Group A to H strains were identified as Lactobacillus (L.) plantarum subsp. plantarum (species ratio: 8.1%), L. casei (5.1%), Leuconostoc (Ln.) pseudomesenteroides (11.1%), Pediococcus (P.) pentosaceus (24.2%), Enterococcus (E.) mundtii (12.1%), Lactococcus (Lc.) garvieae (15.2%), E. faecium (9.1%) and Lc. lactis subsp. lactis (15.2%) based on sequence analyses of their 16S rRNA and recA genes. P. pentosaceus was the most abundant member of the LAB population in the paddy rice silage. A selected strain, namely L. casei R 465, was found to be able to grow under low pH conditions and to improve the silage quality with low pH and a relatively high content of lactic acid. This study demonstrated that forage paddy rice silage contains abundant LAB species and its silage can be well preserved by inoculation with LAB, and that strain R 465 can be a potentially excellent inoculant for paddy rice silage.     

Identification and probiotic potential of lactic acid bacteria from camel milk

In the present study, a total of 80 presumed lactic acid bacteria (LAB) were isolated from camel milk. Selected LAB were identified as Lactococcus lactis (cam 12), Enterococcus lactis (cam 14) and Lactobacillus plantarum (cam 15) and their potential were tested by tolerance & de-conjugation of bile salts, antimicrobial activity, surface hydrophobicity and adhesion potential) along with this of probiotics were evaluated for curd formation and assessed for sensory properties and syneresis. Selected LABs showed antimicrobial activity against wide range of pathogenic bacteria (Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus cereus and Escherchiaia. coli). LAB (cam 12, cam 14 and cam15) were highly sceptible to chloramphenicol, vancomycin, and tetracyclin. In vitro adhesion studies with Caco-2 cells demonstrated strong adhesion activity with hydrophobicity (99%) was observed. Acute oral toxicity of E. lactis and L. plantarum showed non-toxic, non-virulent and safe for industrial application. The study provides potential LAB which may act as a substitute of functional food, synthetic feed and industrial curd formulation with in the shortest span (240 min at 28–32 °C).     

A dynamic,genome-scale flux model of Lactococcus lactis to increase specific recombinant protein expression

Recently, lactic acid bacteria (LAB) have attracted a great deal of interest because of their potential to serve as oral delivery vehicles for recombinant protein vaccines. An important limitation to their use is the typically low level of heterologous expression obtained in LAB. To address this, a dynamic flux balance analysis (DFBA) model was used to identify gene targets for increasing specific expression of Green Fluorescent Protein (GFP), a model heterologous protein, in Lactococcus lactis IL1403. Two strains, each targeting one of the top model-identified genes, were constructed and tested in vivo. Data show that both strains, by a conservative estimate, achieved 15% higher GFP per cell than the control strain, a qualitative confirmation of the model predictions. A genome-scale DFBA model for L. lactis growing on M17 medium is presented along with the procedure for screening gene targets and a powerful method for visualizing fluxes in genome-scale metabolic networks.     

Development of Lactococcus lactis encoding fluorescent proteins,GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system

Fluorescent proteins are useful reporter molecules for a variety of biological systems. We present an alternative strategy for cloning reporter genes that are regulated by the nisin-controlled gene expression (NICE) system. Lactoccocus lactis was genetically engineered to express green fluorescent protein (GFP), mCherry or near-infrared fluorescent protein (iRFP). The reporter gene sequences were optimized to be expressed by L. lactis using inducible promoter pNis within the pNZ8048 vector. Expression of constructions that carry mCherry or GFP was observed by fluorescence microscopy 2 h after induction with nisin. Expression of iRFP was evaluated at 700 nm using an infrared scanner; cultures induced for 6 h showed greater iRFP expression than non-induced cultures or those expressing GFP. We demonstrated that L. lactis can express efficiently GFP, mCherry and iRFP fluorescent proteins using an inducible expression system. These strains will be useful for live cell imaging studies in vitro or for imaging studies in vivo in the case of iRFP.     

Lactococcus lactis Expressing either Staphylococcus aureus Fibronectin-Binding Protein A or Listeria monocytogenes Internalin A Can Efficiently Internalize and Deliver DNA in Human Epithelial Cells

Lactococci are noninvasive bacteria frequently used as protein delivery vectors and, more recently, as in vitro and in vivo DNA delivery vehicles. We previously showed that a functional eukaryotic enhanced green fluorescent protein (eGFP) expression plasmid vector was delivered in epithelial cells by Lactococcus lactis producing Listeria monocytogenes internalin A (L. lactis InlA⁺), but this strategy is limited in vivo to transgenic mice and guinea pigs. In this study, we compare the internalization ability of L. lactis InlA⁺ and L. lactis producing either the fibronectin-binding protein A of Staphylococcus aureus (L. lactis FnBPA⁺) or its fibronectin binding domains C and D (L. lactis CD⁺). L. lactis FnBPA⁺ and L. lactis InlA⁺ showed comparable internalization rates in Caco-2 cells, while the internalization rate observed with L. lactis CD⁺ was lower. As visualized by conventional and confocal fluorescence microscopy, large clusters of L. lactis FnBPA⁺, L. lactis CD⁺, and L. lactis InlA⁺ were present in the cytoplasm of Caco-2 cells after internalization. Moreover, the internalization rates of Lactobacillus acidophilus NCFM and of an NCFM mutant strain with the gene coding for the fibronectin-binding protein (fbpA) inactivated were also evaluated in Caco-2 cells. Similar low internalization rates were observed for both wild-type L. acidophilus NCFM and the fbpA mutant, suggesting that commensal fibronectin binding proteins have a role in adhesion but not in invasion. L. lactis FnBPA⁺, L. lactis CD⁺, and L. lactis InlA⁺ were then used to deliver a eukaryotic eGFP expression plasmid in Caco-2 cells: flow cytometry analysis showed that the highest percentage of green fluorescent Caco-2 cells was observed after coculture with either L. lactis FnBPA⁺ or L. lactis InlA⁺. Analysis of the in vivo efficiency of these invasive recombinant strains is currently in progress to validate their potential as DNA vaccine delivery vehicles.The mucosal administration of bacterial carriers to deliver antigens and plasmid DNA constitutes a promising vaccination strategy. Pathogenic bacteria that have the capacity to invade cells, such as Listeria, Salmonella, and Shigella strains, have been used to deliver DNA constructs into mammalian cells (23). Nevertheless, the risk associated with possible reversion to a virulent phenotype of these pathogens is a major concern (5). Lactococcus lactis, the food-grade, gram-positive, noninvasive model bacterium, has been intensively used to deliver antigens and cytokines at the mucosal level (30). We previously showed (i) that native L. lactis can deliver a eukaryotic expression cassette coding for the bovine β-lactoglobulin (BLG), one of the major cow''s milk allergens, into mammalian epithelial Caco-2 cells, and (ii) that these cells were able to express and secrete BLG protein in its native conformation (10). Recently, we demonstrated the ability of native noninvasive L. lactis to deliver a fully functional plasmid to murine intestinal cells in vivo (2).The internalization of the bacterial carrier is a fundamental step to achieve efficient DNA delivery in eukaryotic cells (7). In order to increase DNA delivery by lactic acid bacteria (LAB), invasin genes were expressed in L. lactis. Due to the safety profile of LAB, recombinant lactococci expressing invasin genes from intracellular bacteria are attractive as potential DNA delivery vectors compared to the attenuated pathogens presently used.In this field, we previously demonstrated that L. lactis bacteria expressing the main Listeria monocytogenes invasin, internalin A (L. lactis InlA⁺), were able to invade eukaryotic cells and efficiently deliver a functional green fluorescent protein (GFP) expression plasmid into epithelial/endothelial cells (9). Even though attractive, the experimental use of lactococci expressing InlA in a mouse model has a major bottleneck: InlA, which binds to human E-cadherin (15), does not interact with murine E-cadherin. Consequently, in vivo experimental studies using lactococci expressing InlA as DNA delivery vehicles are limited to transgenic mice expressing human E-cadherin or to guinea pigs (13).Fibronectin binding protein A (FnBPA) of Staphylococcus aureus is another bacterial invasin that is involved in intracellular spreading of S. aureus in the host (27). It is a multifunctional adhesion protein having both fibrinogen and fibronectin binding capacities (24). Its N-terminal part, also called domain A, is responsible for fibrinogen (29) and elastin (20) binding, whereas its C-terminal part, including domains B, C, and D, binds to fibronectin (25). FnBPA is known to mediate adhesion to host tissue and bacterial uptake into nonphagocytic host cells (27). Its expression by L. lactis was previously shown to be sufficient to confer the ability to invade nonphagocytic cells in vitro and in vivo, while the expression of domains C and D confers invasivity only in vitro (19).In this study, we show that L. lactis bacteria expressing full-length FnBPA of S. aureus (L. lactis FnBPA⁺) or a truncated form encompassing only its C and D domains (L. lactis CD⁺) are internalized as efficiently as L. lactis InlA⁺ in the human intestinal cell line Caco-2. We also provide, for the first time, direct microscopic evidence of the intracellular location of the internalized lactococci, showing that the bacteria are heterogeneously distributed in the cell monolayer and that their number per cell can reach a surprisingly high level. However, we demonstrate that FbpA, a fibronectin binding protein from the commensal Lactobacillus acidophilus NCFM, does not mediate bacterial internalization: no difference in invasivity was observed between the wild-type (wt) strain and the mutant with fbpA inactivated. This result indicates that, although widely distributed among bacteria, fibronectin binding proteins are not universal mediators of bacterial internalization, even at low levels. Finally, we also demonstrate that, similarly to L. lactis InlA⁺, L. lactis FnBPA⁺ and L. lactis CD⁺ can efficiently deliver a eukaryotic GFP expression plasmid in Caco-2 cells and trigger GFP expression in these cells. Consequently, L. lactis FnBPA⁺ can be used for further DNA delivery experiments in vivo.     

Lactobacillus rhamnosus BFE 5264 and Lactobacillus plantarum NR74 Promote Cholesterol Excretion Through the Up-Regulation of ABCG5/8 in Caco-2 Cells

The effect of two putative probiotic strains, Lactobacillus rhamnosus BFE5264 and Lactobacillus plantarum NR74, on the control of cholesterol efflux in enterocytes was assessed by focusing on the promotion of ATP-binding cassette sub-family G members 5 and 8 (ABCG5 and ABCG8). Differentiated Caco-2 enterocytes were treated with live bacteria, heat-killed bacteria, a bacterial cell wall fraction, and metabolites and were subjected to cholesterol uptake assay, mRNA analysis, and protein analyses. Following LXR-transfection by incubation with CHO-K1 cells in DNA-lipofectin added media, the luciferase assay was conducted for LXR analysis. Treatment of Caco-2 cells with L. rhamnosus BFE5264 (isolated from traditional fermented Maasai milk) and L. plantarum NR74 (isolated from Korean kimchi) resulted in the up-regulation of LXR, concomitantly with the elevated expression of ABCG5 and ABCG8. This was associated with the promotion of cholesterol efflux at significantly higher levels compared to the positive control strain L. rhamnosus GG (LGG). The experiment with CHO-K1 cells confirmed up-regulation of LXR-beta by the test strains, and treatment with the live L. rhamnosus BFE5264 and L. plantarum NR74 strains significantly increased cholesterol efflux. Heat-killed cells and cell wall fractions of both LAB strains induced the upregulation of ABCG5/8 through LXR activation. By contrast, LAB metabolites did not show any effect on ABCG5/8 and LXR expression. Data from this study suggest that LAB strains, such as L. rhamnosus BFE5264 and L. plantarum NR74, may promote cholesterol efflux in enterocytes, and thus potentially contribute to the prevention of hypercholesterolemia and atherosclerosis.     

Species-specific FISH analysis of cecal microflora in rats administered with lactic acid bacteria

Summary We investigated the effects of lactic acid bacterial (LAB) cells in rats using fluorescence in situ hybridization (FISH) targeting 16S rRNA to identify the cecal microbial community. We designed a novel species-specific 16S rDNA probe to detect Lactobacillus rhamnosus (Lrham454). Subtractive technique using the LAC722 probe (Sakai et al. 2004 Journal of Bioscience and Bioengineering 98, 48) under different hybridization stringency (LAC722(L-H)) was applied to identify Lactococcus lactis. We also applied Lplan447 and LAC722(L) to detect Lactobacillus plantarum and a wide range of LAB (total LAB), respectively. We optimized the hybridization and washing conditions and then quantified L. rhamnosus, L. plantarum, and L. lactis cells in rat cecal contents. We monitored increases in individual bacterial populations and in total LAB caused by the administration of the corresponding LAB cells. Growth, food efficiency and internal disorders did not significantly differ among the rats administered with LABs. Rats administered with polydextrose (POL) developed diarrhea, which decreased the total numbers of cecal bacteria, whereas the simultaneous administration of POL and L. rhamnosus KY-3 eased this symptom, and recovered the numbers of total LAB and of L. rhamnosus.     

Lactobacillus plantarum WCFS1 Electron Transport Chains

Lactobacillus plantarum WCFS1 requires both heme and menaquinone to induce respiration-like behavior under aerobic conditions. The addition of these compounds enhanced both biomass production, without progressive acidification, and the oxygen consumption rate. When both heme and menaquinone were present, L. plantarum WCFS1 was also able to reduce nitrate. The ability to reduce nitrate was severely inhibited by the glucose levels that are typically found in L. plantarum growth media (1 to 2% [vol/vol] glucose). In contrast, comparable mannitol levels did not inhibit the reduction of nitrate. L. plantarum reduced nitrate with concomitant formation of nitrite and ammonia. Genes that encode a bd-type cytochrome (cydABCD) and a nitrate reductase (narGHJI) were identified in the genome of L. plantarum. The narGHJI operon is part of a cluster of genes that includes the molybdopterin cofactor biosynthesis genes and narK. Besides a menaquinone source, isogenic mutants revealed that cydA and ndh1 are required for the aerobic-respiration-like response and narG for nitrate reduction. The ndh1 mutant was still able to reduce nitrate. The existence of a nonredundant branched electron transport chain in L. plantarum WCFS1 that is capable of using oxygen or nitrate as a terminal electron acceptor is proposed.Lactic acid bacteria (LAB) are extensively used for the production of fermented foods from dairy, meat, fruit, and vegetable sources. These fermented foods are valued for their enhanced shelf life, flavor, and structural properties. LAB have been exploited for this purpose for millennia and generally behave as facultative anaerobic, obligate fermentative bacteria.However, the production of cytochromes, typical constituents of respiratory chains, has been observed in several LAB species when they are grown in the presence of heme. These include Lactococcus lactis (Streptococcus lactis), Leuconostoc mesenteroides, and Enterococcus faecalis (36, 42).Recently, in L. lactis, generation of a proton motive force by a heme-dependent aerobic electron transport chain was demonstrated (9). In other words, heme induces respiration in L. lactis. L. lactis cells grown under these respiration-permissive conditions have enhanced biomass yields and are more robust (more resistant to oxygen, acid, and cold-storage stress) (15, 18, 31). Respiration-like behavior has also been reported for Streptococcus agalactiae and Oenococcus oeni (43; A. Gruss, unpublished results). However, there have been no published reports of heme-induced respiration-like behavior in any member of the genus Lactobacillus. This genus contains many species that are used extensively in food fermentation, such as Lactobacillus plantarum.L. plantarum has been isolated from the human gastrointestinal tract and plant surfaces. It is an economically important starter culture bacterium, to initiate food fermentation, and certain strains are even sold as probiotics (2, 3, 13, 40). Improvements in the efficiency of biomass formation and robustness, which are associated with respiration in L. lactis, are desirable traits for starter cultures, as well as probiotics.In this work, we investigated whether functional electron transport chains are present in L. plantarum. We analyzed the genome for components of electron transport chains and investigated the ability of L. plantarum to exploit extracellular electron acceptors.     

Use of the mCherry Fluorescent Protein To Study Intestinal Colonization by Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 in Mice

Lactic acid bacteria (LAB) are natural inhabitants of the gastrointestinal tract (GIT) of humans and animals, and some LAB species receive considerable attention due to their health benefits. Although many papers have been published on probiotic LAB, only a few reports have been published on the migration and colonization of the cells in the GIT. This is due mostly to the lack of efficient reporter systems. In this study, we report on the application of the fluorescent mCherry protein in the in vivo tagging of the probiotic strains Enterococcus mundtii ST4SA and Lactobacillus plantarum 423. The mCherry gene, encoding a red fluorescent protein (RFP), was integrated into a nonfunctional region on the genome of L. plantarum 423 by homologous recombination. In the case of E. mundtii ST4SA, the mCherry gene was cloned into the pGKV223D LAB/Escherichia coli expression vector. Expression of the mCherry gene did not alter the growth rate of the two strains and had no effect on bacteriocin production. Both strains colonized the cecum and colon of mice.     

Synthesis of γ-Aminobutyric Acid by Lactic Acid Bacteria Isolated from a Variety of Italian Cheeses

The concentrations of γ-aminobutyric acid (GABA) in 22 Italian cheese varieties that differ in several technological traits markedly varied from 0.26 to 391 mg kg⁻¹. Presumptive lactic acid bacteria were isolated from each cheese variety (total of 440 isolates) and screened for the capacity to synthesize GABA. Only 61 isolates showed this activity and were identified by partial sequencing of the 16S rRNA gene. Twelve species were found. Lactobacillus paracasei PF6, Lactobacillus delbrueckii subsp. bulgaricus PR1, Lactococcus lactis PU1, Lactobacillus plantarum C48, and Lactobacillus brevis PM17 were the best GABA-producing strains during fermentation of reconstituted skimmed milk. Except for L. plantarum C48, all these strains were isolated from cheeses with the highest concentrations of GABA. A core fragment of glutamate decarboxylase (GAD) DNA was isolated from L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48 by using primers based on two highly conserved regions of GAD. A PCR product of ca. 540 bp was found for all the strains. The amino acid sequences deduced from nucleotide sequence analysis showed 98, 99, 90, and 85% identity to GadB of L. plantarum WCFS1 for L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48, respectively. Except for L. lactis PU1, the three lactobacillus strains survived and synthesized GABA under simulated gastrointestinal conditions. The findings of this study provide a potential basis for exploiting selected cheese-related lactobacilli to develop health-promoting dairy products enriched in GABA.     

Applications for green fluorescent protein (GFP) in the study of hostpathogen interactions

The green fluorescent protein (GFP) from Aequorea victoria is a novel fluorescent marker that has potential use in the study of bacterial pathogenicity. To explore some of the potential applications of GFP to the study of host-parasite interactions, we constructed two GFP expression vectors suitable for different facultative intracellular bacterial pathogens. The first expression vector was tested in the enteric pathogens, Salmonella typhimurium and Yersinia pseudotuberculosis, and the second vector tested in Mycobacterium marinum (Mm). Both expression vectors were found to be stable and to direct high levels of GFP synthesis. Standard epifluorescence microscopy was used to detect all three bacterial pathogenic species during the early and late stages of infection of live mammalian cells. Mm expressing gfp was also visualized in infected animal tissues, gfp expression did not adversely affect bacterial survival, nor did it compromise entry into mammalian cells or their survival within macrophages. In addition, all three gfp-expressing bacterial pathogens could be detected and sorted in a flow cytometer, either alone or in association with epithelial cells or macrophages. Therefore, GFP not only provides a convenient tool to image pathogenic bacteria, but allows the quantitative measurement of bacterial association with mammalian cells.     

Construction of theta-type shuttle vector for Leuconostoc and other lactic acid bacteria using pCB42 isolated from kimchi

The pCB42 plasmid from Leuconostoc citreum CB2567, a strain isolated from kimchi, was characterized, and a shuttle vector for Escherichia coli and lactic acid bacteria (LAB) was constructed. The pCB42 plasmid has a circular structure of 4312 bp, a low G + C content, and no single-stranded DNA intermediates during replication, which indicates that pCB42 replicates via the theta-type replication mechanism. In silico analysis of this plasmid revealed 6 open reading frames: 1 transposase gene, 1 DNA-binding gene, 2 putative replication genes, and 2 unknown genes. The fragment encompassing ORF5 contains a functional plasmid replicon. This plasmid was capable of replicating in various LAB, including L. citreum, L. mesenteroides, Lactobacillus plantarum, Lb. reuteri, Lactococcus lactis, Streptococcus thermophilus, Weissella confusa, and Oenococcus oeni. The LAB-E. coli shuttle vector was constructed by ligating pCB42 and pEK104, and the resulting shuttle vector, pLeuCM42, showed a high segregational stability in L. citreum CB2567 after 100 generations of cell division. By using this shuttle vector, the β-gal gene from Lb. plantarum was successfully expressed in the host strain, L. citreum CB2567. The pLeuCM42 shuttle vector can serve as a useful gene-delivery and expression tool for the genetic study or metabolic engineering of various strains of LAB.     

Phylogeny of the Defined Murine Microbiota: Altered Schaedler Flora

The “altered Schaedler flora” (ASF) was developed for colonizing germfree rodents with a standardized microbiota. The purpose of this study was to identify each of the eight ASF strains by 16S rRNA sequence analysis. Three strains were previously identified as Lactobacillus acidophilus (strain ASF 360), Lactobacillus salivarius (strain ASF 361), and Bacteroides distasonis (strain ASF 519) based on phenotypic criteria. 16S rRNA analysis indicated that each of the strains differed from its presumptive identity. The 16S rRNA sequence of strain ASF 361 is essentially identical to the 16S rRNA sequences of the type strains of Lactobacillus murinis and Lactobacillus animalis (both isolated from mice), and all of these strains probably belong to a single species. Strain ASF 360 is a novel lactobacillus that clusters with L. acidophilus and Lactobacillus lactis. Strain ASF 519 falls into an unnamed genus containing [Bacteroides] distasonis, [Bacteroides] merdae, [Bacteroides] forsythus, and CDC group DF-3. This unnamed genus is in the Cytophaga-Flavobacterium-Bacteroides phylum and is most closely related to the genus Porphyromonas. The spiral-shaped strain, strain ASF 457, is in the Flexistipes phylum and exhibits sequence identity with rodent isolates of Robertson. The remaining four ASF strains, which are extremely oxygen-sensitive fusiform bacteria, group phylogenetically with the low-G+C-content gram-positive bacteria (Firmicutes, Bacillus-Clostridium group). ASF 356, ASF 492, and ASF 502 fall into Clostridium cluster XIV of Collins et al. Morphologically, ASF 492 resembles members of this cluster, Roseburia cecicola, and Eubacterium plexicaudatum. The 16S rRNA sequence of ASF 492 is identical to that of E. plexicaudatum. Since the type strain and other viable original isolates of E. plexicaudatum have been lost, strain ASF 492 is a candidate for a neotype strain. Strain ASF 500 branches deeply in the low-G+C-content gram-positive phylogenetic tree but is not closely related to any organisms whose 16S rRNA sequences are currently in the GenBank database. The 16S rRNA sequence information determined in the present study should allow rapid identification of ASF strains and should permit detailed analysis of the interactions of ASF organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms.     

In Situ Determination of the Intracellular pH of Lactococcus lactis and Lactobacillus plantarum during Pressure Treatment

Hydrostatic pressure may affect the intracellular pH of microorganisms by (i) enhancing the dissociation of weak organic acids and (ii) increasing the permeability of the cytoplasmic membrane and inactivation of enzymes required for pH homeostasis. The internal pHs of Lactococcus lactis and Lactobacillus plantarum during and after pressure treatment at 200 and 300 MPa and at pH values ranging from 4.0 to 6.5 were determined. Pressure treatment at 200 MPa for up to 20 min did not reduce the viability of either strain at pH 6.5. Pressure treatment at pH 6.5 and 300 MPa reduced viable cell counts of Lactococcus lactis and Lactobacillus plantarum by 5 log after 20 and 120 min, respectively. Pressure inactivation was faster at pH 5 or 4. At ambient pressure, both strains maintained a transmembrane pH gradient of 1 pH unit at neutral pH and about 2 pH units at pH 4.0. During pressure treatment at 200 and 300 MPa, the internal pH of L. lactis was decreased to the value of the extracellular pH during compression. The same result was observed during treatment of Lactobacillus plantarum at 300 MPa. Lactobacillus plantarum was unable to restore the internal pH after a compression-decompression cycle at 300 MPa and pH 6.5. Lactococcus lactis lost the ability to restore its internal pH after 20 and 4 min of pressure treatment at 200 and 300 MPa, respectively. As a consequence, pressure-mediated stress reactions and cell death may be considered secondary effects promoted by pH and other environmental conditions.     

Effect of Lactobacillus plantarum isolated from digestive tract of wild shrimp on growth and survival of white shrimp (Litopenaeus vannamei) challenged with Vibrio harveyi

Two hundred and two strains of lactic acid bacteria (LAB) isolated from digestive tracts of cultivated and wild adult shrimp, including Litopenaeus vannamei, Metapenaeus brevicornis and Penaeus merguiensis were selected based on their antibacterial activity against Vibrio harveyi. LAB strain of MRO3.12 exhibiting highest reduction of V. harveyi was identified as Lactobacillus plantarum MRO3.12 based on the nucleotide sequence of its 16S rDNA, which showed 99% (780/786 bp) homology to L. plantarum strain L5 (GenBank accession number DQ 239698.1). Co-cultivation of V. harveyi and L. plantarum MRO3.12 showed complete reduction of V. harveyi at 24 h under aerobic and anaerobic conditions, whereas L. plantarum increased from 5.29 to 9.47 log CFU ml⁻¹. After 6-week feeding trial with L. plantarum supplemented diet, white shrimp (L. vannamei) exhibited significant differences (p < 0.05) in relative growth rate (% RGR), feed conversion ratio (FCR) and survival compared to the control group fed with non-supplemented diet. LAB-fed group showed 98.89% survival, whereas only 68.89% survival was observed in the control group. LAB from the digestive tract of probiotic-fed shrimp showed higher level of 5.0 ± 0.14 log CFU/g than the non-supplemented ones (3.34 ± 0.21 log CFU/g). However, total bacterial and non-fermenting vibrios counts decreased in shrimps fed on L. plantarum. Ten days after infection with V. harveyi (5.3-5.5 log CFU ml⁻¹), significant survival (p < 0.05) of 77% was observed in LAB supplemented shrimp, while only 67% survival was observed in the control.