Growth phase-dependent proteomes of the Malaysian isolated Lactococcus lactis dairy strain M4 using label-free qualitative shotgun proteomics analysis.

PubMed

Yap, Theresa Wan Chen; Rabu, Amir; Abu Bakar, Farah Diba; Rahim, Raha Abdul; Mahadi, Nor Muhammad; Illias, Rosli Md; Murad, Abdul Munir Abdul

2014-01-01

Lactococcus lactis is the most studied mesophilic fermentative lactic acid bacterium. It is used extensively in the food industry and plays a pivotal role as a cell factory and also as vaccine delivery platforms. The proteome of the Malaysian isolated L. lactis M4 dairy strain, obtained from the milk of locally bred cows, was studied to elucidate the physiological changes occurring between the growth phases of this bacterium. In this study, ultraperformance liquid chromatography nanoflow electrospray ionization tandem mass spectrometry (UPLC- nano-ESI-MS(E)) approach was used for qualitative proteomic analysis. A total of 100 and 121 proteins were identified from the midexponential and early stationary growth phases, respectively, of the L. lactis strain M4. During the exponential phase, the most important reaction was the generation of sufficient energy, whereas, in the early stationary phase, the metabolic energy pathways decreased and the biosynthesis of proteins became more important. Thus, the metabolism of the cells shifted from energy production in the exponential phase to the synthesis of macromolecules in the stationary phase. The resultant proteomes are essential in providing an improved view of the cellular machinery of L. lactis during the transition of growth phases and hence provide insight into various biotechnological applications.

Evaluation of Lactococcus lactis Isolates from Nondairy Sources with Potential Dairy Applications Reveals Extensive Phenotype-Genotype Disparity and Implications for a Revised Species

PubMed Central

Cavanagh, Daniel; Casey, Aidan; Altermann, Eric; Cotter, Paul D.; Fitzgerald, Gerald F.

2015-01-01

Lactococcus lactis is predominantly associated with dairy fermentations, but evidence suggests that the domesticated organism originated from a plant niche. L. lactis possesses an unusual taxonomic structure whereby strain phenotypes and genotypes often do not correlate, which in turn has led to confusion in L. lactis classification. A bank of L. lactis strains was isolated from various nondairy niches (grass, vegetables, and bovine rumen) and was further characterized on the basis of key technological traits, including growth in milk and key enzyme activities. Phenotypic analysis revealed all strains from nondairy sources to possess an L. lactis subsp. lactis phenotype (lactis phenotype); however, seven of these strains possessed an L. lactis subsp. cremoris genotype (cremoris genotype), determined by two separate PCR assays. Multilocus sequence typing (MLST) showed that strains with lactis and cremoris genotypes clustered together regardless of habitat, but it highlighted the increased diversity that exists among “wild” strains. Calculation of average nucleotide identity (ANI) and tetranucleotide frequency correlation coefficients (TETRA), using the JSpecies software tool, revealed that L. lactis subsp. cremoris and L. lactis subsp. lactis differ in ANI values by ∼14%, below the threshold set for species circumscription. Further analysis of strain TIFN3 and strains from nonindustrial backgrounds revealed TETRA values of <0.99 in addition to ANI values of <95%, implicating that these two groups are separate species. These findings suggest the requirement for a revision of L. lactis taxonomy. PMID:25841018

Micrococcus lactis sp. nov., isolated from dairy industry waste.

PubMed

Chittpurna; Singh, Pradip K; Verma, Dipti; Pinnaka, Anil Kumar; Mayilraj, Shanmugam; Korpole, Suresh

2011-12-01

A Gram-positive, yellow-pigmented, actinobacterial strain, DW152(T), was isolated from a dairy industry effluent treatment plant. 16S rRNA gene sequence analysis indicated that strain DW152(T) exhibited low similarity with many species with validly published names belonging to the genera Micrococcus and Arthrobacter. However, phenotypic properties including chemotaxonomic markers affiliated strain DW152(T) to the genus Micrococcus. Strain DW152(T) had ai-C(15:0) and i-C(15:0) as major cellular fatty acids, and MK-8(H(2)) as the major menaquinone. The cell-wall peptidoglycan of strain DW152(T) had l-lysine as the diagnostic amino acid and the type was A4α. The DNA G+C content of strain DW152(T) was 68.0 mol%. In 16S rRNA gene sequence analysis, strain DW152(T) exhibited significant similarity with Micrococcus terreus NBRC 104258(T), but the mean value of DNA-DNA relatedness between these strains was only 42.3%. Moreover, strain DW152(T) differed in biochemical and chemotaxonomic characteristics from M. terreus and other species of the genus Micrococcus. Based on the above differences, we conclude that strain DW152(T) should be treated as a novel species of the genus Micrococcus, for which the name Micrococcus lactis sp. nov. is proposed. The type strain of Micrococcus lactis sp. nov. is DW152(T) (=MTCC10523(T) =DSM 23694(T)).

Genome Sequence of the Cheese-Starter Strain Lactobacillus delbrueckii subsp. lactis CRL 581.

PubMed

Hebert, Elvira María; Raya, Raúl R; Brown, Lucía; Font de Valdez, Graciela; Savoy de Giori, Graciela; Taranto, María Pía

2013-08-08

We report the genome sequence of Lactobacillus delbrueckii subsp. lactis CRL 581 (1,911,137 bp, GC 49.7%), a proteolytic strain isolated from a homemade Argentinian hard cheese which has a key role in bacterial nutrition and releases bioactive health-beneficial peptides from milk proteins.

Identification and characterization of tetracycline resistance in Lactococcus lactis isolated from Polish raw milk and fermented artisanal products.

PubMed

Zycka-Krzesinska, Joanna; Boguslawska, Joanna; Aleksandrzak-Piekarczyk, Tamara; Jopek, Jakub; Bardowski, Jacek K

2015-10-15

To assess the occurrence of antibiotic-resistant Lactic Acid Bacteria (LAB) in Polish raw milk and fermented artisanal products, a collection comprising 500 isolates from these products was screened. Among these isolates, six strains (IBB28, IBB160, IBB161, IBB224, IBB477 and IBB487) resistant to tetracycline were identified. The strains showing atypical tetracycline resistance were classified as Lactococcus lactis: three of them were identified as L. lactis subsp. cremoris (IBB224, IBB477 and IBB487) and the other three (IBB28, IBB160, IBB161) were identified as L. lactis subsp. lactis. The mechanism involving Ribosomal Protection Proteins (RPP) was identified as responsible for tetracycline resistance. Three of the tested strains (IBB28, IBB160 and IBB224) had genes encoding the TetS protein, whereas the remaining three (IBB161, IBB477 and IBB487) expressed TetM. The results also demonstrated that the genes encoding these proteins were located on genetic mobile elements. The tet(S) gene was found to be located on plasmids, whereas tet(M) was found within the Tn916 transposon. Copyright © 2015 Elsevier B.V. All rights reserved.

Antihypertensive and hypolipidemic effect of milk fermented by specific Lactococcus lactis strains.

PubMed

Rodríguez-Figueroa, J C; González-Córdova, A F; Astiazaran-García, H; Hernández-Mendoza, A; Vallejo-Cordoba, B

2013-07-01

The antihypertensive and hypolipidemic effects of milk fermented by specific Lactococcus lactis strains in spontaneously hypertensive rats (SHR) were investigated. The SHR were fed ad libitum milk fermented by Lc. lactis NRRL B-50571, Lc. lactis NRRL B-50572, Captopril (40mg/kg of body weight, Sigma-Aldrich Co., St. Louis, MO) or purified water for 4 wk. Results suggested that Lc. lactis fermented milks presented a significant blood pressure-lowering effect. No significant difference was noted among milk fermented by Lc. lactis NRRL B-50571 and Captopril by the second and third week of treatment. Additionally, milk fermented by Lc. lactis strains modified SHR lipid profiles. Milk fermented by Lc. lactis NRRL B-50571 and B-50572 were able to reduce plasma low-density lipoprotein cholesterol and triglyceride contents. Thus, milk fermented by Lc. lactis strains may be a coadjuvant in the reduction of hypertension and hyperlipidemia and may be used as a functional food for better cardiovascular health. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Draft Genome Sequence of the Putrescine-Producing Strain Lactococcus lactis subsp. lactis 1AA59

PubMed Central

del Rio, Beatriz; Linares, Daniel M.; Fernandez, María; Mayo, Baltasar; Martín, M. Cruz

2015-01-01

We report here the 2,576,542-bp genome annotated draft assembly sequence of Lactococcus lactis subsp. lactis 1AA59. This strain—isolated from a traditional cheese—produces putrescine, one of the most frequently biogenic amines found in dairy products. PMID:26089428

Safety characterisation and inhibition of fungi and bacteria by a novel multiple enterocin-producing Enterococcus lactis 4CP3 strain.

PubMed

Ben Braïek, Olfa; Cremonesi, Paola; Morandi, Stefano; Smaoui, Slim; Hani, Khaled; Ghrairi, Taoufik

2018-03-07

This study aims to characterise a potential bacteriocinogenic lactic acid bacterial strain isolated from a raw pink shrimp (Palaemon serratus) and evaluate its safety aspect. The strain designated as 4CP3 was noted to display antibacterial activities (P < 0.05) against Gram-positive and Gram-negative foodborne pathogens (Listeria monocytogenes and Pseudomonas aeruginosa) and some filamentous fungi (e.g. Aspergillus niger A79). Phenotypic and molecular techniques as well as phylogenetic analysis identified the isolate 4CP3 as Enterococcus lactis. Its produced antimicrobial substance was determined as a bacteriocin that was stable over a wide range of pH (2-10) and after heating at 100 °C for 15 min. The maximum bacteriocin production was 1400 AU/ml recorded after 12 h of incubation in de Man, Rogosa and Sharpe (MRS) broth medium at 30 °C. The mode of action of the bacteriocin produced by 4CP3 strain was identified as bactericidal against L. monocytogenes EGDe 107776 and P. aeruginosa ATCC 27853. By specific PCR amplifications, E. lactis 4CP3 was shown to produce the enterocins A, B and P. To our knowledge, this feature is newly described for E. lactis strain isolated from raw shrimps. Regarding safety aspect of E. lactis 4CP3, it has been demonstrated that this strain was not haemolytic, gelatinase negative, sensitive to vancomycin, and free of common antibiotic resistance genes and virulence factors. Therefore, it may be useful as a safe natural agent in preservation of foods or as a new probiotic strain in food and feed. Copyright © 2018 Elsevier Ltd. All rights reserved.

Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Application in the control of Listeria monocytogenes in fresh Minas-type goat cheese

PubMed Central

Furtado, Danielle N.; Todorov, Svetoslav D.; Landgraf, Mariza; Destro, Maria T.; Franco, Bernadette D.G.M.

2015-01-01

Listeria monocytogenes is a pathogen frequently found in dairy products. Its control in fresh cheeses is difficult, due to the psychrotrophic properties and salt tolerance. Bacteriocinogenic lactic acid bacteria (LAB) with proven in vitro antilisterial activity can be an innovative technological approach but their application needs to be evaluated by means of in situ tests. In this study, a novel bacteriocinogenic Lactococcus lactis strain ( Lc . lactis DF4Mi), isolated from raw goat milk, was tested for control of growth of L. monocytogenes in artificially contaminated fresh Minas type goat cheese during storage under refrigeration. A bacteriostatic effect was achieved, and counts after 10 days were 3 log lower than in control cheeses with no added LAB. However, this effect did not differ significantly from that obtained with a non-bacteriocinogenic Lc. lactis strain. Addition of nisin (12.5 mg/kg) caused a rapid decrease in the number of viable L. monocytogenes in the cheeses, suggesting that further studies with the purified bacteriocin DF4Mi may open new possibilities for this strain as biopreservative in dairy products. PMID:26221109

Combination of Metabolomic and Proteomic Analysis Revealed Different Features among Lactobacillus delbrueckii Subspecies bulgaricus and lactis Strains While In Vivo Testing in the Model Organism Caenorhabditis elegans Highlighted Probiotic Properties

PubMed Central

Zanni, Elena; Schifano, Emily; Motta, Sara; Sciubba, Fabio; Palleschi, Claudio; Mauri, Pierluigi; Perozzi, Giuditta; Uccelletti, Daniela; Devirgiliis, Chiara; Miccheli, Alfredo

2017-01-01

Lactobacillus delbrueckii represents a technologically relevant member of lactic acid bacteria, since the two subspecies bulgaricus and lactis are widely associated with fermented dairy products. In the present work, we report the characterization of two commercial strains belonging to L. delbrueckii subspecies bulgaricus, lactis and a novel strain previously isolated from a traditional fermented fresh cheese. A phenomic approach was performed by combining metabolomic and proteomic analysis of the three strains, which were subsequently supplemented as food source to the model organism Caenorhabditis elegans, with the final aim to evaluate their possible probiotic effects. Restriction analysis of 16S ribosomal DNA revealed that the novel foodborne strain belonged to L. delbrueckii subspecies lactis. Proteomic and metabolomic approaches showed differences in folate, aminoacid and sugar metabolic pathways among the three strains. Moreover, evaluation of C. elegans lifespan, larval development, brood size, and bacterial colonization capacity demonstrated that L. delbrueckii subsp. bulgaricus diet exerted beneficial effects on nematodes. On the other hand, both L. delbrueckii subsp. lactis strains affected lifespan and larval development. We have characterized three strains belonging to L. delbrueckii subspecies bulgaricus and lactis highlighting their divergent origin. In particular, the two closely related isolates L. delbrueckii subspecies lactis display different galactose metabolic capabilities. Moreover, the L. delbrueckii subspecies bulgaricus strain demonstrated potential probiotic features. Combination of omic platforms coupled with in vivo screening in the simple model organism C. elegans is a powerful tool to characterize industrially relevant bacterial isolates. PMID:28702021

Combination of Metabolomic and Proteomic Analysis Revealed Different Features among Lactobacillus delbrueckii Subspecies bulgaricus and lactis Strains While In Vivo Testing in the Model Organism Caenorhabditis elegans Highlighted Probiotic Properties.

PubMed

Zanni, Elena; Schifano, Emily; Motta, Sara; Sciubba, Fabio; Palleschi, Claudio; Mauri, Pierluigi; Perozzi, Giuditta; Uccelletti, Daniela; Devirgiliis, Chiara; Miccheli, Alfredo

2017-01-01

Lactobacillus delbrueckii represents a technologically relevant member of lactic acid bacteria, since the two subspecies bulgaricus and lactis are widely associated with fermented dairy products. In the present work, we report the characterization of two commercial strains belonging to L. delbrueckii subspecies bulgaricus , lactis and a novel strain previously isolated from a traditional fermented fresh cheese. A phenomic approach was performed by combining metabolomic and proteomic analysis of the three strains, which were subsequently supplemented as food source to the model organism Caenorhabditis elegans , with the final aim to evaluate their possible probiotic effects. Restriction analysis of 16S ribosomal DNA revealed that the novel foodborne strain belonged to L. delbrueckii subspecies lactis . Proteomic and metabolomic approaches showed differences in folate, aminoacid and sugar metabolic pathways among the three strains. Moreover, evaluation of C. elegans lifespan, larval development, brood size, and bacterial colonization capacity demonstrated that L. delbrueckii subsp. bulgaricus diet exerted beneficial effects on nematodes. On the other hand, both L. delbrueckii subsp. lactis strains affected lifespan and larval development. We have characterized three strains belonging to L. delbrueckii subspecies bulgaricus and lactis highlighting their divergent origin. In particular, the two closely related isolates L. delbrueckii subspecies lactis display different galactose metabolic capabilities. Moreover, the L. delbrueckii subspecies bulgaricus strain demonstrated potential probiotic features. Combination of omic platforms coupled with in vivo screening in the simple model organism C. elegans is a powerful tool to characterize industrially relevant bacterial isolates.

Specific Identification and Targeted Characterization of Bifidobacterium lactis from Different Environmental Isolates by a Combined Multiplex-PCR Approach

PubMed Central

Ventura, Marco; Reniero, Roberto; Zink, Ralf

2001-01-01

The species Bifidobacterium lactis, with its main representative strain Bb12 (DSM 10140), is a yoghurt isolate used as a probiotic strain and is commercially applied in different types of yoghurts and infant formulas. In order to ensure the genetic identity and safety of this bacterial isolate, species- and strain-specific molecular tools for genetic fingerprinting must be available to identify isolated bifidobacteria or lactic acid bacteria from, e.g., various clinical environments of relevance in medical microbiology. Two opposing rRNA gene-targeted primers have been developed for specific detection of this microorganism by PCR. The specificity of this approach was evaluated and verified with DNA samples isolated from single and mixed cultures of bifidobacteria and lactobacilli (48 isolates, including the type strains of 29 Bifidobacterium and 9 Lactobacillus species). Furthermore, we performed a Multiplex-PCR using oligonucleotide primers targeting a specific region of the 16S rRNA gene for the genus Bifidobacterium and a conserved eubacterial 16S rDNA sequence. The specificity and sensitivity of this detection with a pure culture of B. lactis were, respectively, 100 bacteria/ml after 25 cycles of PCR and 1 to 10 bacteria/ml after a 50-cycle nested-PCR approach. PMID:11375192

Complete Genome Sequence of Lactococcus lactis Strain AI06, an Endophyte of the Amazonian Açaí Palm

PubMed Central

de Oliveira, Viviane Matoso; de Almeida Pina, André Vicioli; Pérez-Chaparro, Paula Juliana; de Almeida, Lara Mendes; de Vasconcelos, Janaina Mota; de Oliveira, Layanna Freitas; da Silva, Daisy Elaine Andrade; Rogez, Hervé Louis Ghislain; Cretenet, Marina; Mamizuka, Elsa Masae; Nunes, Marcio Roberto Teixeira

2014-01-01

We report the genome, in a single chromosome, of Lactococcus lactis strain AI06, isolated from the mesocarp of the açaí fruit (Euterpe oleracea) in eastern Amazonia, Brazil. This strain is an endophyte of the açaí palm and also a component of the microbiota of the edible food product. PMID:25414513

Lactococcus lactis subsp. cremoris strain JFR1 attenuates Salmonella adhesion to human intestinal cells in vitro.

PubMed

Zhang, Justina Su; Guri, Anilda; Corredig, Milena; Morales-Rayas, Rocio; Hassan, Ashraf; Griffiths, Mansel; LaPointe, Gisèle

2016-12-01

Lactococcus lactis subsp. cremoris JFR1 has been studied in reduced fat cheese due to its ability to produce exopolysaccharides (EPS) in situ, contributing to improved textural and organoleptic properties. In this study, the effect of strain JFR1 on virulence gene expression and attachment of Salmonella to HT-29 human colon carcinoma cells was investigated. Overnight cultures of L. lactis subsp. cremoris JFR1 containing EPS, grown in M17 media with 0.5% glucose supplementation, decreased attachment as well as down regulated virulence gene expression in Salmonella enterica subsp. enterica when tested on HT-29 cells. However, EPS isolated from milk fermented with L. lactis subsp. cremoris JFR1 did not affect Salmonella virulence gene expression or attachment to HT-29 cells. These results suggest that EPS does not contribute to the attachment of Salmonella to human intestinal cells. However, the possibility that the isolation process may have affected the structural features of EPS cannot be ruled out. Copyright © 2016 Elsevier Ltd. All rights reserved.

Purification and partial characterization of bacteriocin produced by Lactococcus lactis ssp. lactis LL171.

PubMed

Kumari, Archana; Akkoç, Nefise; Akçelik, Mustafa

2012-04-01

Lactic acid bacteria (LAB) are possessing ability to synthesize antimicrobial compounds (like bacteriocin) during their growth. In this regard, novel bacteriocin compound secreting capability of LAB isolated from Tulum Cheese in Turkey was demonstrated. The synthesized bacteriocin was purified by ammonium sulphate precipitation, dialysis and gel filtration. The molecular weight (≈3.4 kDa) of obtained bacteriocin was confirmed by SDS-PAGE, which revealed single peptide band. Molecular identification of LAB strain isolated from Tulum Cheese was conducted using 16S rDNA gene sequencing as Lactococcus lactis ssp. lactis LL171. The amino acid sequences (KKIDTRTGKTMEKTEKKIELSLKNMKTAT) of the bacteriocin from Lactococcus lactis ssp. lactis LL171 was found unique and novel than reported bacteriocins. Further, the bacteriocin was possessed the thermostable property and active at wide range of pH values from 1 to 11. Thus, bacteriocin reported in this study has the potential applications property as food preservative agent.

Strain-Dependent Transcriptome Signatures for Robustness in Lactococcus lactis

PubMed Central

Dijkstra, Annereinou R.; Alkema, Wynand; Starrenburg, Marjo J. C.; van Hijum, Sacha A. F. T.; Bron, Peter A.

2016-01-01

Recently, we demonstrated that fermentation conditions have a strong impact on subsequent survival of Lactococcus lactis strain MG1363 during heat and oxidative stress, two important parameters during spray drying. Moreover, employment of a transcriptome-phenotype matching approach revealed groups of genes associated with robustness towards heat and/or oxidative stress. To investigate if other strains have similar or distinct transcriptome signatures for robustness, we applied an identical transcriptome-robustness phenotype matching approach on the L. lactis strains IL1403, KF147 and SK11, which have previously been demonstrated to display highly diverse robustness phenotypes. These strains were subjected to an identical fermentation regime as was performed earlier for strain MG1363 and consisted of twelve conditions, varying in the level of salt and/or oxygen, as well as fermentation temperature and pH. In the exponential phase of growth, cells were harvested for transcriptome analysis and assessment of heat and oxidative stress survival phenotypes. The variation in fermentation conditions resulted in differences in heat and oxidative stress survival of up to five 10-log units. Effects of the fermentation conditions on stress survival of the L. lactis strains were typically strain-dependent, although the fermentation conditions had mainly similar effects on the growth characteristics of the different strains. By association of the transcriptomes and robustness phenotypes highly strain-specific transcriptome signatures for robustness towards heat and oxidative stress were identified, indicating that multiple mechanisms exist to increase robustness and, as a consequence, robustness of each strain requires individual optimization. However, a relatively small overlap in the transcriptome responses of the strains was also identified and this generic transcriptome signature included genes previously associated with stress (ctsR and lplL) and novel genes, including nan

Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures

PubMed Central

Kelly, William J.; Ward, Lawrence J. H.; Leahy, Sinead C.

2010-01-01

A large collection of Lactococcus lactis strains, including wild-type isolates and dairy starter cultures, were screened on the basis of their phenotype and the macrorestriction patterns produced from pulsed-field gel electrophoresis (PFGE) analysis of SmaI digests of genomic DNA. Three groups of dairy starter cultures, used for different purposes in the dairy industry, and a fourth group made up of strains isolated from the environment were selected for analysis of their chromosomal diversity using the endonuclease I-CeuI. Chromosome architecture was largely conserved with each strain having six copies of the rRNA genes, and the chromosome size of individual strains ranged between 2,240 and 2,688 kb. The origin of L. lactis strains showed the greatest correlation with chromosome size, and dairy strains, particularly those with the cremoris phenotype, had smaller chromosomes than wild-type strains. Overall, this study, coupled with analysis of the sequenced L. lactis genomes, provides evidence that defined strain dairy starter cultures have arisen from plant L. lactis strains. Adaptation of these strains to the dairy environment has involved loss of functions resulting in smaller chromosomes and acquisition of genes (usually plasmid associated) that facilitate growth in milk. We conclude that dairy starter cultures generally and the industrially used cremoris and diacetylactis phenotype strains in particular comprise a specialized group of L. lactis strains that have been selected to become an essential component of industrial processes and have evolved accordingly, so that they are no longer fit to survive outside the dairy environment. PMID:20847124

Lactococcus lactis Metabolism and Gene Expression during Growth on Plant Tissues

PubMed Central

Golomb, Benjamin L.

2014-01-01

Lactic acid bacteria have been isolated from living, harvested, and fermented plant materials; however, the adaptations these bacteria possess for growth on plant tissues are largely unknown. In this study, we investigated plant habitat-specific traits of Lactococcus lactis during growth in an Arabidopsis thaliana leaf tissue lysate (ATL). L. lactis KF147, a strain originally isolated from plants, exhibited a higher growth rate and reached 7.9-fold-greater cell densities during growth in ATL than the dairy-associated strain L. lactis IL1403. Transcriptome profiling (RNA-seq) of KF147 identified 853 induced and 264 repressed genes during growth in ATL compared to that in GM17 laboratory culture medium. Genes induced in ATL included those involved in the arginine deiminase pathway and a total of 140 carbohydrate transport and metabolism genes, many of which are involved in xylose, arabinose, cellobiose, and hemicellulose metabolism. The induction of those genes corresponded with L. lactis KF147 nutrient consumption and production of metabolic end products in ATL as measured by gas chromatography-time of flight mass spectrometry (GC-TOF/MS) untargeted metabolomic profiling. To assess the importance of specific plant-inducible genes for L. lactis growth in ATL, xylose metabolism was targeted for gene knockout mutagenesis. Wild-type L. lactis strain KF147 but not an xylA deletion mutant was able to grow using xylose as the sole carbon source. However, both strains grew to similarly high levels in ATL, indicating redundancy in L. lactis carbohydrate metabolism on plant tissues. These findings show that certain strains of L. lactis are well adapted for growth on plants and possess specific traits relevant for plant-based food, fuel, and feed fermentations. PMID:25384484

Bifidobacterium animalis subsp. lactis ATCC 27673 Is a Genomically Unique Strain within Its Conserved Subspecies

PubMed Central

Loquasto, Joseph R.; Barrangou, Rodolphe; Dudley, Edward G.; Stahl, Buffy; Chen, Chun

2013-01-01

Many strains of Bifidobacterium animalis subsp. lactis are considered health-promoting probiotic microorganisms and are commonly formulated into fermented dairy foods. Analyses of previously sequenced genomes of B. animalis subsp. lactis have revealed little genetic diversity, suggesting that it is a monomorphic subspecies. However, during a multilocus sequence typing survey of Bifidobacterium, it was revealed that B. animalis subsp. lactis ATCC 27673 gave a profile distinct from that of the other strains of the subspecies. As part of an ongoing study designed to understand the genetic diversity of this subspecies, the genome of this strain was sequenced and compared to other sequenced genomes of B. animalis subsp. lactis and B. animalis subsp. animalis. The complete genome of ATCC 27673 was 1,963,012 bp, contained 1,616 genes and 4 rRNA operons, and had a G+C content of 61.55%. Comparative analyses revealed that the genome of ATCC 27673 contained six distinct genomic islands encoding 83 open reading frames not found in other strains of the same subspecies. In four islands, either phage or mobile genetic elements were identified. In island 6, a novel clustered regularly interspaced short palindromic repeat (CRISPR) locus which contained 81 unique spacers was identified. This type I-E CRISPR-cas system differs from the type I-C systems previously identified in this subspecies, representing the first identification of a different system in B. animalis subsp. lactis. This study revealed that ATCC 27673 is a strain of B. animalis subsp. lactis with novel genetic content and suggests that the lack of genetic variability observed is likely due to the repeated sequencing of a limited number of widely distributed commercial strains. PMID:23995933

Strain improvement of Lactobacillus lactis for D-lactic acid production.

PubMed

Joshi, D S; Singhvi, M S; Khire, J M; Gokhale, D V

2010-04-01

Three mutants, isolated by repeated UV mutagenesis of Lactobacillus lactis NCIM 2368, produced increased D: -lactic acid concentrations. These mutants were compared with the wild type using 100 g hydrolyzed cane sugar/l in the fermentation medium. One mutant, RM2-24, produced 81 g lactic acid/l which was over three times that of the wild type. The highest D: -lactic acid (110 g/l) in batch fermentation was obtained with 150 g cane sugar/l with a 73% lactic acid yield. The mutant utilizes cellobiose efficiently, converting it into D-lactic acid suggesting the presence of cellobiase. Thus, this strain could be used to obtain D-lactic acid from cellulosic materials that are pre-hydrolyzed with cellulase.

Nisin-Producing Lactococcus lactis Strains Isolated from Human Milk

PubMed Central

Beasley, Shea S.; Saris, Per E. J.

2004-01-01

Characterization by partial 16S rRNA gene sequencing, ribotyping, and green fluorescent protein-based nisin bioassay revealed that 6 of 20 human milk samples contained nisin-producing Lactococcus lactis bacteria. This suggests that the history of humans consuming nisin is older than the tradition of consuming fermented milk products. PMID:15294850

Spray-drying process preserves the protective capacity of a breast milk-derived Bifidobacterium lactis strain on acute and chronic colitis in mice

PubMed Central

Burns, Patricia; Alard, Jeanne; Hrdỳ, Jiri; Boutillier, Denise; Páez, Roxana; Reinheimer, Jorge; Pot, Bruno; Vinderola, Gabriel; Grangette, Corinne

2017-01-01

Gut microbiota dysbiosis plays a central role in the development and perpetuation of chronic inflammation in inflammatory bowel disease (IBD) and therefore is key target for interventions with high quality and functional probiotics. The local production of stable probiotic formulations at limited cost is considered an advantage as it reduces transportation cost and time, thereby increasing the effective period at the consumer side. In the present study, we compared the anti-inflammatory capacities of the Bifidobacterium animalis subsp. lactis (B. lactis) INL1, a probiotic strain isolated in Argentina from human breast milk, with the commercial strain B. animalis subsp. lactis BB12. The impact of spray-drying, a low-cost alternative of bacterial dehydration, on the functionality of both bifidobacteria was also investigated. We showed for both bacteria that the spray-drying process did not impact on bacterial survival nor on their protective capacities against acute and chronic colitis in mice, opening future perspectives for the use of strain INL1 in populations with IBD. PMID:28233848

Phage-resistance linked to cell heterogeneity in the commercial strain Lactobacillus delbrueckii subsp. lactis Ab1.

PubMed

Suárez, Viviana B; Maciel, Natalia; Guglielmotti, Daniela; Zago, Miriam; Giraffa, Giorgio; Reinheimer, Jorge

2008-12-10

The aim of this work was to study the relationship between the cell morphological heterogeneity and the phage-resistance in the commercial strain Lactobacillus delbrueckii subsp. lactis Ab1. Two morphological variants (named C and T) were isolated from this strain. Phage-resistant derivatives were isolated from them and the percentage of occurrence of confirmed phage-resistant cells was 0.001% of the total cellular population. Within these phage-resistant cell derivatives there were T (3 out of 4 total isolates) and C (1 out of 4 total isolates) variants. The study of some technological properties (e.g. proteolytic and acidifying activities) demonstrated that most of phage-resistant derivatives were not as good as the parental strain. However, for one derivative (a T variant), the technological properties were better than those of the parental strain. On the other hand, it was possible to determinate that the system of phage-resistance in the T variants was interference in adsorption step, with adsorption rates <15%. For the C variant derivative it was possible to demonstrate the presence of a restriction/modification system and, moreover, to determinate that this system could be Type I R/M.

Complete Genome Sequence of Kluyveromyces lactis Strain GG799, a Common Yeast Host for Heterologous Protein Expression

PubMed Central

Chuzel, Léa; Ganatra, Mehul B.; Schermerhorn, Kelly M.; Gardner, Andrew F.; Anton, Brian P.

2017-01-01

ABSTRACT We report the genome sequence of the dairy yeast Kluyveromyces lactis strain GG799 obtained using the Pacific Biosciences RS II platform. K. lactis strain GG799 is a common host for the expression of proteins at both laboratory and industrial scales. PMID:28751387

Novel angiotensin I-converting enzyme inhibitory peptides produced in fermented milk by specific wild Lactococcus lactis strains.

PubMed

Rodríguez-Figueroa, J C; González-Córdova, A F; Torres-Llanez, M J; Garcia, H S; Vallejo-Cordoba, B

2012-10-01

The ability of specific wild Lactococcus lactis strains to hydrolyze milk proteins to release angiotensin I-converting enzyme (ACE) inhibitory peptides was evaluated. The peptide profiles were obtained from the <3 kDa water-soluble extract and subsequently fractionated by reversed-phase HPLC. The fractions with the lowest half-maximal inhibitory concentration estimated values (peptide concentration necessary to inhibit ACE activity by 50%) were Lc. lactis NRRL B-50571 fraction (F)1 (0.034 ± 0.002 μg/mL; mean ± SD) and Lc. lactis NRRL B-50572B F 0005 (0.041 ± 0.003 μg/mL; mean ± SD). All peptide fractions were analyzed by reversed-phase HPLC tandem mass spectrometry. Twenty-one novel peptide sequences associated with ACE inhibitory (ACEI) activity were identified. Several novel ACEI peptides presented peptides encrypted with proven hypotensive activity. In conclusion, specific wild Lc. lactis strains were able to hydrolyze milk proteins to generate potent ACEI peptides. However, further studies are necessary to find out the relationship between Lc. lactis strain proteolytic systems and their ability to biogenerate hypotensive peptides. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Unleashing Natural Competence in Lactococcus lactis by Induction of the Competence Regulator ComX

PubMed Central

Mulder, Joyce; Wels, Michiel; Kuipers, Oscar P.; Bron, Peter A.

2017-01-01

ABSTRACT In biotechnological workhorses like Streptococcus thermophilus and Bacillus subtilis, natural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starter Lactococcus lactis, natural competence has not been established to date. However, in silico analysis of the complete genome sequences of 43 L. lactis strains revealed complete late competence gene sets in 2 L. lactis subsp. cremoris strains (KW2 and KW10) and at least 10 L. lactis subsp. lactis strains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulator comX in L. lactis subsp. lactis KF147 resulted in the induction of expression of the canonical competence regulon and elicited a state of natural competence in this strain. In contrast, comX expression in L. lactis NZ9000, which was predicted to encode an incomplete competence gene set, failed to induce natural competence. Moreover, mutagenesis of the comEA-EC operon in strain KF147 abolished the comX-driven natural competence, underlining the involvement of the competence machinery. Finally, introduction of nisin-inducible comX expression into nisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence in these strains also, expanding this phenotype to other L. lactis strains of both subspecies. IMPORTANCE Specific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the species Lactococcus lactis are very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end product. The activation of natural competence in this industrially

Analysis of volatile compounds produced by 2 strains of Lactococcus lactis isolated from leben (Tunisian fermented milk) using solid-phase microextraction-gas chromatography.

PubMed

Ziadi, M; Wathelet, J P; Marlier, M; Hamdi, M; Thonart, P

2008-08-01

The volatile compounds that characterize Leben during fermentation with 2 Lactococcus lactis strains (SLT6 and SLT10) in flasks, in a 100-L fermentor, and during storage at 4 degrees C, were investigated and compared to those from commercial Leben. Volatile compounds from Leben were concentrated by a Carboxen-PDMS fiber and analyzed by GC-MS. These compounds include acids, alcohols, aldehydes, ketones, sulfur compounds, and hydrocarbons. Commercial Leben presented a poor volatile profile compared to the laboratory-made Leben. The mixed culture of 2 Lactococcus lactis strains resulted in higher volatile compound formation than the single strain culture. The GC volatile profiles of Leben produced in flask and in the 100-L fermentor were similar. Changes in volatile compounds were observed during storage at 4 degrees C. The effect of culture conditions on production of volatiles by SLT6 strain was studied. Aeration (0.1 mL/min) and agitation enhanced the production of diacetyl, acetoin, 3-methylbutanal, and 3-methylbutanol. Fermentation at pH 5 had no effect on volatile production.

Isolation of a bacteriocin-producing Lactococcus lactis subsp. lactis and application to control Listeria monocytogenes in Moroccan jben.

PubMed

Benkerroum, N; Oubel, H; Zahar, M; Dlia, S; Filali-Maltouf, A

2000-12-01

Use of a bacteriocin-producing lactococcal strain to control Listeria monocytogenes in jben. A Lactococcus lactis strain isolated from lben was shown, by the spot technique, to produce a bacteriocin different from nisin. Inhibitory activity of the bacteriocin-producing strain against Listeria monocytogenes was investigated in jben, made from cow's milk fermented with the producer organism and contaminated with 104 or 107 cfu ml-1. Listeria counts were monitored during manufacture, and during conservation at room and at refrigeration temperatures. Results showed that the pathogen was reduced by 2.7 logarithmic units after 30 h of jben processing when the initial inoculum of 107 cfu ml(-1) was used. For the initial inoculum of 104 cfu ml(-1), the bacterium was completely eliminated at 24 h. Furthermore, the use of the bacteriocin-producing starter culture extended the shelf-life of jben by 5 days. In situ production of the lactococcal bacteriocin is an efficient biological means of controlling L. monocytogenes in jben and of allowing shelf-life extension. The proposed technology will essentially benefit minimally processed dairy products and those made with raw milk.

A Food-Grade Cloning System for Industrial Strains of Lactococcus lactis

PubMed Central

Sørensen, Kim I.; Larsen, Rasmus; Kibenich, Annette; Junge, Mette P.; Johansen, Eric

2000-01-01

We have previously reported the construction of a food-grade cloning vector for Lactococcus using the ochre suppressor, supB, as the selective marker. This vector, pFG1, causes only a slight growth inhibition in the laboratory strain MG1363 but is unstable in the industrial strains tested. As supB suppresses both amber and ochre stop codons, which are present in 82% of all known lactococcal genes, this undesirable finding may result from the accumulation of elongated mistranslated polypeptides. Here, we report the development of a new food-grade cloning vector, pFG200, which is suitable for overexpressing a variety of genes in industrial strains of Lactococcus lactis. The vector uses an amber suppressor, supD, as selectable marker and consists entirely of Lactococcus DNA, with the exception of a small polylinker region. Using suppressible pyrimidine auxotrophs, selection and maintenance are efficient in any pyrimidine-free medium including milk. Importantly, the presence of this vector in a variety of industrial strains has no significant effect on the growth rate or the rate of acidification in milk, making this an ideal system for food-grade modification of industrially relevant L. lactis strains. The usefulness of this system is demonstrated by overexpressing the pepN gene in a number of industrial backgrounds. PMID:10742196

Allergenicity reduction of bovine milk β-lactoglobulin by proteolytic activity of lactococcus lactis BMC12C and BMC19H isolated from Iranian dairy products.

PubMed

Kazemi, Rezvan; Taheri-Kafrani, Asghar; Motahari, Ahmad; Kordesedehi, Reihane

2018-06-01

Nowadays health benefits of bioactive food constituents, known as probiotic microorganisms, are a growing awareness. Cow's milk is a nutritious food containing probiotic bacteria. However, milk allergenicity is one of the most common food allergies. The milk protein, β-lactoglobulin (BLG), is in about 80% of all main cases of milk allergies for children and infants. With the aim of screening proteolytic strains of lactic acid bacteria to evaluate their potential for the reduction of allergenicity of the major bovine milk proteins, we isolated new proteolytic strains of cocci lactic acid bacteria from traditional Iranian dairy products. The proteases produced by these strains had strong proteolytic activity against BLG. Proteolysis of BLG, observed after sodium dodecyl sulfate-PAGE, was confirmed by the analysis of the peptide profiles by reversed-phase HPLC. The two isolates were submitted to 16S rDNA sequencing and identified as Lactcoccus lactis subsp. cremoris and Lactcoccus lactis subsp. hordniea. The competitive ELISA experiments confirmed that these isolates, with high proteolytic activity, reduce significantly the allergenicity of BLG. Accordingly, these isolates can reduce the immunoreactivity of bovine milk proteins, which can be helpful for the production of low-allergic dairy products. Copyright © 2018 Elsevier B.V. All rights reserved.

An application in cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147.

PubMed Central

Ryan, M P; Rea, M C; Hill, C; Ross, R P

1996-01-01

Lactococcus lactis DPC3147, a strain isolated from an Irish kefir grain, produces a bacteriocin with a broad spectrum of inhibition. The bacteriocin produced is heat stable, particularly at a low pH, and inhibits nisin-producing (Nip+) lactococci. On the basis of the observation that the nisin structural gene (nisA) does not hybridize to DPC3147 genomic DNA, the bacteriocin produced was considered novel and designated lacticin 3147. The genetic determinants which encode lacticin 3147 are contained on a 63-kb plasmid, which was conjugally mobilized to a commercial cheese starter, L. lactis subsp. cremoris DPC4268. The resultant transconjugant, DPC4275, both produces and is immune to lacticin 3147. The ability of lacticin 3147-producing lactococci to perform as cheddar cheese starters was subsequently investigated in cheesemaking trials. Bacteriocin-producing starters (which included the transconjugant strain DPC4275) produced acid at rates similar to those of commercial strains. The level of lacticin 3147 produced in cheese remained constant over 6 months of ripening and correlated with a significant reduction in the levels of nonstarter lactic acid bacteria. Such results suggest that these starters provide a means of controlling developing microflora in ripened fermented products. PMID:8593062

Characterization of lactococci isolated from milk produced in the Camembert region of Normandy.

PubMed

Desmasures, N; Mangin, I; Corroler, D; Guéguen, M

1998-12-01

Thirty-eight Lactococcus strains, isolated from raw milk produced in two dairy areas in Normandy, were identified at the phenotypic level. Only Lactococcus lactis strains with the lactis phenotype were found in the milk samples. Most strains fermented lactose (97%) and showed proteinase activity (76%). Isolates were characterized by RAPD technique and rRNA gene restriction analysis. More L. lactis strains with the lactis genotype were found in the first area, while L. lactis strains with the cremoris genotype predominated in the second area. RAPD was more efficient than rRNA gene restriction analysis in differentiating between strains with the subsp. lactis genotype. For L. lactis with the subsp. cremoris genotype, the second method gave a better result but there was poor discrimination between strains. Plasmid profiles were determined. Patterns ranged in size from 1.3 to 16.5 kbp, and 29 different profiles were found. Six groups of strains were determined, five of which were specific for the area of origin. It is suggested that the region of manufacture could influence organoleptic properties of cheeses because of different Lactococcus strains in the raw milk used for cheese making.

Strain improvement and metabolic flux analysis in the wild-type and a mutant Lactobacillus lactis strain for L(+)-lactic acid production.

PubMed

Bai, Dong-Mei; Zhao, Xue-Ming; Li, Xin-Gang; Xu, Shi-Min

2004-12-20

The effects of initial glucose concentration and calcium lactate concentration on the lactic acid production by the parent strain, Lactobacillus lactis BME5-18, were studied. The results of the experiments indicated that glucose and lactate repressed the cell growth and the lactic acid production by Lactobacillus lactis BME5-18. A L(+)-lactic acid overproducing strain, Lactobacillus lactis BME5-18M, was screened by mutagenizing the parent strain with ultraviolet (UV) light irradiation and selecting the high glucose and lactate calcium concentration repression resistant mutant. Starting with a concentration of 100g L(-1) glucose, the mutant produced 98.6 g L(-1) lactic acid after 60 h in flasks, 73.9% higher than that of the parent strain. The L(+)-lactic acid purity was 98.1% by weight based on the amount of total lactic acid. The culture of the parent strain could not be analyzed well by conventional metabolic flux analysis techniques, since some pyruvate were accumulated intracellularly. Therefore, a revised flux analysis method was proposed by introducing intracellular pyruvate pool. Further studies demonstrate that there is a high level of NADH oxidase activity (12.11 mmol mg(-1) min(-1)) in the parent strain. The molecular mechanisms of the strain improvement were proposed, i.e., the high level of NADH oxidase activity was eliminated and the uptake rate of glucose was increased from 82.1 C-mmol (g DW h)(-1) to 98.9 C-mmol (g DW h)(-1) by mutagenizing the parent strain with UV, and therefore the mutant strain converts mostly pyruvate to lactic acid with a higher productivity (1.76 g L(-1) h(-1)) than the parent strain (0.95 g L(-1) h(-1)).

An Ecological Study of Lactococci Isolated from Raw Milk in the Camembert Cheese Registered Designation of Origin Area

PubMed Central

Corroler, D.; Mangin, I.; Desmasures, N.; Gueguen, M.

1998-01-01

The genetic diversity of lactococci isolated from raw milk in the Camembert cheese Registered Designation of Origin area was studied. Two seasonal samples (winter and summer) of raw milk were obtained from six farms in two areas (Bessin and Bocage Falaisien) of Normandy. All of the strains analyzed had a Lactococcus lactis subsp. lactis phenotype, whereas the randomly amplified polymorphic DNA (RAPD) technique genotypically identified the strains as members of L. lactis subsp. lactis or L. lactis subsp. cremoris. The genotypes were confirmed by performing standard PCR with primers corresponding to a region of the histidine biosynthesis operon. The geographic distribution of each subspecies of L. lactis was determined; 80% of the Bocage Falaisien strains were members of L. lactis subsp. lactis, and 30.5% of the Bessin strains were members of L. lactis subsp. lactis. A dendrogram was produced from a computer analysis of the RAPD profiles in order to evaluate the diversity of the lactococci below the subspecies level. The coefficient of similarity for 117 of the 139 strains identified as members of L. lactis subsp. cremoris was as high as 66%. The L. lactis subsp. lactis strains were more heterogeneous and formed 10 separate clusters (the level of similarity among the clusters was 18%). Reference strains of L. lactis subsp. lactis fell into 2 of these 10 clusters, demonstrating that lactococcal isolates are clearly different. As determined by the RAPD profiles, some L. lactis subsp. lactis strains were specific to the farms from which they originated and were recovered throughout the year (in both summer and winter). Therefore, the typicality of L. lactis subsp. lactis strains was linked to the farm of origin rather than the area. These findings emphasize the significance of designation of origin and the specificity of “Camembert de Normandie” cheese. PMID:9835555

An ecological study of lactococci isolated from raw milk in the camembert cheese registered designation of origin area.

PubMed

Corroler, D; Mangin, I; Desmasures, N; Gueguen, M

1998-12-01

The genetic diversity of lactococci isolated from raw milk in the Camembert cheese Registered Designation of Origin area was studied. Two seasonal samples (winter and summer) of raw milk were obtained from six farms in two areas (Bessin and Bocage Falaisien) of Normandy. All of the strains analyzed had a Lactococcus lactis subsp. lactis phenotype, whereas the randomly amplified polymorphic DNA (RAPD) technique genotypically identified the strains as members of L. lactis subsp. lactis or L. lactis subsp. cremoris. The genotypes were confirmed by performing standard PCR with primers corresponding to a region of the histidine biosynthesis operon. The geographic distribution of each subspecies of L. lactis was determined; 80% of the Bocage Falaisien strains were members of L. lactis subsp. lactis, and 30.5% of the Bessin strains were members of L. lactis subsp. lactis. A dendrogram was produced from a computer analysis of the RAPD profiles in order to evaluate the diversity of the lactococci below the subspecies level. The coefficient of similarity for 117 of the 139 strains identified as members of L. lactis subsp. cremoris was as high as 66%. The L. lactis subsp. lactis strains were more heterogeneous and formed 10 separate clusters (the level of similarity among the clusters was 18%). Reference strains of L. lactis subsp. lactis fell into 2 of these 10 clusters, demonstrating that lactococcal isolates are clearly different. As determined by the RAPD profiles, some L. lactis subsp. lactis strains were specific to the farms from which they originated and were recovered throughout the year (in both summer and winter). Therefore, the typicality of L. lactis subsp. lactis strains was linked to the farm of origin rather than the area. These findings emphasize the significance of designation of origin and the specificity of "Camembert de Normandie" cheese.

Differentiation of Streptococcus lactis var. maltigenes from Other Lactic Streptococci1

PubMed Central

Gordon, D. F.; Morgan, M. E.; Tucker, J. S.

1963-01-01

Strains of lactic streptococci isolated from samples of raw milk which had developed a malty aroma were subjected to the cultural, physiological, and serological tests commonly employed in the classification of streptococci. None of the strains could be differentiated from Streptococcus lactis by these tests. Resting cells of strains which produced an organoleptically detectable malty aroma when cultured in milk were usually found to possess an active α-ketoacid decarboxylase, indicating the presence of the mechanism responsible for the characteristic aroma production. This decarboxylase activity was either weak or nonexistent in the nonmalty strains, and no activity was detected in known strains of S. lactis, S. cremoris, or S. diacetilactis. The malty strains usually produced higher acidities in milk than did the nonmalty strains, and, in most instances, they developed a granular type of growth sediment in broth, as opposed to a viscid sediment. Many of them gave weakly positive Voges-Proskauer tests in glucose broth with or without added citrate and appeared to be somewhat more resistant to nisin than the nonmalty strains. PMID:13949187

Assessment of stress tolerance acquisition in the heat-tolerant derivative strains of Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus rhamnosus GG.

PubMed

Aakko, J; Sánchez, B; Gueimonde, M; Salminen, S

2014-07-01

The purpose of this study was to investigate the heat-shock response at molecular level in Lactobacillus rhamnosus GG, Bifidobacterium animalis subsp. lactis BB-12 and their heat-tolerant derivatives and to characterize the changes that make the derivatives more robust in terms of heat stress. The study strains were exposed for 2 h to a heat-shock treatment, Bif. animalis subsp. lactis BB-12 and its derivative at 50°C and the Lact. rhamnosus GG and its derivative at 60°C. Protein synthesis before and after heat shock was examined using proteomics and RT-qPCR. The analysis revealed that the regulation of seven proteins in both strain pairs was modified as a response to heat or between the original and the derivative strain. The comparison of wild-type strains and the heat-tolerant derivatives suggests that the acquisition of heat tolerance in the Bif. animalis subsp. lactis BB-12 derivative is due to a slightly increased constitutive level of chaperones, while in Lact. rhamnosus GG derivative, the main reason seems to be a higher ability to induce the production of chaperones. This study revealed possible markers of heat tolerance in B. lactis and Lact. rhamnosus strains. This study increases our knowledge on how Lactobacillus and Bifidobacterium strains may acquire heat tolerance. These findings may be useful for improving the heat tolerance of existing probiotic strains as well as screening new heat-tolerant strains. © 2014 The Society for Applied Microbiology.

A novel multi-locus sequence typing (MLST) protocol for Leuconostoc lactis isolates from traditional dairy products in China and Mongolia.

PubMed

Dan, Tong; Liu, Wenjun; Sun, Zhihong; Lv, Qiang; Xu, Haiyan; Song, Yuqin; Zhang, Heping

2014-06-09

Economically, Leuconostoc lactis is one of the most important species in the genus Leuconostoc. It plays an important role in the food industry including the production of dextrans and bacteriocins. Currently, traditional molecular typing approaches for characterisation of this species at the isolate level are either unavailable or are not sufficiently reliable for practical use. Multilocus sequence typing (MLST) is a robust and reliable method for characterising bacterial and fungal species at the molecular level. In this study, a novel MLST protocol was developed for 50 L. lactis isolates from Mongolia and China. Sequences from eight targeted genes (groEL, carB, recA, pheS, murC, pyrG, rpoB and uvrC) were obtained. Sequence analysis indicated 20 different sequence types (STs), with 13 of them being represented by a single isolate. Phylogenetic analysis based on the sequences of eight MLST loci indicated that the isolates belonged to two major groups, A (34 isolates) and B (16 isolates). Linkage disequilibrium analyses indicated that recombination occurred at a low frequency in L. lactis, indicating a clonal population structure. Split-decomposition analysis indicated that intraspecies recombination played a role in generating genotypic diversity amongst isolates. Our results indicated that MLST is a valuable tool for typing L. lactis isolates that can be used for further monitoring of evolutionary changes and population genetics.

A novel multi-locus sequence typing (MLST) protocol for Leuconostoc lactis isolates from traditional dairy products in China and Mongolia

PubMed Central

2014-01-01

Background Economically, Leuconostoc lactis is one of the most important species in the genus Leuconostoc. It plays an important role in the food industry including the production of dextrans and bacteriocins. Currently, traditional molecular typing approaches for characterisation of this species at the isolate level are either unavailable or are not sufficiently reliable for practical use. Multilocus sequence typing (MLST) is a robust and reliable method for characterising bacterial and fungal species at the molecular level. In this study, a novel MLST protocol was developed for 50 L. lactis isolates from Mongolia and China. Results Sequences from eight targeted genes (groEL, carB, recA, pheS, murC, pyrG, rpoB and uvrC) were obtained. Sequence analysis indicated 20 different sequence types (STs), with 13 of them being represented by a single isolate. Phylogenetic analysis based on the sequences of eight MLST loci indicated that the isolates belonged to two major groups, A (34 isolates) and B (16 isolates). Linkage disequilibrium analyses indicated that recombination occurred at a low frequency in L. lactis, indicating a clonal population structure. Split-decomposition analysis indicated that intraspecies recombination played a role in generating genotypic diversity amongst isolates. Conclusions Our results indicated that MLST is a valuable tool for typing L. lactis isolates that can be used for further monitoring of evolutionary changes and population genetics. PMID:24912963

Improved bioethanol production in an engineered Kluyveromyces lactis strain shifted from respiratory to fermentative metabolism by deletion of NDI1

PubMed Central

González-Siso, María Isabel; Touriño, Alba; Vizoso, Ángel; Pereira-Rodríguez, Ángel; Rodríguez-Belmonte, Esther; Becerra, Manuel; Cerdán, María Esperanza

2015-01-01

In this paper, we report the metabolic engineering of the respiratory yeast Kluyveromyces lactis by construction and characterization of a null mutant (Δklndi1) in the single gene encoding a mitochondrial alternative internal dehydrogenase. Isolated mitochondria of the Δklndi1 mutant show unaffected rate of oxidation of exogenous NADH, but no oxidation of matrix NADH; this confirms that KlNdi1p is the only internal NADH dehydrogenase in K. lactis mitochondria. Permeabilized cells of the Δklndi1 mutant do not show oxidation of matrix NADH, which suggests that shuttle systems to transfer the NADH from mitochondrial matrix to cytosol, for being oxidized by external dehydrogenases, are not functional. The Δklndi1 mutation decreases the chronological life span in absence of nutrients. The expression of KlNDI1 is increased by glutathione reductase depletion. The Δklndi1 mutation shifts the K. lactis metabolism from respiratory to fermentative: the Δklndi1 strain shows reduced respiration rate and increased ethanol production from glucose, while it does not grow in non-fermentable carbon sources such as lactate. The biotechnological benefit of the Δklndi1 mutant for bioethanol production from waste cheese whey lactose was proved. PMID:25186243

Lactococcus lactis Diversity in Undefined Mixed Dairy Starter Cultures as Revealed by Comparative Genome Analyses and Targeted Amplicon Sequencing of epsD.

PubMed

Frantzen, Cyril A; Kleppen, Hans Petter; Holo, Helge

2018-02-01

Undefined mesophilic mixed (DL) starter cultures are used in the production of continental cheeses and contain unknown strain mixtures of Lactococcus lactis and leuconostocs. The choice of starter culture affects the taste, aroma, and quality of the final product. To gain insight into the diversity of Lactococcus lactis strains in starter cultures, we whole-genome sequenced 95 isolates from three different starter cultures. Pan-genomic analyses, which included 30 publically available complete genomes, grouped the strains into 21 L. lactis subsp . lactis and 28 L. lactis subsp. cremoris lineages. Only one of the 95 isolates grouped with previously sequenced strains, and the three starter cultures showed no overlap in lineage distributions. The culture diversity was assessed by targeted amplicon sequencing using purR , a core gene, and epsD , present in 93 of the 95 starter culture isolates but absent in most of the reference strains. This enabled an unprecedented discrimination of starter culture Lactococcus lactis and revealed substantial differences between the three starter cultures and compositional shifts during the cultivation of cultures in milk. IMPORTANCE In contemporary cheese production, standardized frozen seed stock starter cultures are used to ensure production stability, reproducibility, and quality control of the product. The dairy industry experiences significant disruptions of cheese production due to phage attacks, and one commonly used countermeasure to phage attack is to employ a starter rotation strategy, in which two or more starters with minimal overlap in phage sensitivity are used alternately. A culture-independent analysis of the lactococcal diversity in complex undefined starter cultures revealed large differences between the three starter cultures and temporal shifts in lactococcal composition during the production of bulk starters. A better understanding of the lactococcal diversity in starter cultures will enable the development of

Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) stimulates murine macrophages infected with Citrobacter rodentium.

PubMed

Hugo, Ayelén A; Rolny, Ivanna S; Romanin, David; Pérez, Pablo F

2017-03-01

Citrobacter rodentium is a specific murine enteropathogen which causes diarrheal disease characterized by colonic hyperplasia and intestinal inflammation. Recruitment of neutrophils and macrophages constitute a key step to control the infection. Since modulation of the activity of professional phagocytic cells could contribute to improve host´s defences against C. rodentium, we investigated the effect of Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) on the interaction between murine macrophages (RAW 264.7) and C. rodentium. Phagocytosis, surface molecules and inducible nitric oxide synthase (iNOs) expression were determined by flow cytometry. Reactive oxygen species (ROS) were assessed by fluorescence microscopy. The presence of lactobacilli increased phagocytosis of C. rodentium whereas C. rodentium had no effect on lactobacilli internalization. Survival of internalized C. rodentium diminished when strain CIDCA 133 was present. CD-86, MHCII, iNOs expression and nitrite production were increased when C. rodentium and lactobacilli were present even though strain CIDCA 133 alone had no effect. Strain CIDCA 133 led to a strong induction of ROS activity which was not modified by C. rodentium. Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) is able to increase the activation of murine macrophages infected with C. rodentium. The sole presence of lactobacilli is enough to modify some stimulation markers (e.g. ROS induction) whereas other markers require the presence of both bacteria; thus, indicating a synergistic effect.

Draft Genome Sequence of Bifidobacterium animalis subsp. lactis Strain CECT 8145, Able To Improve Metabolic Syndrome In Vivo.

PubMed

Chenoll, E; Codoñer, F M; Silva, A; Martinez-Blanch, J F; Martorell, P; Ramón, D; Genovés, S

2014-03-27

Bifidobacterium animalis subsp. lactis strain CECT 8145 is able to reduce body fat content and improve metabolic syndrome biomarkers. Here, we report the draft genome sequence of this strain, which may provide insights into its safety status and functional role.

Purification and characterization of two new cell-bound bioactive compounds produced by wild Lactococcus lactis strain.

PubMed

Saraiva, Margarete Alice Fontes; Brede, Dag Anders; Nes, Ingolf Figved; Baracat-Pereira, Maria Cristina; de Queiroz, Marisa Vieira; de Moraes, Célia Alencar

2017-07-03

Novel compounds and innovative methods are required considering that antibiotic resistance has reached a crisis point. In the study, two cell-bound antimicrobial compounds produced by Lactococcus lactis ID1.5 were isolated and partially characterized. Following purification by cationic exchange and a solid-phase C18 column, antimicrobial activity was recovered after three runs of RPC using 60% (v/v) and 100% (v/v) of 2-propanol for elution, suggesting that more than one antimicrobial compound were produced by L. lactis ID1.5, which were in this study called compounds AI and AII. The mass spectrum of AI and AII showed major intensity ions at m/z 1070.05 and 955.9 Da, respectively. The compound AI showed a spectrum of antimicrobial activity mainly against L. lactis species, while the organisms most sensitive to compound AII were Bacillus subtilis, Listeria innocua, Streptococcus pneumoniae and Pseudomonas aeruginosa. The antimicrobial activity of both compounds was suppressed by treatment with Tween 80. Nevertheless, both compounds showed high stability to heat and proteases treatments. The isolated compounds, AI and AII, showed distinct properties from other antimicrobial substances already reported as produced by L. lactis, and have a significant inhibitory effect against two clinically important respiratory pathogens. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Selection of a Bifidobacterium strain to complement resistant starch in a synbiotic yoghurt.

PubMed

Crittenden, R G; Morris, L F; Harvey, M L; Tran, L T; Mitchell, H L; Playne, M J

2001-02-01

To employ an in vitro screening regime to select a probiotic Bifidobacterium strain to complement resistant starch (Hi-maizetrade mark) in a synbiotic yoghurt. Of 40 Bifidobacterium isolates examined, only B. lactis Laftitrade mark B94 possessed all of the required characteristics. This isolate hydrolysed Hi-maizetrade mark, survived well in conditions simulating passage through the gastrointestinal tract and possessed technological properties suitable for yoghurt manufacture. It grew well at temperatures up to 45 degrees C, and grew to a high cell yield in an industrial growth medium. In addition to resistant starch, the organism was able to utilize a range of prebiotics including inulin, and fructo-, galacto-, soybean- and xylo-oligosaccharides. Pulse field gel electrophoresis of restriction enzyme cut chromosomal DNA revealed that B. lactis Laftitrade mark B94 was very closely related to the B. lactis Type Strain (DSM 10140), and to the commercial strains B. lactis Bb-12 and B. lactis DS 920. However, B. lactis Laftitrade mark B94 was the only one of these isolates that could hydrolyse Hi-maizetrade mark. This phenotypic difference did not appear to be due to the presence of plasmid encoded amylase. Bifidobacterium lactis Laftitrade mark B94 survived without substantial loss of viability in synbiotic yoghurt containing Hi-maizetrade mark during storage at 4 degrees C for six weeks. Bifidobacterium lactis Laftitrade mark B94 is a promising new yoghurt culture that warrants further investigation to assess its probiotic potential. In vitro screening procedures can be used to integrate complementary probiotic and prebiotic ingredients for new synbiotic functional food products.

Characterization of certain bacterial strains for potential use as starter or probiotic cultures in dairy products.

PubMed

Monteagudo-Mera, A; Caro, I; Rodríguez-Aparicio, L B; Rúa, J; Ferrero, M A; García-Armesto, M R

2011-08-01

The present work was aimed at characterizing 12 strains of lactic acid bacteria (LAB) to obtain improved potential starter or probiotic cultures that could be used for making dairy products from ewe's milk and cow's milk. Eight strains with antimicrobial properties, isolated from ewe's milk and from cheese made from ewe's and/or cow's milk, were studied. They were identified as Enterococcus faecalis (five strains), Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides, and Lactobacillus paracasei subsp. paracasei (one strain of each species). Additionally, four strains were obtained from the American Type Culture Collection: Lactobacillus casei 393 (isolated from cheese), L. lactis subsp. lactis 11454 (origin nonspecified and a producer of nisin), and two strains isolated from human feces (L. paracasei subsp. paracasei 27092 and Lactobacillus rhamnosus 53103, antibacterial agent producer). All E. faecalis strains showed at least one virulence factor (either hemolysin or gelatinase), which emphasizes the importance of these studies in this species. Both L. lactis strains and most Lactobacillus spp. were good acidifiers in ewe's milk and cow's milk at 30°C. High β-galactosidase activity, as well as aminopeptidase activities that favor the development of desirable flavors in cheese, were detected in all Lactobacillus spp. strains. Furthermore, L. rhamnosus ATCC 53103 showed α-fucosidase activity (thought to help colonization of the intestine) and lack of α-glucosidase activity (a trait considered positive for diabetic and obese humans). This last enzymatic activity was also lacking in L. lactis ATCC 11454. L. mesenteroides was the only strain D(2)-lactic acid producer. The selection of any particular strain for probiotic or dairy cultures should be performed according to the technological and/or functional abilities needed.

Bile resistance in Lactococcus lactis strains varies with cellular fatty acid composition: analysis by using different growth media.

PubMed

Kimoto-Nira, Hiromi; Kobayashi, Miho; Nomura, Masaru; Sasaki, Keisuke; Suzuki, Chise

2009-05-31

Bile resistance is one of the basic characteristics of probiotic bacteria. The aim of this study was to investigate the characteristics of bile resistance in lactococci by studying the relationship between bile resistance and cellular fatty acid composition in lactococcci grown on different media. We determined the bile resistance of 14 strains in lactose-free M17 medium supplemented with either glucose only (GM17) or lactose only (LM17). Gas chromatographic analyses of free lipids extracted from the tested strains were used for determining their fatty acid composition. A correlation analysis of all strains grown in both media revealed significant positive correlations between bile resistance and relative contents of hexadecanoic acid and octadecenoic acid, and negative correlations between bile resistance and relative contents of hexadecenoic acid and C-19 cyclopropane fatty acid. It is also a fact that the fatty acids associated with bile resistance depended on species, strain, and/or growth medium. In L. lactis subsp. cremoris strains grown in GM17 medium, the bile-resistant strains had significantly more octadecenoic acid than the bile-sensitive strains. In LM17 medium, bile-resistant strains had significantly more octadecenoic acid and significantly less C-19 cyclopropane fatty acid than the bile-sensitive strains. In L. lactis subsp. lactis strains, bile resistances of some of the tested strains were altered by growth medium. Some strains were resistant to bile in GM17 medium but sensitive to bile in LM17 medium. Some strains were resistant in both media tested. The strains grown in GM17 medium had significantly more hexadecanoic acid and octadecenoic acid, and significantly less tetradecanoic acid, octadecadienoic acid and C-19 cyclopropane fatty acid than the strains grown in LM17 medium. In conclusion, the fatty acid compositions of the bile-resistant lactococci differed from those of the bile-sensitive ones. More importantly, our data suggest that

Determination of the cell wall polysaccharide and teichoic acid structures from Lactococcus lactis IL1403.

PubMed

Vinogradov, Evgeny; Sadovskaya, Irina; Courtin, Pascal; Kulakauskas, Saulius; Grard, Thierry; Mahony, Jennifer; van Sinderen, Douwe; Chapot-Chartier, Marie-Pierre

2018-06-15

In the lactic acid bacterium Lactococcus lactis, a cell wall polysaccharide (CWPS) is the bacterial receptor of the majority of infecting bacteriophages. The diversity of CWPS structures between strains explains, at least partially, the narrow host range of lactococcal phages. In the present work, we studied the polysaccharide components of the cell wall of the prototype L. lactis subsp. lactis strain IL1403. We identified a rhamnose-rich complex polysaccharide, carrying a glycerophosphate substitution, as the major component. Its structure was analyzed by 2D NMR spectroscopy, methylation analysis and MALDI-TOF MS and shown to be distinctly different from currently known lactococcal CWPS structures. It contains a linear backbone of repeated α-l-Rha disaccharide subunits, which is irregularly substituted with a trisaccharide occasionally bearing a glycerophosphate group. A poly (glycerol phosphate) teichoic acid, another important carbohydrate component of the IL1403 cell wall, was also isolated and structurally characterized. Copyright © 2018 Elsevier Ltd. All rights reserved.

Short communication: Genotypic and phenotypic identification of environmental streptococci and association of Lactococcus lactis ssp. lactis with intramammary infections among different dairy farms.

PubMed

Werner, B; Moroni, P; Gioia, G; Lavín-Alconero, L; Yousaf, A; Charter, M E; Carter, B Moslock; Bennett, J; Nydam, D V; Welcome, F; Schukken, Y H

2014-11-01

Lactococcus species are counted among a large and closely related group of environmental streptococci and streptococci-like bacteria that include bovine mastitis pathogenic Streptococcus, Enterococcus, and Aerococcus species. Phenotypic and biochemical identification methods can be inaccurate and unreliable for species within this group, particularly for Lactococcus spp. As a result, the incidence of Lactococcus spp. on the farm may have been historically underreported and consequently little is known about the clinical importance of this genus as a mastitis pathogen. We used molecular genetic identification methods to accurately differentiate 60 environmental streptococci and streptococci-like bacteria isolated from cows with high somatic cell count and chronic intramammary infection (IMI; >2 somatic cell scores above 4) among 5 geographically distinct farms in New York and Minnesota that exhibited an observed increase in IMI. These isolates were phenotypically identified as Streptococcus uberis and Streptococcus spp. Genetic methods identified 42 isolates (70%) as Lactococcus lactis ssp. lactis, including all 10 isolates originally phenotypically identified as Streptococcus uberis. Antibiotic inhibition testing of all Lc. lactis ssp. lactis showed that 7 isolates were resistant to tetracycline. In the present study, a predominance of Lc. lactis ssp. lactis was identified in association with chronic, clinical bovine IMI among all 5 farms and characterized antimicrobial resistance for treatment therapies. Routine use by mastitis testing labs of molecular identification methods for environmental streptococci and streptococci-like bacteria can further define the role and prevalence of Lc. lactis ssp. lactis in association with bovine IMI and may lead to more targeted therapies. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A General Method for Selection of α-Acetolactate Decarboxylase-Deficient Lactococcus lactis Mutants To Improve Diacetyl Formation

PubMed Central

Curic, Mirjana; Stuer-Lauridsen, Birgitte; Renault, Pierre; Nilsson, Dan

1999-01-01

The enzyme acetolactate decarboxylase (Ald) plays a key role in the regulation of the α-acetolactate pool in both pyruvate catabolism and the biosynthesis of the branched-chain amino acids, isoleucine, leucine, and valine (ILV). This dual role of Ald, due to allosteric activation by leucine, was used as a strategy for the isolation of Ald-deficient mutants of Lactococcus lactis subsp. lactis biovar diacetylactis. Such mutants can be selected as leucine-resistant mutants in ILV- or IV-prototrophic strains. Most dairy lactococcus strains are auxotrophic for the three amino acids. Therefore, the plasmid pMC004 containing the ilv genes (encoding the enzymes involved in the biosynthesis of IV) of L. lactis NCDO2118 was constructed. Introduction of pMC004 into ILV-auxotrophic dairy strains resulted in an isoleucine-prototrophic phenotype. By plating the strains on a chemically defined medium supplemented with leucine but not valine and isoleucine, spontaneous leucine-resistant mutants were obtained. These mutants were screened by Western blotting with Ald-specific antibodies for the presence of Ald. Selected mutants lacking Ald were subsequently cured of pMC004. Except for a defect in the expression of Ald, the resulting strain, MC010, was identical to the wild-type strain, as shown by Southern blotting and DNA fingerprinting. The mutation resulting in the lack of Ald in MC010 occurred spontaneously, and the strain does not contain foreign DNA; thus, it can be regarded as food grade. Nevertheless, its application in dairy products depends on the regulation of genetically modified organisms. These results establish a strategy to select spontaneous Ald-deficient mutants from transformable L. lactis strains. PMID:10049884

Multilocus sequence typing of Lactococcus lactis from naturally fermented milk foods in ethnic minority areas of China.

PubMed

Xu, Haiyan; Sun, Zhihong; Liu, Wenjun; Yu, Jie; Song, Yuqin; Lv, Qiang; Zhang, Jiachao; Shao, Yuyu; Menghe, Bilige; Zhang, Heping

2014-05-01

To determine the genetic diversity and phylogenetic relationships among Lactococcus lactis isolates, 197 strains isolated from naturally homemade yogurt in 9 ethnic minority areas of 6 provinces of China were subjected to multilocus sequence typing (MLST). The MLST analysis was performed using internal fragment sequences of 12 housekeeping genes (carB, clpX, dnaA, groEL, murC, murE, pepN, pepX, pyrG, recA, rpoB, and pheS). Six (dnaA) to 8 (murC) different alleles were detected for these genes, which ranged from 33.62 (clpX) to 41.95% (recA) GC (guanine-cytosine) content. The nucleotide diversity (π) ranged from 0.00362 (murE) to 0.08439 (carB). Despite this limited allelic diversity, the allele combinations of each strain revealed 72 different sequence types, which denoted significant genotypic diversity. The dN/dS ratios (where dS is the number of synonymous substitutions per synonymous site, and dN is the number of nonsynonymous substitutions per nonsynonymous site) were lower than 1, suggesting potential negative selection for these genes. The standardized index of association of the alleles IA(S)=0.3038 supported the clonality of Lc. lactis, but the presence of network structure revealed by the split decomposition analysis of the concatenated sequence was strong evidence for intraspecies recombination. Therefore, this suggests that recombination contributed to the evolution of Lc. lactis. A minimum spanning tree analysis of the 197 isolates identified 14 clonal complexes and 23 singletons. Phylogenetic trees were constructed based on the sequence types, using the minimum evolution algorithm, and on the concatenated sequence (6,192 bp), using the unweighted pair-group method with arithmetic mean, and these trees indicated that the evolution of our Lc. lactis population was correlated with geographic origin. Taken together, our results demonstrated that MLST could provide a better understanding of Lc. lactis genome evolution, as well as useful information for

Safety of Bifidobacterium animalis Subsp. Lactis (B. lactis) Strain BB-12-Supplemented Yogurt in Healthy Children.

PubMed

Tan, Tina P; Ba, Zhaoyong; Sanders, Mary E; D'Amico, Frank J; Roberts, Robert F; Smith, Keisha H; Merenstein, Daniel J

2017-02-01

Probiotics are live microorganisms that may provide health benefits to the individual when consumed in sufficient quantities. For studies conducted on health or disease endpoints on probiotics in the United States, the Food and Administration has required those studies to be conducted as investigational new drugs. This phase I, double-blinded, randomized, controlled safety study represents the first requirement of this pathway. The purpose of the study was to determine the safety of Bifidobacterium animalis subsp. lactis (B lactis) strain BB-12 (BB-12)-supplemented yogurt when consumed by a generally healthy group of children. The secondary aim was to assess the effect of BB-12-supplemented yogurt on the gut microbiota of the children. Sixty children ages 1 to 5 years were randomly assigned to consume 4 ounces of either BB-12-supplemented yogurt or nonsupplemented control yogurt daily for 10 days. The primary outcome was to assess safety and tolerability, as determined by the number of reported adverse events. A total of 186 nonserious adverse events were reported, with no significant differences between the control and BB-12 groups. No significant changes due to probiotic treatment were observed in the gut microbiota of the study cohort. BB-12-supplemented yogurt is safe and well-tolerated when consumed by healthy children. The present study will form the basis for future randomized clinical trials investigating the potential effects of BB-12-supplemented yogurt in different disease states.

Carpoglyphus lactis (Acari: Astigmata) from various dried fruits differed in associated micro-organisms.

PubMed

Hubert, J; Nesvorná, M; Kopecký, J; Ságová-Marečková, M; Poltronieri, P

2015-02-01

Carpoglyphus lactis is a stored product mite infesting saccharide-rich stored commodities including dried fruits, wine, beer, milk products, jams and honey. The association with micro-organisms can improve the survival of mites on dried fruits. The microbial communities associated with C. lactis were studied in specimens originating from the packages of dried apricot, plums and figs and compared to the laboratory strain reared on house dust mite diet (HDMd). Clone libraries of bacterial 16S rRNA gene and fungal internal transcribed spacer (ITS) region were constructed and analysed by operational taxonomic unit (OTU) approach. The 16S rRNA gene libraries differed among the compared diets. The sequences classified to the genera Leuconostoc, Elizabethkingia, Ewingella, Erwinia, Bacillus and Serratia were prevailing in mites sampled from the dried fruits. The ITS library showed smaller differences between the laboratory strain on HDMd and the isolates from dried fruits packages, with the exception of the mite strain from dried plums. The population growth was used as an indirect indicator of fitness and decreased in the order from yeast diet to HDMd and dried fruits. The treatment and pretreatment of mites by antibiotics did not reveal the presence of antagonistic bacteria which might slow down the C. lactis population growth. The shifts of the microbial community in the gut of C. lactis were induced by the diet changes. The identified yeasts and bacteria are suggested as the main food source of stored product mites on dried fruits. The study describes the adaptation of C. lactis to feeding on dried fruits including the interaction with micro-organisms. We also identified potentially pathogenic bacteria carried by the mites to dried fruits for human consumption. © 2014 The Society for Applied Microbiology.

The investigation of probiotic potential of lactic acid bacteria isolated from traditional Mongolian dairy products.

PubMed

Takeda, Shiro; Yamasaki, Keiko; Takeshita, Masahiko; Kikuchi, Yukiharu; Tsend-Ayush, Chuluunbat; Dashnyam, Bumbein; Ahhmed, Abdulatef M; Kawahara, Satoshi; Muguruma, Michio

2011-08-01

The aims of this study were to investigate the diversity of lactic acid bacteria (LAB) isolated from traditional Mongolian dairy products, and to estimate the probiotic potential of the isolated strains. We collected 66 samples of the traditional Mongolian dairy products tarag (n = 45), airag (n = 7), aaruul (n = 8), byasulag (n = 1) and eezgii (n = 5), from which 543 LAB strains were isolated and identified based on 16S ribosomal DNA sequence. The predominant species of those products were Lactobacillus (L.) delbrueckii ssp. bulgaricus, L. helveticus, L. fermentum, L. delbrueckii ssp. lactis and Lactococcus lactis ssp. lactis. However, we could not detect any LAB strains from eezgii. All LAB isolates were screened for tolerance to low pH and to bile acid, gas production from glucose, and adherence to Caco-2 cells. In vitro, we found 10 strains possess probiotic properties, and almost identified them as L. plantarum or L. paracasei subspecies, based on 16S ribosomal DNA and carbohydrate fermentation pattern. These strains were differentiated from each other individually by randomly amplified polymorphic DNA analysis. Additionally, it was notable that 6/10 strains were isolated from camel milk tarag from the Dornogovi province. 2011 The Authors. Animal Science Journal © 2011 Japanese Society of Animal Science.

Development of a rapid SNP-typing assay to differentiate Bifidobacterium animalis ssp. lactis strains used in probiotic-supplemented dairy products.

PubMed

Lomonaco, Sara; Furumoto, Emily J; Loquasto, Joseph R; Morra, Patrizia; Grassi, Ausilia; Roberts, Robert F

2015-02-01

Identification at the genus, species, and strain levels is desirable when a probiotic microorganism is added to foods. Strains of Bifidobacterium animalis ssp. lactis (BAL) are commonly used worldwide in dairy products supplemented with probiotic strains. However, strain discrimination is difficult because of the high degree of genome identity (99.975%) between different genomes of this subspecies. Typing of monomorphic species can be carried out efficiently by targeting informative single nucleotide polymorphisms (SNP). Findings from a previous study analyzing both reference and commercial strains of BAL identified SNP that could be used to discriminate common strains into 8 groups. This paper describes development of a minisequencing assay based on the primer extension reaction (PER) targeting multiple SNP that can allow strain differentiation of BAL. Based on previous data, 6 informative SNP were selected for further testing, and a multiplex preliminary PCR was optimized to amplify the DNA regions containing the selected SNP. Extension primers (EP) annealing immediately adjacent to the selected SNP were developed and tested in simplex and multiplex PER to evaluate their performance. Twenty-five strains belonging to 9 distinct genomic clusters of B. animalis ssp. lactis were selected and analyzed using the developed minisequencing assay, simultaneously targeting the 6 selected SNP. Fragment analysis was subsequently carried out in duplicate and demonstrated that the assay yielded 8 specific profiles separating the most commonly used commercial strains. This novel multiplex PER approach provides a simple, rapid, flexible SNP-based subtyping method for proper characterization and identification of commercial probiotic strains of BAL from fermented dairy products. To assess the usefulness of this method, DNA was extracted from yogurt manufactured with and without the addition of B. animalis ssp. lactis BB-12. Extracted DNA was then subjected to the minisequencing

Novel conjugative plasmids from the natural isolate Lactococcus lactis subspecies cremoris DPC3758: a repository of genes for the potential improvement of dairy starters.

PubMed

Fallico, V; Ross, R P; Fitzgerald, G F; McAuliffe, O

2012-07-01

A collection of 17 natural lactococcal isolates from raw milk cheeses were studied in terms of their plasmid distribution, content, and diversity. All strains in the collection harbored an abundance of plasmids, including Lactococcus lactis ssp. cremoris DPC3758, whose 8-plasmid complement was selected for sequencing. The complete sequences of pAF22 (22,388 kb), pAF14 (14,419 kb), pAF12 (12,067 kb), pAF07 (7,435 kb), and pAF04 (3,801 kb) were obtained, whereas gene functions of technological interest were mapped to pAF65 (65 kb) and pAF45 (45 kb) by PCR. The plasmids of L. lactis DPC3758 were found to encode many genes with the potential to improve the technological properties of dairy starters. These included 3 anti-phage restriction/modification (R/M) systems (1 of type I and 2 of type II) and genes for immunity/resistance to nisin, lacticin 481, cadmium, and copper. Regions encoding conjugative/mobilization functions were present in 6 of the 8 plasmids, including those containing the R/M systems, thus enabling the food-grade transfer of these mechanisms to industrial strains. Using cadmium selection, the sequential stacking of the R/M plasmids into a plasmid-free host provided the recipient with increased protection against 936- and c2-type phages. The association of food-grade selectable markers and mobilization functions on L. lactis DPC3758 plasmids will facilitate their exploitation to obtain industrial strains with enhanced phage protection and robustness. These natural plasmids also provide another example of the major role of plasmids in contributing to host fitness and preservation within its ecological niche. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Microbiota of Minas cheese as influenced by the nisin producer Lactococcus lactis subsp. lactis GLc05.

PubMed

Perin, Luana Martins; Dal Bello, Barbara; Belviso, Simona; Zeppa, Giuseppe; Carvalho, Antônio Fernandes de; Cocolin, Luca; Nero, Luís Augusto

2015-12-02

Minas cheese is a popular dairy product in Brazil that is traditionally produced using raw or pasteurized cow milk. This study proposed an alternative production of Minas cheese using raw goat milk added of a nisin producer Lactococcus lactis subsp. lactis GLc05. An in situ investigation was carried on to evaluate the interactions between the L. lactis subsp. lactis GLc05 and the autochthonous microbiota of a Minas cheese during the ripening; production of biogenic amines (BAs) was assessed as a safety aspect. Minas cheese was produced in two treatments (A, by adding L. lactis subsp. lactis GLc05, and B, without adding this strain), in three independent repetitions (R1, R2, and R3). Culture dependent (direct plating) and independent (rep-PCR and PCR-DGGE) methods were employed to characterize the microbiota and to assess the possible interferences caused by L. lactis subsp. lactis GLc05. BA amounts were measured using HPLC. A significant decrease in coagulase-positive cocci was observed in the cheeses produced by adding L. lactis subsp. lactis GLc05 (cheese A). The rep-PCR and PCR-DGGE highlighted the differences in the microbiota of both cheeses, separating them into two different clusters. Lactococcus sp. was found as the main microorganism in both cheeses, and the microbiota of cheese A presented a higher number of species. High concentrations of tyramine were found in both cheeses and, at specific ripening times, the BA amounts in cheese B were significantly higher than in cheese A (p<0.05). The interaction of nisin producer L. lactis subsp. lactis GLc05 was demonstrated in situ, by demonstration of its influence in the complex microbiota naturally present in a raw goat milk cheese and by controlling the growth of coagulase-positive cocci. L. lactis subsp. lactis GLc05 influenced also the production of BA determining that their amounts in the cheeses were maintained at acceptable levels for human consumption. Copyright © 2015 Elsevier B.V. All rights reserved.

Evaluation of technological properties of Enterococcus faecium CECT 8849, a strain isolated from human milk, for the dairy industry.

PubMed

Cárdenas, Nivia; Arroyo, Rebeca; Calzada, Javier; Peirotén, Ángela; Medina, Margarita; Rodríguez, Juan Miguel; Fernández, Leonides

2016-09-01

In this work, a variety of biochemical properties of Enterococcus faecium CECT 8849, which had been isolated from breast milk, were analyzed. Its acidifying capacity and proteolytic activity were low but, in contrast, remarkable peptidase and esterase activities were observed. Ethanol and 3-hydroxy-2-butanone were the most abundant volatile compounds found in experimental model cheese manufactured with E. faecium CECT 8849. This strain inhibited the growth of several Listeria monocytogenes and Listeria innocua strains in vitro. Enterocin A and B structural genes were detected in E. faecium CECT 8849. Model fermented milk and cheeses were manufactured from milk inoculated or not with L. innocua CECT 8848 (2.5-3 log10 colony forming units mL(-1)) using E. faecium CECT 8849 or Lactococcus lactis ESI 153 as starter cultures. Although E. faecium CECT 8849 controlled Listeria growth in both dairy models, it led to lower reduction in Listeria counts when compared with L. lactis ESI 153.

Assessment of probiotic properties of lactic acid bacteria isolated from Indonesian naturally fermented milk

NASA Astrophysics Data System (ADS)

Jatmiko, Yoga Dwi; Howarth, Gordon S.; Barton, Mary D.

2017-11-01

This study aimed to characterize the probiotic properties of lactic acid bacteria from the naturally fermented milk of Indonesia, namely dangke and dadih. Fifty-one representative lactic acid bacteria belonging to the species Lactobacillus Plantarum, Lactococcus lactis subsp. lactis and Enterococcus faecium were evaluated in vitro for potential probiotic properties based on their bile salt resistance, low pH tolerance, antimicrobial activity, antibiotic susceptibility and adherence to Caco-2 colon cancer cells. In addition, bacteriocin related gene (plantaricin A), bile salt hydrolase (bsh) and mannose-specific adhesin (msa) genes in the genome of lactobacilli were also examined. None of the dangke isolates, which belonged to the species L. lactis subsp. lactis tolerated low pH. However, eight of the isolates were able to grow in the presence of bile salts. It was observed that L. plantarum strain S1.30 and SL2.7 from dadih tolerated low pH, survived bile salt concentrations and were resistant to vancomycin. Furthermore, these strains also contained bacteriocin regulating gene (plantaricin A) and msa and bsh genes in their genome. However, only the strain S1.30 exhibited optimal antimicrobial activity against the selected pathogens and was able to adhere to Caco-2 cells by up to 82.24±0.14%. Antagonistic activity of L. lactis subsp. lactis from dadih and dangke was not detected. However, 73.94±1.26% adherence to Caco-2 cells was demonstrated by L. lactis subsp. lactis strain SL3.34 sourced from dangke. These results suggest that Lactobacillus plantarum strain S1.30 associated with dadih fulfilled the in vitro probiotic criteria and could be exploited for further in vivo evaluation. In addition, dadih was an effective probiotic carrier compared to dangke.

Transcriptome analysis and related databases of Lactococcus lactis.

PubMed

Kuipers, Oscar P; de Jong, Anne; Baerends, Richard J S; van Hijum, Sacha A F T; Zomer, Aldert L; Karsens, Harma A; den Hengst, Chris D; Kramer, Naomi E; Buist, Girbe; Kok, Jan

2002-08-01

Several complete genome sequences of Lactococcus lactis and their annotations will become available in the near future, next to the already published genome sequence of L. lactis ssp. lactis IL 1403. This will allow intraspecies comparative genomics studies as well as functional genomics studies aimed at a better understanding of physiological processes and regulatory networks operating in lactococci. This paper describes the initial set-up of a DNA-microarray facility in our group, to enable transcriptome analysis of various Gram-positive bacteria, including a ssp. lactis and a ssp. cremoris strain of Lactococcus lactis. Moreover a global description will be given of the hardware and software requirements for such a set-up, highlighting the crucial integration of relevant bioinformatics tools and methods. This includes the development of MolGenIS, an information system for transcriptome data storage and retrieval, and LactococCye, a metabolic pathway/genome database of Lactococcus lactis.

Effect of autochthonous bacteriocin-producing Lactococcus lactis on bacterial population dynamics and growth of halotolerant bacteria in Brazilian charqui.

PubMed

Biscola, Vanessa; Abriouel, Hikmate; Todorov, Svetoslav Dimitrov; Capuano, Verena Sant'Anna Cabral; Gálvez, Antonio; Franco, Bernadette Dora Gombossy de Melo

2014-12-01

Charqui is a fermented, salted and sun-dried meat product, widely consumed in Brazil and exported to several countries. Growth of microorganisms in this product is unlikely due to reduced Aw, but halophilic and halotolerant bacteria may grow and cause spoilage. Charqui is a good source of lactic acid bacteria able to produce antimicrobial bacteriocins. In this study, an autochthonous bacteriocinogenic strain (Lactococcus lactis subsp. lactis 69), isolated from charqui, was added to the meat used for charqui manufacture and evaluated for its capability to prevent the growth of spoilage bacteria during storage up to 45 days. The influence of L. lactis 69 on the bacterial diversity during the manufacturing of the product was also studied, using denaturing gradient gel electrophoresis (DGGE). L. lactis 69 did not affect the counts and diversity of lactic acid bacteria during manufacturing and storage, but influenced negatively the populations of halotolerant microorganisms, reducing the spoilage potential. The majority of tested virulence genes was absent, evidencing the safety and potential technological application of this strain as an additional hurdle to inhibit undesirable microbial growth in this and similar fermented meat products. Copyright © 2014 Elsevier Ltd. All rights reserved.

Three new insertion sequence elements ISLdl2, ISLdl3, and ISLdl4 in Lactobacillus delbrueckii: isolation, molecular characterization, and potential use for strain identification.

PubMed

Ravin, Victor; Alatossava, Tapani

2003-05-01

A group of new insertion sequence (IS) elements, ISLdl2, ISLdl3, and ISLdl4, from Lactobacillus delbrueckii subsp. lactis ATCC 15808 was isolated, characterized, and used for strain identification together with ISLdl1, recently characterized as an L. delbrueckii IS element belonging to the ISL3 family. ISLdl2 was 1367 bp in size and had a 24 bp IR and an 8 bp DR. The single ORF of ISLdl2 encoded a protein of 392 aa similar to transposases of the IS256 family. ISLdl3 had a single ORF encoding a protein of 343 aa similar to transposases of the IS30 family. Finally, ISLdl4 had a single ORF encoding a protein of 406 aa and displayed homology to the transposases of the IS110 family. ISLdl4 was only slight different from ISL4 (Accession No. AY040213). ISLdl1, ISLdl2, and ISLdl4 were present in all of the 10 L. delbrueckii subsp. lactis and subsp. delbrueckii strains tested, as well as in three of the 11 L. delbrueckii subsp. bulgaricus strains tested. ISLdl3 was present only in four closely related strains of L. delbrueckii subsp. lactis. These IS elements were not observed in Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus helveticus, or Lactobacillus plantarum. A cluster of IS elements, ISLdl1, ISLdl2, ISLdl3, ISLdl4, and ISL6, was observed in L. delbrueckii subsp. lactis strain ATCC 15808. Within this cluster, ISLdl4 was inserted into ISLdl1 between the left IR and the start codon of ORF455, encoding a putative transposase. Most of the integration sites of the IS elements were strain-specific. We have observed that IS elements can migrate from one strain to another as integral parts of bacterial DNA by using phage LL-H as a vehicle. We demonstrate for the first time that inverse PCR and vectorette PCR methods with primers based on sequences of the IS elements could be used for identification of L. delbrueckii strains.

Safety of Bifidobacterium animalis subsp. lactis (B. lactis) strain BB-12®-supplemented yogurt in healthy children

PubMed Central

Tan, Tina P.; Ba, Zhaoyong; Sanders, Mary Ellen; D’Amico, Frank J.; Roberts, Robert F.; Smith, Keisha Herbin; Merenstein, Daniel J.

2016-01-01

Objectives Probiotics are live microorganisms that may provide health benefits to the individual when consumed in sufficient quantities. For studies conducted on health or disease endpoints on probiotics in the United States, the Food and Administration (FDA) has required those studies to be conducted as investigational new drugs. This phase I, double-blinded, randomized, controlled safety study represents the first requirement of this pathway. The purpose of the study was to determine the safety of Bifidobacterium animalis subsp. lactis (B. lactis) strain BB-12® (BB-12®)-supplemented yogurt when consumed by a generally healthy group of children. The secondary aim was to assess the effect of BB-12®-supplemented yogurt on the gut microbiota of the children. Methods Sixty children aged 1–5 years were randomly assigned to consume four ounces of either BB-12®-supplemented yogurt or non-supplemented control yogurt daily for 10 days. The primary outcome was to assess safety and tolerability, as determined by the number of reported adverse events. Results A total of 186 non-serious adverse events were reported, with no significant differences between the control and BB-12® groups. No significant changes due to probiotic treatment were observed in the gut microbiota of the study cohort. Conclusions BB-12®-supplemented yogurt is safe and well-tolerated when consumed by healthy children. This study will form the basis for future randomized clinical trials investigating the potential effects of BB-12®-supplemented yogurt in different disease states. PMID:28114246

Detection and Viability of Lactococcus lactis throughout Cheese Ripening

PubMed Central

Cocolin, Luca

2014-01-01

Recent evidences highlighted the presence of Lactococcus lactis during late cheese ripening. For this reason, the role of this microorganism, well known as dairy starter, should be reconsidered throughout cheese manufacturing and ripening. Thus, the main objective of this study was to develop a RT-qPCR protocol for the detection, quantification and determination of the viability of L. lactis in ripened cheese samples by direct analysis of microbial nucleic acids. Standard curves were constructed for the specific quantification of L. lactis in cheese matrices and good results in terms of selectivity, correlation coefficient and efficiency were obtained. Thirty-three ripened cheeses were analyzed and, on the basis of RNA analysis, twelve samples showed 106 to 108 CFU of L. lactis per gram of product, thirteen from 103 to 105 CFU/g, and in eight cheeses, L. lactis was not detected. Traditional plating on M17 medium led to loads ranging from 105 to 109 CFU/g, including the cheese samples where no L. lactis was found by RT-qPCR. From these cheeses, none of the colonies isolated on M17 medium was identified as L. lactis species. These data could be interpreted as a lack of selectivity of M17 medium where colony growth is not always related to lactococcal species. At the same time, the absence or low abundance of L. lactis isolates on M17 medium from cheese where L. lactis was detected by RT-qPCR support the hypothesis that L. lactis starter populations are mainly present in viable but not culturable state during ripening and, for this reason, culture-dependent methods have to be supplemented with direct analysis of cheese. PMID:25503474

Potential of functional strains, isolated from traditional Maasai milk, as starters for the production of fermented milks.

PubMed

Patrignani, Francesca; Lanciotti, Rosalba; Mathara, Julius Maina; Guerzoni, Maria Elisabetta; Holzapfel, Wilhelm H

2006-03-01

The purpose of this research was the evaluation of technological features and of the ability of functional LAB strains with desirable sensory characteristics, to produce fermented milk. Eight strains of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus paracasei and Lactococcus lactis, isolated from Maasai traditional fermented milk in Kenya and previously tested for their probiotic properties, were selected for this investigation. Technological features such as growth kinetics in fresh heat-treated whole milk medium and survival in the final product during storage at 4 degrees C, were studied. The strains Lb. acidophilus BFE 6,059, Lb. paracasei BFE 5,264 and Lc. lactis BFE 6,049 showed the best potential and were thus selected for use as starter cultures in further trials with the objective to improve their technological performance and to optimise the sensory features of fermented milk obtained. The effects of fat (F), non-fat milk solids (S) and fermentation temperature (T), modulated according to a Central Composite Design, on fermentation rates and viability losses during refrigerated storage of the chosen starters, and on product texture parameters, were studied. From the data analysis, it was possible to select optimum conditions for enhancing positive sensory traits of final products and for improving the survival of these potentially probiotic cultures.

Yeast Kluyveromyces lactis as host for expression of the bacterial lipase: cloning and adaptation of the new lipase gene from Serratia sp.

PubMed

Šiekštelė, Rimantas; Veteikytė, Aušra; Tvaska, Bronius; Matijošytė, Inga

2015-10-01

Many microbial lipases have been successfully expressed in yeasts, but not in industrially attractive Kluyveromyces lactis, which among other benefits can be cultivated on a medium supplemented with whey--cheap and easily available industrial waste. A new bacterial lipase from Serratia sp. was isolated and for the first time expressed into the yeast Kluyveromyces lactis by heterologous protein expression system based on a strong promoter of Kluyveromyces marxianus triosephosphate isomerase gene and signal peptide of Kluyveromyces marxianus endopolygalacturonase gene. In addition, the bacterial lipase gene was synthesized de novo by taking into account a codon usage bias optimal for K. lactis and was expressed into the yeast K. lactis also. Both resulting strains were characterized by high output level of the target protein secreted extracellularly. Secreted lipases were characterized for activity and stability.

Construction of upp deletion mutant strains of Lactobacillus casei and Lactococcus lactis based on counterselective system using temperature-sensitive plasmid.

PubMed

Song, Li; Cui, Hongyu; Tang, Lijie; Qiao, Xinyuan; Liu, Min; Jiang, Yanping; Cui, Wen; Li, Yijing

2014-07-01

Integration plasmids are often used in constructing chromosomal mutations, as it enables the alternation of genes at any location by integration or replacement. Food-grade integration vectors can integrate into the host genome without introducing any selectable markers or residual bases, and the recombination often happens in non-coding region. In this study we used the temperature-sensitive pWV01 replicon to construct 2 chloramphenicol-resistant integration plasmids (pGBHC32-upp) containing the uracil phosphoribosyl transferase (upp) gene as a counterselective marker for Lactobacillus casei (L. casei) ATCC393 and Lactococcus lactis (L. lactis) MG1363. We then ligated the designed homologous arms to the pGBHC32-upp plasmids to allow their integration to the bacterial chromosome, and selected upp deletion mutants of L. casei ATCC393 and L. lactis MG1363 in the presence of 5-fluorouracil (5-FU). Analysis of genetic stability, growth curve, carbon utilization and scanning electronic microscopy showed that, except for 5-FU resistance, there were no significant differences between the wild type and mutant lactic acid bacteria. The integration system and the upp deletion strains could be used in the insertion or deletion of genes at any location of the chromosome of both L. casei ATCC 393 and L. lactis MG1363, and the homologous recombination would not introduce any selectable markers or residual bases. These mutant strains can be further investigated for heterologous protein expression and construction of a live mucosal vaccine carrier. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification of Lactococcus-Specific Bacteriocins Produced by Lactococcal Isolates, and the Discovery of a Novel Bacteriocin, Lactococcin Z.

PubMed

Ishibashi, Naoki; Seto, Hiromi; Koga, Shoko; Zendo, Takeshi; Sonomoto, Kenji

2015-09-01

Lactic acid bacteria that produce Lactococcus-specific bacteriocins were isolated and identified as Lactococcus lactis from fresh corn or lettuce. Among them, four isolates were identified as lactococcin Q producers. Seven isolates showed antimicrobial activity against a lactococcin Q producer, L. lactis QU 4, as well as against nisin Z and lacticin Q producers belonging to L. lactis. Strain QU 7 was selected as a standard strain and showed no cross-immunity to lactococcin Q or other lactococcal bacteriocins. The bacteriocin produced by strain QU 7 was purified in three chromatographic steps, and its molecular mass was determined to be 5041.35 Da. The amino acid sequence analysis revealed that it is a novel class IId bacteriocin, referred to as lactococcin Z. It consisted of 45 amino acid residues. The lczA gene encoding the prepeptide of lactococcin Z showed homology to lactococcins A, B, and M. Thus, this report demonstrates a new example of Lactococcus-specific bacteriocins.

Technological characterization and survival of the exopolysaccharide-producing strain Lactobacillus delbrueckii subsp. lactis 193 and its bile-resistant derivative 193+ in simulated gastric and intestinal juices.

PubMed

Burns, Patricia; Vinderola, Gabriel; Reinheimer, Jorge; Cuesta, Isabel; de Los Reyes-Gavilán, Clara G; Ruas-Madiedo, Patricia

2011-08-01

The capacity of lactic acid bacteria to produce exopolysaccharides (EPS) conferring microorganisms a ropy phenotype could be an interesting feature from a technological point of view. Progressive adaptation to bile salts might render some lactobacilli able to overcome physiological gut barriers but could also modify functional properties of the strain, including the production of EPS. In this work some technological properties and the survival ability in simulated gastrointestinal conditions of Lactobacillus delbrueckii subsp. lactis 193, and Lb. delbrueckii subsp. lactis 193+, a strain with stable bile-resistant phenotype derived thereof, were characterized in milk in order to know whether the acquisition of resistance to bile could modify some characteristics of the microorganism. Both strains were able to grow and acidify milk similarly; however the production of ethanol increased at the expense of the aroma compound acetaldehyde in milk fermented by the strain 193+, with respect to milk fermented by the strain 193. Both microorganisms produced a heteropolysaccharide composed of glucose and galactose, and were able to increase the viscosity of fermented milks. In spite of the higher production yield of EPS by the bile-resistant strain 193+, it displayed a lower ability to increase viscosity than Lb. delbrueckii subsp. lactis 193. Milk increased survival in simulated gastric juice; the presence of bile improved adhesion to the intestinal cell line HT29-MTX in both strains. However, the acquisition of a stable resistance phenotype did not improve survival in simulated gastric and intestinal conditions or the adhesion to the intestinal cell line HT29-MTX. Thus, Lb. delbrueckii subsp. lactis 193 presents suitable technological properties for the manufacture of fermented dairy products; the acquisition of a stable bile-resistant phenotype modified some properties of the microorganism. This suggests that the possible use of bile-resistant derivative strains should be

Some Factors Influencing Acid Production by an Oxytetracycline-Resistant Strain of Streptococcus lactis1

PubMed Central

Mikolajcik, E. M.; Harper, W. J.; Gould, I. A.

1963-01-01

Induction of oxytetracycline resistance in a strain of Streptococcus lactis caused this organism to display reduced acid production, salt tolerance, pyruvate synthesis, growth at alkaline pH, and a loss in ability to produce ammonia from arginine. α-Ketoglutaric and oxaloacetic acids were found to accumulate in the growth medium of resistant cells, in contrast to none in the medium of susceptible cells. No free arginine could be detected in the intracellular fraction of resistant cells, but arginine was present in the intracellular fraction of susceptible cells and decreased in concentration upon the addition of oxytetracycline to the growth medium. Depressed acid production in milk by the oxytetracycline resistant strain is evidently a consequence of the inability of this organism to metabolize arginine effectively. PMID:14063784

[Characteristics and identification of bacteriocins produced by Lactococcus lactis subsp. lactis 194-K].

PubMed

Ustiugova, E A; Timofeeva, A V; Stoianova, L G; Netrusov, A I; Katrukha, G S

2012-01-01

The Lactococcus lactis subsp. lactis 194-K strain has been established to be able to produce two bacteriocins, one of which was identified as the known lantibiotic nisin A, and the other 194-D bacteriocin represents a polypeptide with a 2589-Da molecular mass and comprises 20 amino acid residues. Both bacteriocins were produced in varying proportions in all of the studied nutrient media, which support the growth of the producer. Depending on the cultivation medium, the nisin A content was 380- to 1123-fold lower in the 194-K stain culture fluid than that of the 194-D peptide. In comparision to to nisin A Bacteriocin 194-D possessed a wide range of antibacterial activity and suppressed the growth of both Gram-positive and Gram-negative bacteria. An optimal medium for 194-D bacteriocin synthesis was shown to be a fermentation medium which contained yeast extract, casein hydrolysate, and potassium phosphate. The biosynthesis ofbacteriocin 194-D by the 194-K strain in these media occurred parallel to producer growth, and its maximal accumulation in the culture fluid was observed at 14-20 h of the strain's growth.

Long-term administration of pDC-Stimulative Lactococcus lactis strain decelerates senescence and prolongs the lifespan of mice.

PubMed

Sugimura, Tetsu; Jounai, Kenta; Ohshio, Konomi; Suzuki, Hiroaki; Kirisako, Takayoshi; Sugihara, Yoshihiko; Fujiwara, Daisuke

2018-05-01

The decline in immune function caused by aging increases the risk of infectious diseases, tumorigeneses and chronic inflammation, resulting in accelerating senescence. We previously reported a lactic acid bacteria, Lactococcus lactis strain Plasma (synonym of Lactococcus lactis subsp. lactis JCM 5805, Lc-Plasma), that stimulates plasmacytoid dendritic cells (pDCs), which play a crucial role in phylaxis from viral infection. In this study, we investigated the anti-aging effects of long-term oral administration of Lc-Plasma in a senescence-accelerated mouse strain, SAMP6. Mice given Lc-Plasma showed a significant improvement in survival rate at 82 weeks and a decreased senescence score as compared with control mice throughout this study. Anatomic analysis at 82 weeks revealed that the frequency of altered hepatocellular foci was significantly lower, and the incidence of other pathological findings in the liver and lungs tended to be lower in Lc-Plasma mice than in control mice. Transcription level of the IL-1β gene in lungs also tended to be lower in Lc-Plasma mice. Furthermore, the thinning of skin and age-related decrease in muscle mass were also significantly suppressed in the Lc-Plasma group as compared with the control group. Consistent with these phenotypic features, pDCs activity was significantly higher in Lc-Plasma mice than in control mice. In conclusion, long-term administration of Lc-Plasma can decelerate senescence and prolong lifespan via maintenance of the immune system due to activation of pDCs. Copyright © 2018 Elsevier B.V. All rights reserved.

Expression of six peptidases from Lactobacillus helveticus in Lactococcus lactis.

PubMed

Luoma, S; Peltoniemi, K; Joutsjoki, V; Rantanen, T; Tamminen, M; Heikkinen, I; Palva, A

2001-03-01

For development of novel starter strains with improved proteolytic properties, the ability of Lactococcus lactis to produce Lactobacillus helveticus aminopeptidase N (PepN), aminopeptidase C (PepC), X-prolyl dipeptidyl aminopeptidase (PepX), proline iminopeptidase (PepI), prolinase (PepR), and dipeptidase (PepD) was studied by introducing the genes encoding these enzymes into L. lactis MG1363 and its derivatives. According to Northern analyses and enzyme activity measurements, the L. helveticus aminopeptidase genes pepN, pepC, and pepX are expressed under the control of their own promoters in L. lactis. The highest expression level, using a low-copy-number vector, was obtained with the L. helveticus pepN gene, which resulted in a 25-fold increase in PepN activity compared to that of wild-type L. lactis. The L. helveticus pepI gene, residing as a third gene in an operon in its host, was expressed in L. lactis under the control of the L. helveticus pepX promoter. The genetic background of the L. lactis derivatives tested did not affect the expression level of any of the L. helveticus peptidases studied. However, the growth medium used affected both the recombinant peptidase profiles in transformant strains and the resident peptidase activities. The levels of expression of the L. helveticus pepD and pepR clones under the control of their own promoters were below the detection limit in L. lactis. However, substantial amounts of recombinant pepD and PepR activities were obtained in L. lactis when pepD and pepR were expressed under the control of the inducible lactococcal nisA promoter at an optimized nisin concentration.

Expression of Six Peptidases from Lactobacillus helveticus in Lactococcus lactis

PubMed Central

Luoma, Susanna; Peltoniemi, Kirsi; Joutsjoki, Vesa; Rantanen, Terhi; Tamminen, Marja; Heikkinen, Inka; Palva, Airi

2001-01-01

For development of novel starter strains with improved proteolytic properties, the ability of Lactococcus lactis to produce Lactobacillus helveticus aminopeptidase N (PepN), aminopeptidase C (PepC), X-prolyl dipeptidyl aminopeptidase (PepX), proline iminopeptidase (PepI), prolinase (PepR), and dipeptidase (PepD) was studied by introducing the genes encoding these enzymes into L. lactis MG1363 and its derivatives. According to Northern analyses and enzyme activity measurements, the L. helveticus aminopeptidase genes pepN, pepC, and pepX are expressed under the control of their own promoters in L. lactis. The highest expression level, using a low-copy-number vector, was obtained with the L. helveticus pepN gene, which resulted in a 25-fold increase in PepN activity compared to that of wild-type L. lactis. The L. helveticus pepI gene, residing as a third gene in an operon in its host, was expressed in L. lactis under the control of the L. helveticus pepX promoter. The genetic background of the L. lactis derivatives tested did not affect the expression level of any of the L. helveticus peptidases studied. However, the growth medium used affected both the recombinant peptidase profiles in transformant strains and the resident peptidase activities. The levels of expression of the L. helveticus pepD and pepR clones under the control of their own promoters were below the detection limit in L. lactis. However, substantial amounts of recombinant pepD and PepR activities were obtained in L. lactis when pepD and pepR were expressed under the control of the inducible lactococcal nisA promoter at an optimized nisin concentration. PMID:11229915

The Prophylactic Effect of Probiotic Enterococcus lactis IW5 against Different Human Cancer Cells

PubMed Central

Nami, Yousef; Haghshenas, Babak; Haghshenas, Minoo; Abdullah, Norhafizah; Yari Khosroushahi, Ahmad

2015-01-01

Enterococcus lactis IW5 was obtained from human gut and the potential probiotic characteristics of this organism were then evaluated. Results showed that this strain was highly resistant to low pH and high bile salt and adhered strongly to Caco-2 human epithelial colorectal cell lines. The supernatant of E. lactis IW5 strongly inhibited the growth of several pathogenic bacteria and decreased the viability of different cancer cells, such as HeLa, MCF-7, AGS, HT-29, and Caco-2. Conversely, E. lactis IW5 did not inhibit the viability of normal FHs-74 cells. This strain did not generate toxic enzymes, including β-glucosidase, β-glucuronidase, and N-acetyl-β-glucosaminidase and was highly susceptible to ampicillin, gentamycin, penicillin, vancomycin, clindamycin, sulfamethoxazol, and chloramphenicol but resistant to erythromycin and tetracyclin. This study provided evidence for the effect of E. lactis IW5 on cancer cells. Therefore, E. lactis IW5, as a bioactive therapeutics, should be subjected to other relevant tests to verify the therapeutic suitability of this strain for clinical applications. PMID:26635778

Licheniocin 50.2 and Bacteriocins from Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 Inhibit Biofilms of Coagulase Negative Staphylococci and Listeria monocytogenes Clinical Isolates.

PubMed

Cirkovic, Ivana; Bozic, Dragana D; Draganic, Veselin; Lozo, Jelena; Beric, Tanja; Kojic, Milan; Arsic, Biljana; Garalejic, Eliana; Djukic, Slobodanka; Stankovic, Slavisa

2016-01-01

Coagulase negative staphylococci (CoNS) and Listeria monocytogenes have important roles in pathogenesis of various genital tract infections and fatal foetomaternal infections, respectively. The aim of our study was to investigate the inhibitory effects of two novel bacteriocins on biofilms of CoNS and L. monocytogenes genital isolates. The effects of licheniocin 50.2 from Bacillus licheniformis VPS50.2 and crude extract of bacteriocins produced by Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 (BGBU1-4 crude extract) were evaluated on biofilm formation and formed biofilms of eight CoNS (four S. epidermidis, two S. hominis, one S. lugdunensis and one S. haemolyticus) and 12 L. monocytogenes genital isolates. Licheniocin 50.2 and BGBU1-4 crude extract inhibited the growth of both CoNS and L. monocytogenes isolates, with MIC values in the range between 200-400 AU/ml for licheniocin 50.2 and 400-3200 AU/ml for BGBU1-4 crude extract. Subinhibitory concentrations (1/2 × and 1/4 × MIC) of licheniocin 50.2 inhibited biofilm formation by all CoNS isolates (p < 0.05, respectively), while BGBU1-4 crude extract inhibited biofilm formation by all L. monocytogenes isolates (p < 0.01 and p < 0.05, respectively). Both bacteriocins in concentrations of 100 AU/mL and 200 AU/mL reduced the amount of 24 h old CoNS and L. monocytogenes biofilms (p < 0.05, p < 0.01, p < 0.001). This study suggests that novel bacteriocins have potential to be used for genital application, to prevent biofilm formation and/or to eradicate formed biofilms, and consequently reduce genital and neonatal infections by CoNS and L. monocytogenes.

Structural studies of the cell wall polysaccharide from Lactococcus lactis UC509.9.

PubMed

Vinogradov, Evgeny; Sadovskaya, Irina; Grard, Thierry; Murphy, James; Mahony, Jennifer; Chapot-Chartier, Marie-Pierre; van Sinderen, Douwe

2018-05-22

Lactococcus lactis is the most widely utilised starter bacterial species in dairy fermentations. The L. lactis cell envelope contains polysaccharides, which, among other known functions, serve as bacteriophage receptors. Our previous studies have highlighted the structural diversity of these so-called cell wall polysaccharides (CWPSs) among L. lactis strains that could account for the narrow host range of most lactococcal bacteriophages. In the present work, we studied the CWPS of L. lactis strain UC509.9, an Irish dairy starter strain that is host to the temperate and well-characterized P335-type phage Tuc2009. The UC509.9 CWPS structure was analyzed by methylation, deacetylation/deamination, Smith degradation and 2D NMR spectroscopy. The CWPS consists of a linear backbone composed of a tetrasaccharide repeat unit, partially substituted with a branched phosphorylated oligosaccharide having a common trisaccharide and three non-stoichiometric substitutions. Copyright © 2018 Elsevier Ltd. All rights reserved.

Casein Hydrolysates by Lactobacillus brevis and Lactococcus lactis Proteases: Peptide Profile Discriminates Strain-Dependent Enzyme Specificity.

PubMed

Bounouala, Fatima Zohra; Roudj, Salima; Karam, Nour-Eddine; Recio, Isidra; Miralles, Beatriz

2017-10-25

Casein from ovine and bovine milk were hydrolyzed with two extracellular protease preparations from Lactobacillus brevis and Lactococcus lactis. The hydrolysates were analyzed by HPLC-MS/MS for peptide identification. A strain-dependent peptide profile could be observed, regardless of the casein origin, and the specificity of these two proteases could be computationally ascribed. The cleavage pattern yielding phenylalanine, leucine, or tyrosine at C-terminal appeared both at L. lactis and Lb. brevis hydrolysates. However, the cleavage C-terminal to lysine was favored with Lb. brevis protease. The hydrolysates showed ACE-inhibitory activity with IC 50 in the 16-70 μg/mL range. Ovine casein hydrolysates yielded greater ACE-inhibitory activity. Previously described antihypertensive and opioid peptides were found in these ovine and bovine casein hydrolysates and prediction of the antihypertensive activity of the sequences based on quantitative structure and activity relationship (QSAR) was performed. This approach might represent a useful classification tool regarding health-related properties prior to further purification.

A Zn-Dependent Metallopeptidase Is Responsible for Sensitivity to LsbB, a Class II Leaderless Bacteriocin of Lactococcus lactis subsp. lactis BGMN1-5

PubMed Central

Uzelac, Gordana; Lozo, Jelena; Aleksandrzak-Piekarczyk, Tamara; Gabrielsen, Christina; Kristensen, Tom; Nes, Ingolf F.; Diep, Dzung B.; Topisirovic, Ljubisa

2013-01-01

Lactococcus lactis subsp. lactis BGMN1-5 produces a leaderless class II bacteriocin called LsbB. To identify the receptor for LsbB, a cosmid library of the LsbB-sensitive strain BGMN1-596 was constructed. About 150 cosmid clones were individually isolated and transferred to LsbB-resistant mutants of BGMN1-596. Cosmid pAZILcos/MN2, carrying a 40-kb insert, was found to restore LsbB sensitivity in LsbB-resistant mutants. Further subcloning revealed that a 1.9-kb fragment, containing only one open reading frame, was sufficient to restore sensitivity. The fragment contains the gene yvjB coding for a Zn-dependent membrane-bound metallopeptidase, suggesting that this gene may serve as the receptor for LsbB. Further support for this notion derives from several independent experiments: (i) whole-genome sequencing confirmed that all LsbB-resistant mutants contain mutations in yvjB; (ii) disruption of yvjB by direct gene knockout rendered sensitive strains BGMN1-596 and IL1403 resistant to LsbB; and (iii) most compellingly, heterologous expression of yvjB in naturally resistant strains of other species, such as Lactobacillus paracasei and Enterococcus faecalis, also rendered them sensitive to the bacteriocin. To our knowledge, this is the first time a membrane-bound peptidase gene has been shown to be involved in bacteriocin sensitivity in target cells. We also demonstrated a novel successful approach for identifying bacteriocin receptors. PMID:24123824

Safety of Bifidobacterium animalis subsp. lactis (B. lactis) strain BB-12-supplemented yogurt in healthy adults on antibiotics: a phase I safety study

PubMed Central

Merenstein, Daniel J; Tan, Tina P; Molokin, Aleksey; Smith, Keisha Herbin; Roberts, Robert F; Shara, Nawar M; Mete, Mihriye; Sanders, Mary Ellen; Solano-Aguilar, Gloria

2015-01-01

Probiotics are live microorganisms that, when administered in sufficient doses, provide health benefits on the host. The United States Food and Drug Administration (FDA) requires phase I safety studies for probiotics when the intended use of the product is as a drug. The purpose of the study was to determine the safety of Bifidobacterium animalis subsp lactis (B. lactis) strain BB-12 (BB-12)-supplemented yogurt when consumed by a generally healthy group of adults who were prescribed a 10-day course of antibiotics for a respiratory infection. Secondary aims were to assess the ability of BB-12 to affect the expression of whole blood immune markers associated with cell activation and inflammatory response. A phase I, double-blinded, randomized controlled study was conducted in compliance with FDA guidelines for an Investigational New Drug (IND). Forty participants were randomly assigned to consume 4 ounces of either BB-12 -supplemented yogurt or non-supplemented control yogurt daily for 10 d. The primary outcome was to assess safety and tolerability, assessed by the number of reported adverse events. A total of 165 non-serious adverse events were reported, with no differences between the control and BB-12 groups. When compared to the control group, B lactis fecal levels were modestly higher in the BB-12-supplemented group. In a small subset of patients, changes in whole blood expression of genes associated with regulation and activation of immune cells were detected in the BB-12-supplemented group. BB-12-supplemented yogurt is safe and well tolerated when consumed by healthy adults concurrently taking antibiotics. This study will form the basis for future randomized clinical trials investigating the potential immunomodulatory effects of BB-12-supplemented yogurt in a variety of disease states. PMID:25569274

Safety of Bifidobacterium animalis subsp. lactis (B. lactis) strain BB-12-supplemented yogurt in healthy adults on antibiotics: a phase I safety study.

PubMed

Merenstein, Daniel J; Tan, Tina P; Molokin, Aleksey; Smith, Keisha Herbin; Roberts, Robert F; Shara, Nawar M; Mete, Mihriye; Sanders, Mary Ellen; Solano-Aguilar, Gloria

2015-01-01

Probiotics are live microorganisms that, when administered in sufficient doses, provide health benefits on the host. The United States Food and Drug Administration (FDA) requires phase I safety studies for probiotics when the intended use of the product is as a drug. The purpose of the study was to determine the safety of Bifidobacterium animalis subsp lactis (B. lactis) strain BB-12 (BB-12)-supplemented yogurt when consumed by a generally healthy group of adults who were prescribed a 10-day course of antibiotics for a respiratory infection. Secondary aims were to assess the ability of BB-12 to affect the expression of whole blood immune markers associated with cell activation and inflammatory response. A phase I, double-blinded, randomized controlled study was conducted in compliance with FDA guidelines for an Investigational New Drug (IND). Forty participants were randomly assigned to consume 4 ounces of either BB-12 -supplemented yogurt or non-supplemented control yogurt daily for 10 d. The primary outcome was to assess safety and tolerability, assessed by the number of reported adverse events. A total of 165 non-serious adverse events were reported, with no differences between the control and BB-12 groups. When compared to the control group, B lactis fecal levels were modestly higher in the BB-12-supplemented group. In a small subset of patients, changes in whole blood expression of genes associated with regulation and activation of immune cells were detected in the BB-12-supplemented group. BB-12-supplemented yogurt is safe and well tolerated when consumed by healthy adults concurrently taking antibiotics. This study will form the basis for future randomized clinical trials investigating the potential immunomodulatory effects of BB-12-supplemented yogurt in a variety of disease states.

Licheniocin 50.2 and Bacteriocins from Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 Inhibit Biofilms of Coagulase Negative Staphylococci and Listeria monocytogenes Clinical Isolates

PubMed Central

Draganic, Veselin; Lozo, Jelena; Beric, Tanja; Kojic, Milan; Arsic, Biljana; Garalejic, Eliana; Djukic, Slobodanka; Stankovic, Slavisa

2016-01-01

Background Coagulase negative staphylococci (CoNS) and Listeria monocytogenes have important roles in pathogenesis of various genital tract infections and fatal foetomaternal infections, respectively. The aim of our study was to investigate the inhibitory effects of two novel bacteriocins on biofilms of CoNS and L. monocytogenes genital isolates. Methods The effects of licheniocin 50.2 from Bacillus licheniformis VPS50.2 and crude extract of bacteriocins produced by Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 (BGBU1-4 crude extract) were evaluated on biofilm formation and formed biofilms of eight CoNS (four S. epidermidis, two S. hominis, one S. lugdunensis and one S. haemolyticus) and 12 L. monocytogenes genital isolates. Results Licheniocin 50.2 and BGBU1-4 crude extract inhibited the growth of both CoNS and L. monocytogenes isolates, with MIC values in the range between 200–400 AU/ml for licheniocin 50.2 and 400–3200 AU/ml for BGBU1-4 crude extract. Subinhibitory concentrations (1/2 × and 1/4 × MIC) of licheniocin 50.2 inhibited biofilm formation by all CoNS isolates (p < 0.05, respectively), while BGBU1-4 crude extract inhibited biofilm formation by all L. monocytogenes isolates (p < 0.01 and p < 0.05, respectively). Both bacteriocins in concentrations of 100 AU/mL and 200 AU/mL reduced the amount of 24 h old CoNS and L. monocytogenes biofilms (p < 0.05, p < 0.01, p < 0.001). Conclusions This study suggests that novel bacteriocins have potential to be used for genital application, to prevent biofilm formation and/or to eradicate formed biofilms, and consequently reduce genital and neonatal infections by CoNS and L. monocytogenes. PMID:27930711

Lacticin LC14, a new bacteriocin produced by Lactococcus lactis BMG6.14: isolation, purification and partial characterization.

PubMed

Lasta, Samar; Ouzari, Hadda; Andreotti, Nicolas; Fajloun, Ziad; Mansuelle, Pascal; Boudabous, Abdellatif; Sampieri, Francois; Sabatier, Jean Marc

2012-08-01

A new bacteriocin, lacticin LC14, produced by Lactococcus lactis BMG6.14, was isolated and characterized. It was purified to homogeneity from overnight broth culture by ammonium sulfate precipitation, Sep-Pak chromatography, and two steps of reversed-phase HPLC. Lacticin LC14 showed bactericidal-type antimicrobial activity against several lactic acid bacteria and pathogenic strains including Listeria monocytogenes. It was inactivated by proteinase K and pronase E, but was resistant to papain, lysozyme, lipase and catalase. Lacticin LC14 was heat resistant, stable over a wide range of pH (2-10) and after treatment by solvents and detergents. Its N-terminal end was found unreactive towards Edman sequencing. Based on MALDI-TOF mass spectrometry, its molecular mass was 3333.7 Da. LC14 amino acid composition revealed a high proportion of hydrophobic residues, but no modified ones. LC14 may be able to challenge other well known other bacteriocins in probiotic and therapeutic applications.

Analyses of the probiotic property and stress resistance-related genes of Lactococcus lactis subsp. lactis NCDO 2118 through comparative genomics and in vitro assays

PubMed Central

Saraiva, Tessália D. L.; Silva, Wanderson M.; Pereira, Ulisses P.; Campos, Bruno C.; Benevides, Leandro J.; Rocha, Flávia S.; Figueiredo, Henrique C. P.; Azevedo, Vasco; Soares, Siomar C.

2017-01-01

Lactococcus lactis subsp. lactis NCDO 2118 was recently reported to alleviate colitis symptoms via its anti-inflammatory and immunomodulatory activities, which are exerted by exported proteins that are not produced by L. lactis subsp. lactis IL1403. Here, we used in vitro and in silico approaches to characterize the genomic structure, the safety aspects, and the immunomodulatory activity of this strain. Through comparative genomics, we identified genomic islands, phage regions, bile salt and acid stress resistance genes, bacteriocins, adhesion-related and antibiotic resistance genes, and genes encoding proteins that are putatively secreted, expressed in vitro and absent from IL1403. The high degree of similarity between all Lactococcus suggests that the Symbiotic Islands commonly shared by both NCDO 2118 and KF147 may be responsible for their close relationship and their adaptation to plants. The predicted bacteriocins may play an important role against the invasion of competing strains. The genes related to the acid and bile salt stresses may play important roles in gastrointestinal tract survival, whereas the adhesion proteins are important for persistence in the gut, culminating in the competitive exclusion of other bacteria. Finally, the five secreted and expressed proteins may be important targets for studies of new anti-inflammatory and immunomodulatory proteins. Altogether, the analyses performed here highlight the potential use of this strain as a target for the future development of probiotic foods. PMID:28384209

L+-lactic acid production from starch by a novel amylolytic Lactococcus lactis subsp. lactis B84.

PubMed

Petrov, Kaloyan; Urshev, Zoltan; Petrova, Penka

2008-06-01

A new Lactococcus lactis subsp. lactis B84, capable of utilizing starch as a sole carbon source and producing L(+)-lactate, was isolated from spontaneously fermented rye sourdough. Aiming at maximum lactic acid productivity, the components of the media and the cultivation conditions were varied. In MRS-starch medium (with absence of yeast and meat extracts), at 33 degrees C, agitation 200 rpm and pH 6.0 for 6 days complete starch hydrolysis occurred and 5.5 gl(-1) lactic acid were produced from 18 gl(-1) starch. The identification of strain B84 was based on genetic criteria. Amplified ribosomal DNA restriction analysis (ARDRA), PCR with species-specific primers and sequencing of the 16S rDNA proved its species affiliation. Four genes for enzymes, involved in starch degradation were detected in B84 genome: amyL, amyY, glgP and apu, coding cytoplasmic and extracellular alpha-amylases, glycogen phosphorylase and amylopullulanase, respectively. Reverse transcription PCR experiments showed that both genes, encoding alpha-amylases (amyL and amyY) were expressed into mRNAs, whereas apu and glgP were not. Amylase activity assay was performed at different pH and temperatures. The cell-bond amylase proved to be the key enzyme, involved in the starch hydrolysis with maximum activity at 45 degrees C and pH 5.4.

Isolation, biochemical characterization, and cloning of a bacteriocin from the poultry-associated Staphylococcus aureus strain CH-91.

PubMed

Wladyka, Benedykt; Wielebska, Katarzyna; Wloka, Marcin; Bochenska, Oliwia; Dubin, Grzegorz; Dubin, Adam; Mak, Pawel

2013-08-01

Staphylococcus aureus strain CH-91, isolated from a broiler chicken with atopic dermatitis, has a highly proteolytic phenotype that is correlated with the disease. We describe the isolation and biochemical and molecular characterization of the AI-type lantibiotic BacCH91 from S. aureus CH-91 culture medium. The bacteriocin was purified using a three-stage procedure comprising precipitation with ammonium sulfate, extraction with organic solvents, and reversed-phase HPLC. The BacCH91 peptide is thermostable and highly resistant to cleavage by both prokaryotic and eukaryotic peptidases. The MIC for the Gram-positive bacteria ranged from 2.5 nM for Microococcus luteus through 1.3-6.0 μM for staphylococcal strains up to more than 100 μM for Lactococcus lactis. BacCH91 was ineffective against the Gram-negative strains tested at the maximal concentration (100 μM). The amino acid sequence of BacCH91 is similar to that of epidermin and gallidermin. The encoding gene (bacCH91) occurred in two allelic variants distinguishable in the restriction fragment length polymorphism assay. Variant I, identified in S. aureus CH-91, dominated in S. aureus strains of poultry origin, although strains with variant II were also identified in this group. S. aureus strains of human origin were characterized exclusively by variant II.

Safety evaluation of Lactobacillus delbrueckii subsp. lactis UO 004, a probiotic bacterium.

PubMed

Fernández, M Fernanda; Boris, Soledad; Barbés, Covadonga

2005-03-01

Lactobacillus delbrueckii subsp. lactis UO 004 was evaluated for its use as a potential probiotic from a safety point of view. The strain did not exhibit mucinolytic or other enzymatic activities that might be detrimental, such as those involving glycosidases (beta-D-glucosaminidase or alpha-D-galactosidase) or arylamidases (factor Xa and quimotrypsin-like activities), frequently present in Lactobacillus strains isolated from patients with endocarditis, although it was able to express protein Ca and kallikrein-like activities. On the other hand, the presence of the strain did not interfere with the growth of certain species of normal intestinal microbiota, such as Enterococcus fecalis, Escherichia coli, Bifidobacterium bifidum or Bacteroides fragilis. Moreover, the potential probiotic strain UO 004 is sensitive to antibiotics with transmissible resistance mechanisms in Lactobacillus such as chloramphenicol, erythromycin, tetracycline and vancomycin. In addition, strain L. delbrueckii UO 004 was not able to translocate towards the intestinal barrier of mice or produce changes in their activity or general health status.

Introduction of Peptidase Genes from Lactobacillus delbrueckii subsp. lactis into Lactococcus lactis and Controlled Expression

PubMed Central

Wegmann, U.; Klein, J. R.; Drumm, I.; Kuipers, O. P.; Henrich, B.

1999-01-01

Peptidases PepI, PepL, PepW, and PepG from Lactobacillus delbrueckii subsp. lactis, which have no counterparts in Lactococcus lactis, and peptidase PepQ were examined to determine their potential to confer new peptidolytic properties to lactococci. Controllable expression of the corresponding genes (pep genes) was achieved by constructing translational fusions with the promoter of the nisA gene (PnisA). A suitable host strain, UKLc10, was constructed by chromosomal integration of the genes encoding the NisRK two-component system into the fivefold peptidase-deficient mutant IM16 of L. lactis. Recombinants of this strain were used to analyze growth, peptidase activities, peptide utilization, and intracellular protein cleavage products. After nisin induction of PnisA::pep fusions, all of the peptidases were visible as distinct bands in protein gels. Despite the fact that identical transcription and translation signals were used to express the pep genes, the relative amounts of individual peptidases varied considerably. All of the peptidases exhibited activities in extracts of recombinant UKLc10 clones, but only PepL and PepG allowed the clones to utilize specific peptide substrates as sources of essential amino acids. In milk medium, induction of pepG and induction of pepW resulted in growth acceleration. The activities of all five peptidases during growth in milk medium were revealed by high-performance liquid chromatography analyses of intracellular amino acid and peptide pools. PMID:10543778

Sequencing and Transcriptional Analysis of the Biosynthesis Gene Cluster of Putrescine-Producing Lactococcus lactis ▿ †

PubMed Central

Ladero, Victor; Rattray, Fergal P.; Mayo, Baltasar; Martín, María Cruz; Fernández, María; Alvarez, Miguel A.

2011-01-01

Lactococcus lactis is a prokaryotic microorganism with great importance as a culture starter and has become the model species among the lactic acid bacteria. The long and safe history of use of L. lactis in dairy fermentations has resulted in the classification of this species as GRAS (General Regarded As Safe) or QPS (Qualified Presumption of Safety). However, our group has identified several strains of L. lactis subsp. lactis and L. lactis subsp. cremoris that are able to produce putrescine from agmatine via the agmatine deiminase (AGDI) pathway. Putrescine is a biogenic amine that confers undesirable flavor characteristics and may even have toxic effects. The AGDI cluster of L. lactis is composed of a putative regulatory gene, aguR, followed by the genes (aguB, aguD, aguA, and aguC) encoding the catabolic enzymes. These genes are transcribed as an operon that is induced in the presence of agmatine. In some strains, an insertion (IS) element interrupts the transcription of the cluster, which results in a non-putrescine-producing phenotype. Based on this knowledge, a PCR-based test was developed in order to differentiate nonproducing L. lactis strains from those with a functional AGDI cluster. The analysis of the AGDI cluster and their flanking regions revealed that the capacity to produce putrescine via the AGDI pathway could be a specific characteristic that was lost during the adaptation to the milk environment by a process of reductive genome evolution. PMID:21803900

Fermentation of protopanaxadiol type ginsenosides (PD) with probiotic Bifidobacterium lactis and Lactobacillus rhamnosus.

PubMed

Tan, Joanne Sh; Yeo, Chia-Rou; Popovich, David G

2017-07-01

Ginsenosides are believed to be the principal components behind the pharmacological actions of ginseng, and their bioactive properties are closely related to the type, position, and number of sugar moieties attached to the aglycone; thus, modification of the sugar chains may markedly change their biological activities. In this study, major protopanaxadiol type ginsenosides (PD) Rb1, Rc, and Rb2 were isolated from Panax ginseng and were transformed using two probiotic strains namely Bifidobacterium lactis Bi-07 and Lactobacillus rhamnosus HN001 to obtain specific deglycosylated ginsenosides. It was demonstrated that B. lactis transformed ginsenosides Rb1, Rc, and Rb2 to Rd within 1 h of fermentation and rare ginsenoside F2 by the conversion of Rd after 12-h fermentation. The maximum Rd concentration was 147.52 ± 1.45 μg/mL after 48-h fermentation as compared to 45.85 ± 0.71 μg/mL before fermentation. In contrast, L. rhamnosus transformed Rb1, Rc, and Rb2 into Rd as the final metabolite after 72-h fermentation. B. lactis displayed significantly (p < 0.05) higher β-glucosidase activity against p-nitrophenyl-β-glucopyranoside than L. rhamnosus and higher bioconversion efficiency during fermentation. The present study suggests that the fermentation of major PD type ginsenosides with B. lactis Bi-07 may serve as an effective means to afford bioactive deglycosylated ginsenosides and to create novel ginsenoside extracts.

In Vitro Characterization of Lactic Acid Bacteria Isolated from Bovine Milk as Potential Probiotic Strains to Prevent Bovine Mastitis.

PubMed

Pellegrino, Matías S; Frola, Ignacio D; Natanael, Berardo; Gobelli, Dino; Nader-Macias, María E F; Bogni, Cristina I

2018-01-02

Bovine mastitis causes economic losses on dairy farms worldwide. Lactic acid bacteria (LAB) in animal health are an alternative tool to avoid antibiotic therapy on the prevention of bovine mastitis. In previous studies, 12 LAB isolated from bovine milk were selected taking into account some of the following characteristics: hydrophobicity, auto aggregative capability, inhibition of indicator pathogens, hydrogen peroxide, and capsular polysaccharide production. These LAB were considered because of their beneficial properties. In this work, we also analyzed the antimicrobial activity and the co-aggregation against mastitis causing bacteria, auto-inhibition, adhesion to bovine teat canal epithelial cells (BTCEC), and growth kinetic curves for the 12 LAB. Two of them, Lactococcus lactis subsp. lactis CRL 1655 and Lactobacillus perolens CRL 1724, were selected because they had an interesting pattern of adhesion to BTEC, the inhibition of pathogens and the co-aggregation with the 100% of the assayed pathogens. They showed a predictable difference in the PFGE genomic pattern bands. The kinetic growth of these two strains was similar between them and with the rest of the assayed LAB. The strains selected in the present study showed indispensable characteristics for their inclusion in a probiotic formulation to be used at dry-off period for the prevention of bovine mastitis.

Technological properties of bacteriocin-producing lactic acid bacteria isolated from Pico cheese an artisanal cow's milk cheese.

PubMed

Ribeiro, S C; Coelho, M C; Todorov, S D; Franco, B D G M; Dapkevicius, M L E; Silva, C C G

2014-03-01

Evaluate technologically relevant properties from bacteriocin-producing strains to use as starter/adjunct cultures in cheese making. Eight isolates obtained from Pico cheese produced in Azores (Portugal) were found to produce bacteriocins against Listeria monocytogenes and three isolates against Clostridium perfringens. They were identified as Lactococcus lactis and Enterococcus faecalis and submitted to technological tests: growth at different conditions of temperature and salt, acid production, proteolysis, lipolysis, coexistence, enzymatic profile and autolytic capacity. Safety evaluation was performed by evaluating haemolytic, gelatinase and DNase activity, resistance to antibiotics and the presence of virulence genes. Some isolates presented good technological features such as high autolytic activity, acid and diacetyl production. Lactococcus lactis was negative for all virulence genes tested and inhibit the growth of all Lactic acid bacteria (LAB) isolates. Enterococci were positive for the presence of some virulence genes, but none of the isolates were classified as resistant to important antibiotics. The bacteriocin-producing Lc. lactis present good potential for application in food as adjunct culture in cheese production. The study also reveals good technological features for some Enterococcus isolates. Bacteriocin-producing strains presented important technological properties to be exploited as new adjunct culture for the dairy industry, influencing flavour development and improve safety. © 2013 The Society for Applied Microbiology.

Lactolisterin BU, a Novel Class II Broad-Spectrum Bacteriocin from Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4.

PubMed

Lozo, Jelena; Mirkovic, Nemanja; O'Connor, Paula M; Malesevic, Milka; Miljkovic, Marija; Polovic, Natalija; Jovcic, Branko; Cotter, Paul D; Kojic, Milan

2017-11-01

Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 produces a novel bacteriocin, lactolisterin BU, with strong antimicrobial activity against many species of Gram-positive bacteria, including important food spoilage and foodborne pathogens, such as Listeria monocytogenes , Staphylococcus aureus , Bacillus spp., and streptococci. Lactolisterin BU was extracted from the cell surface of BGBU1-4 by 2-propanol and purified to homogeneity by C 18 solid-phase extraction and reversed-phase high-performance liquid chromatography. The molecular mass of the purified lactolisterin BU was 5,160.94 Da, and an internal fragment, AVSWAWQH, as determined by N-terminal sequencing, showed low-level similarity to existing antimicrobial peptides. Curing and transformation experiments revealed the presence of a corresponding bacteriocin operon on the smallest plasmid, pBU6 (6.2 kb), of strain BGBU1-4. Analysis of the bacteriocin operon revealed a leaderless bacteriocin of 43 amino acids that exhibited similarity to bacteriocin BHT-B (63%) from Streptococcus ratti , a bacteriocin with analogy to aureocin A. IMPORTANCE Lactolisterin BU, a broad-spectrum leaderless bacteriocin produced by L. lactis subsp. lactis bv. diacetylactis BGBU1-4, expresses strong antimicrobial activity against food spoilage and foodborne pathogens, such as Listeria monocytogenes , Staphylococcus aureus , Bacillus spp., and streptococci. Lactolisterin BU showed the highest similarity to aureocin-like bacteriocins produced by different bacteria. The operon for synthesis is located on the smallest plasmid, pBU6 (6.2 kb), of strain BGBU1-4, indicating possible horizontal transfer among producers. Copyright © 2017 American Society for Microbiology.

Lactolisterin BU, a Novel Class II Broad-Spectrum Bacteriocin from Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4

PubMed Central

Lozo, Jelena; Mirkovic, Nemanja; O'Connor, Paula M.; Malesevic, Milka; Miljkovic, Marija; Polovic, Natalija; Cotter, Paul D.

2017-01-01

ABSTRACT Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 produces a novel bacteriocin, lactolisterin BU, with strong antimicrobial activity against many species of Gram-positive bacteria, including important food spoilage and foodborne pathogens, such as Listeria monocytogenes, Staphylococcus aureus, Bacillus spp., and streptococci. Lactolisterin BU was extracted from the cell surface of BGBU1-4 by 2-propanol and purified to homogeneity by C18 solid-phase extraction and reversed-phase high-performance liquid chromatography. The molecular mass of the purified lactolisterin BU was 5,160.94 Da, and an internal fragment, AVSWAWQH, as determined by N-terminal sequencing, showed low-level similarity to existing antimicrobial peptides. Curing and transformation experiments revealed the presence of a corresponding bacteriocin operon on the smallest plasmid, pBU6 (6.2 kb), of strain BGBU1-4. Analysis of the bacteriocin operon revealed a leaderless bacteriocin of 43 amino acids that exhibited similarity to bacteriocin BHT-B (63%) from Streptococcus ratti, a bacteriocin with analogy to aureocin A. IMPORTANCE Lactolisterin BU, a broad-spectrum leaderless bacteriocin produced by L. lactis subsp. lactis bv. diacetylactis BGBU1-4, expresses strong antimicrobial activity against food spoilage and foodborne pathogens, such as Listeria monocytogenes, Staphylococcus aureus, Bacillus spp., and streptococci. Lactolisterin BU showed the highest similarity to aureocin-like bacteriocins produced by different bacteria. The operon for synthesis is located on the smallest plasmid, pBU6 (6.2 kb), of strain BGBU1-4, indicating possible horizontal transfer among producers. PMID:28842543

Addition to thermized milk of Lactococcus lactis subsp. cremoris M104, a wild, novel nisin a-producing strain, replaces the natural antilisterial activity of the autochthonous raw milk microbiota reduced by thermization.

PubMed

Lianou, Alexandra; Samelis, John

2014-08-01

Recent research has shown that mild milk thermization treatments routinely used in traditional Greek cheese production are efficient to inactivate Listeria monocytogenes and other pathogenic or undesirable bacteria, but they also inactivate a great part of the autochthonous antagonistic microbiota of raw milk. Therefore, in this study, the antilisterial activity of raw or thermized (63°C, 30 s) milk in the presence or absence of Lactococcus lactis subsp. cremoris M104, a wild, novel, nisin A-producing (Nis-A+) raw milk isolate, was assessed. Bulk milk samples were taken from a local cheese plant before or after thermization and were inoculated with a five-strain cocktail of L. monocytogenes (approximately 4 log CFU/ml) or with the cocktail, as above, plus the Nis-A+ strain (approximately 6 log CFU/ml) as a bioprotective culture. Heat-sterilized (121°C, 5 min) raw milk inoculated with L. monocytogenes was used as a control treatment. All milk samples were incubated at 37°C for 6 h and then at 18°C for an additional 66 h. L. monocytogenes grew abundantly (>8 log CFU/ml) in heat-sterilized milk, whereas its growth was completely inhibited in all raw milk samples. Conversely, in thermized milk, L. monocytogenes increased by 2 log CFU/ml in the absence of strain M104, whereas its growth was completely inhibited in the presence of strain M104. Furthermore, nisin activity was detected only in milk samples inoculated with strain M104. Thus, postthermal supplementation of thermized bulk milk with bioprotective L. lactis subsp. cremoris cultures replaces the natural antilisterial activity of raw milk reduced by thermization.

Assessing the effects of exposure to environmental stress on some functional properties of Bifidobacterium animalis ssp. lactis.

PubMed

Amund, O D; Ouoba, L I I; Sutherland, J P; Ghoddusi, H B

2014-12-01

This study assessed the effects of exposing a strain of Bifidobacterium animalis ssp. lactis to acid, bile and osmotic stresses on antagonistic properties, biofilm formation and antibiotic susceptibility/resistance profile. Exposure to each stress factor appeared to have no significant effect on the antagonism against Escherichia coli NCTC 12900 and Salmonella enterica serovar Enteritidis PT4. No suppression in biofilm formation due to exposure to stress was observed. Bile and osmotic stresses resulted in significantly higher biofilm formation. Expression of an exopolysaccharide synthesis gene, gtf 01207, was significantly higher when the B. animalis ssp. lactis strain was exposed to osmotic stress. Susceptibility of the B. animalis ssp. lactis strain to chloramphenicol, erythromycin, ampicillin and vancomycin, and resistance to tetracycline remained unchanged when exposed to each stress. The expression of a tetracycline resistance gene, tet(W), was significantly higher when exposed to each stress. These results may suggest that the potential for the B. animalis ssp. lactis strain to provide probiotic benefit, after exposure to the stressful conditions of the gastrointestinal tract, remains intact.

Proposal for creation of a new genus Neomicrococcus gen. nov. to accommodate Zhihengliuella aestuarii Baik et al. 2011 and Micrococcus lactis Chittpurna et al. 2011 as Neomicrococcus aestuarii comb. nov. and Neomicrococcus lactis comb. nov.

PubMed

Prakash, Om; Sharma, Avinash; Nimonkar, Yogesh; Shouche, Yogesh S

2015-11-01

Micrococcus lactis and Zhihengliuella aestuarii were described independently in 2011. Their type strains showed high levels of 16S rRNA gene sequence similarity (99.3%). Phylogenetic analysis revealed that M. lactis MCC 2278T and Z. aestuarii JCM 16166T formed a monophyletic group and showed distant relationships to other members of closely related genera such as Micrococcus, Zhihengliuella, Arthrobacter and Citricoccus. The presence of large proportions of iso-C14:0 and iso-C16:0 with small amounts of iso-C15:0 distinguished M. lactis MCC 2278T and Z. aestuarii JCM 16166T from other members of the genera Micrococcus and Zhihengliuella. Unlike other members of the genera Zhihengliuella and Micrococcus, M. lactis MCC 2278T and Z. aestuarii JCM 16166T showed growth at low concentrations of NaCl. Thus, based on distinctive phylogenetic, chemotaxonomic and physiological features of these two organisms in comparison with other members of the genera Micrococcus and Zhihengliuella, it is clear that they do not fit within the existing classification and deserve separate status. DNA-DNA hybridization between the two type strains was 63%, indicating that they represent separate species. In this study, we propose the creation of a novel genus, Neomicrococcus gen. nov., to accommodate the two species with Neomicrococcus aestuarii gen. nov., comb. nov. (type strain JCM 16166T = KCTC 19557T) as the type species. Neomicrococcus lactis comb. nov. (type strain MCC 2278T = DSM 23694T) is also proposed.

The Deletion of the Succinate Dehydrogenase Gene KlSDH1 in Kluyveromyces lactis Does Not Lead to Respiratory Deficiency

PubMed Central

Saliola, Michele; Bartoccioni, Paola Chiara; De Maria, Ilaria; Lodi, Tiziana; Falcone, Claudio

2004-01-01

We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose. PMID:15189981

Enhance nisin yield via improving acid-tolerant capability of Lactococcus lactis F44.

PubMed

Zhang, Jian; Caiyin, Qinggele; Feng, Wenjing; Zhao, Xiuli; Qiao, Bin; Zhao, Guangrong; Qiao, Jianjun

2016-06-16

Traditionally, nisin was produced industrially by using Lactococcus lactis in the neutral fermentation process. However, nisin showed higher activity in the acidic environment. How to balance the pH value for bacterial normal growth and nisin activity might be the key problem. In this study, 17 acid-tolerant genes and 6 lactic acid synthetic genes were introduced in L. lactis F44, respectively. Comparing to the 2810 IU/mL nisin yield of the original strain F44, the nisin titer of the engineered strains over-expressing hdeAB, ldh and murG, increased to 3850, 3979 and 4377 IU/mL, respectively. These engineered strains showed more stable intracellular pH value during the fermentation process. Improvement of lactate production could partly provide the extra energy for the expression of acid tolerance genes during growth. Co-overexpression of hdeAB, murG, and ldh(Z) in strain F44 resulted in the nisin titer of 4913 IU/mL. The engineered strain (ABGL) could grow on plates with pH 4.2, comparing to the surviving pH 4.6 of strain F44. The fed-batch fermentation showed nisin titer of the co-expression L. lactis strain could reach 5563 IU/mL with lower pH condition and longer cultivation time. This work provides a novel strategy of constructing robust strains for use in industry process.

Production of the small heat shock protein Lo18 from Oenococcus oeni in Lactococcus lactis improves its stress tolerance.

PubMed

Weidmann, Stéphanie; Maitre, Magali; Laurent, Julie; Coucheney, Françoise; Rieu, Aurélie; Guzzo, Jean

2017-04-17

Lactococcus lactis is a lactic acid bacterium widely used in cheese and fermented milk production. During fermentation, L. lactis is subjected to acid stress that impairs its growth. The small heat shock protein (sHsp) Lo18 from the acidophilic species Oenococcus oeni was expressed in L. lactis. This sHsp is known to play an important role in protein protection and membrane stabilization in O. oeni. The role of this sHsp could be studied in L. lactis, since no gene encoding for sHsp has been detected in this species. L. lactis subsp. cremoris strain MG1363 was transformed with the pDLhsp18 plasmid, which is derived from pDL278 and contains the hsp18 gene (encoding Lo18) and its own promoter sequence. The production of Lo18 during stress conditions was checked by immunoblotting and the cellular distribution of Lo18 in L. lactis cells after heat shock was determined. Our results clearly indicated a role for Lo18 in cytoplasmic protein protection and membrane stabilization during stress. The production of sHsp in L. lactis improved tolerance to heat and acid conditions in this species. Finally, the improvement of the L. lactis survival in milk medium thanks to Lo18 was highlighted, suggesting an interesting role of this sHsp. These findings suggest that the expression of a sHsp by a L. lactis strain results in greater resistance to stress, and, can consequently enhance the performances of industrial strains. Copyright © 2016 Elsevier B.V. All rights reserved.

Grana Padano cheese whey starters: microbial composition and strain distribution.

PubMed

Rossetti, Lia; Fornasari, Maria Emanuela; Gatti, Monica; Lazzi, Camilla; Neviani, Erasmo; Giraffa, Giorgio

2008-09-30

The aim of this work was to evaluate the species composition and the genotypic strain heterogeneity of dominant lactic acid bacteria (LAB) isolated from whey starter cultures used to manufacture Grana Padano cheese. Twenty-four Grana Padano cheese whey starters collected from dairies located over a wide geographic production area in the north of Italy were analyzed. Total thermophilic LAB streptococci and lactobacilli were quantified by agar plate counting. Population structure of the dominant and metabolically active LAB species present in the starters was profiled by reverse transcriptase, length heterogeneity-PCR (RT-LH-PCR), a culture-independent technique successfully applied to study whey starter ecosystems. The dominant bacterial species were Lactobacillus helveticus, Lactobacillus delbrueckii subsp. lactis, Streptococcus thermophilus, and Lactobacillus fermentum. Diversity in the species composition allowed the whey cultures to be grouped into four main typologies, the one containing L. helveticus, L. delbrueckii subsp. lactis, and S. thermophilus being the most frequent one (45% of the cultures analyzed), followed by that containing only the two lactobacilli (40%). Only a minor fraction of the cultures contained L. helveticus alone (4%) or all the four LAB species (11%). Five hundred and twelve strains were isolated from the 24 cultures and identified by M13-PCR fingerprinting coupled with 16S rRNA gene sequencing. Most of the strains were L. helveticus (190 strains; 37% of the total), L delbrueckii subsp. lactis (90 strains; 18%) and S. thermophilus (215 strains; 42%). This result was in good agreement with the qualitative whey starter composition observed by RT-LH-PCR. M13-PCR fingerprinting indicated a markedly low infra-species diversity, i.e. the same biotypes were often found in more than one culture. The distribution of the biotypes into the different cultures was mainly dairy plant-specific rather than correlated with the different production areas.

Comparative and functional genomics of the Lactococcus lactis taxon; insights into evolution and niche adaptation.

PubMed

Kelleher, Philip; Bottacini, Francesca; Mahony, Jennifer; Kilcawley, Kieran N; van Sinderen, Douwe

2017-03-29

Lactococcus lactis is among the most widely studied lactic acid bacterial species due to its long history of safe use and economic importance to the dairy industry, where it is exploited as a starter culture in cheese production. In the current study, we report on the complete sequencing of 16 L. lactis subsp. lactis and L. lactis subsp. cremoris genomes. The chromosomal features of these 16 L. lactis strains in conjunction with 14 completely sequenced, publicly available lactococcal chromosomes were assessed with particular emphasis on discerning the L. lactis subspecies division, evolution and niche adaptation. The deduced pan-genome of L. lactis was found to be closed, indicating that the representative data sets employed for this analysis are sufficient to fully describe the genetic diversity of the taxon. Niche adaptation appears to play a significant role in governing the genetic content of each L. lactis subspecies, while (differential) genome decay and redundancy in the dairy niche is also highlighted.

Natural DNA transformation is functional in Lactococcus lactis ssp. cremoris KW2.

PubMed

David, Blandine; Radziejwoski, Amandine; Toussaint, Frédéric; Fontaine, Laetitia; Henry de Frahan, Marie; Patout, Cédric; van Dillen, Sabine; Boyaval, Patrick; Horvath, Philippe; Fremaux, Christophe; Hols, Pascal

2017-06-16

Lactococcus lactis is one of the most commonly used lactic acid bacteria in the dairy industry. Activation of competence for natural DNA transformation in this species would greatly improve the selection of novel strains with desired genetic traits. Here, we investigated the activation of natural transformation in L. lactis ssp. cremoris KW2, a strain of plant origin whose genome encodes the master competence regulator ComX and the complete set of proteins usually required for natural transformation. In the absence of knowledge about competence regulation in this species, we constitutively overproduced ComX in a reporter strain of late competence phase activation and showed, by transcriptomic analyses, a ComX-dependent induction of all key competence genes. We further demonstrated that natural DNA transformation is functional in this strain and requires the competence DNA uptake machinery. Since constitutive ComX overproduction is unstable, we alternatively expressed comX under the control of an endogenous xylose-inducible promoter. This regulated system was used to successfully inactivate the adaptor protein MecA and subunits of the Clp proteolytic complex, which were previously shown to be involved in ComX degradation in streptococci. In the presence of a low amount of ComX, the deletion of mecA , clpC , or clpP genes markedly increased the activation of the late competence phase and transformability. Altogether, our results report the functionality of natural DNA transformation in L. lactis and pave the way for the identification of signaling mechanisms that trigger the competence state in this species. IMPORTANCE Lactococcus lactis is a lactic acid bacterium of major importance, which is used as a starter species for milk fermentation, a host for heterologous protein production, and a delivery platform for therapeutic molecules. Here, we report the functionality of natural transformation in L. lactis ssp. cremoris KW2 by the overproduction of the master

Portal vein thrombosis and liver abscess due to Lactococcus lactis.

PubMed

Güz, Galip; Yeğin, Zeynep Arzu; Doğan, Ibrahim; Hizel, Kenan; Bali, Musa; Sindel, Sükrü

2006-06-01

A 26-year-old man was admitted with fever and abdominal pain. Abdominal ultrasonography and Doppler ultrasound eventually revealed portal vein thrombosis and a pyogenic liver abscess (17x11x11 cm). Lactococcus lactis was isolated from a culture of the abscess material. This organism is not a common pathogen in humans. This is the first published description of portal vein thrombosis and pyogenic liver abscess due to L. lactis.

Evaluation of a select strain of Lactobacillus delbrueckii subsp. lactis as a biological control agent for pathogens on fresh-cut vegetables stored at 7 degrees C.

PubMed

Harp, E; Gilliland, S E

2003-06-01

Raw vegetables inoculated with selected pathogenic bacteria were treated with a strain of Lactobacillus delbrueckii subsp. lactis, which was selected for its ability to produce hydrogen peroxide at refrigerated temperatures. The vegetables inoculated included broccoli, cabbage, carrots, and lettuce. Each vegetable was rinsed, chopped, and stored under conditions similar to those used for ready-to-eat vegetables sold at retail. Portions of each vegetable were separately inoculated with one of two pathogenic bacteria, Escherichia coli O157:H7 or Listeria monocytogenes. Prior to packaging, one portion of each inoculated vegetable was treated with a cell suspension of the selected strain of L. delbrueckii subsp. lactis. The vegetables were stored at 7 degrees C for 6 days. The populations of pathogens and lactobacilli on each sample were enumerated on storage days 0, 3, and 6. Although populations of L. delbrueckii subsp. lactis remained at high levels during storage, there was no noticeable antagonistic action against the pathogens under conditions similar to those used for these products at the retail level. Each pathogen survived on all vegetables throughout storage. Further testing revealed that there was apparently sufficient catalase activity in the cut vegetables to destroy enough of the hydrogen peroxide to prevent antagonistic action against the pathogens.

Lactococcus lactis NCC 2287 Alleviates Food Allergic Manifestations in Sensitized Mice by Reducing IL-13 Expression Specifically in the Ileum

PubMed Central

Zuercher, Adrian W.; Weiss, Marietta; Holvoet, Sébastien; Moser, Mireille; Moussu, Hélène; van Overtvelt, Laurence; Horiot, Stéphane; Moingeon, Philippe; Nutten, Sophie; Prioult, Guénolée; Singh, Anurag; Mercenier, Annick

2012-01-01

Objective. Utilizing a food allergy murine model, we have investigated the intrinsic antiallergic potential of the Lactococcus lactis NCC 2287 strain. Methods. BALB/c mice were sensitized at weekly intervals with ovalbumin (OVA) plus cholera toxin (CT) by the oral route for 7 weeks. In this model, an oral challenge with a high dose of OVA at the end of the sensitization period leads to clinical symptoms. Lactococcus lactis NCC 2287 was given to mice via the drinking water during sensitization (prevention phase) or after sensitization (management phase). Results. Lactococcus lactis NCC 2287 administration to sensitized mice strikingly reduced allergic manifestations in the management phase upon challenge, when compared to control mice. No preventive effect was observed with the strain. Lactococcus lactis NCC 2287 significantly decreased relative expression levels of the Th-2 cytokine, IL-13, and associated chemokines CCL11 (eotaxin-1) and CCL17 (TARC) in the ileum. No effect was observed in the jejunum. Conclusion/Significance. These results taken together designate Lactococcus lactis NCC 2287 as a candidate probiotic strain appropriate in the management of allergic symptoms. PMID:21961022

Lactococcus lactis NCC 2287 alleviates food allergic manifestations in sensitized mice by reducing IL-13 expression specifically in the ileum.

PubMed

Zuercher, Adrian W; Weiss, Marietta; Holvoet, Sébastien; Moser, Mireille; Moussu, Hélène; van Overtvelt, Laurence; Horiot, Stéphane; Moingeon, Philippe; Nutten, Sophie; Prioult, Guénolée; Singh, Anurag; Mercenier, Annick

2012-01-01

Utilizing a food allergy murine model, we have investigated the intrinsic antiallergic potential of the Lactococcus lactis NCC 2287 strain. BALB/c mice were sensitized at weekly intervals with ovalbumin (OVA) plus cholera toxin (CT) by the oral route for 7 weeks. In this model, an oral challenge with a high dose of OVA at the end of the sensitization period leads to clinical symptoms. Lactococcus lactis NCC 2287 was given to mice via the drinking water during sensitization (prevention phase) or after sensitization (management phase). Lactococcus lactis NCC 2287 administration to sensitized mice strikingly reduced allergic manifestations in the management phase upon challenge, when compared to control mice. No preventive effect was observed with the strain. Lactococcus lactis NCC 2287 significantly decreased relative expression levels of the Th-2 cytokine, IL-13, and associated chemokines CCL11 (eotaxin-1) and CCL17 (TARC) in the ileum. No effect was observed in the jejunum. These results taken together designate Lactococcus lactis NCC 2287 as a candidate probiotic strain appropriate in the management of allergic symptoms.

Characterization of lactic acid bacteria isolated from a Thai low-salt fermented fish product and the role of garlic as substrate for fermentation.

PubMed

Paludan-Müller, C; Huss, H H; Gram, L

1999-02-18

Lactic acid bacteria (LAB) isolated from raw materials (fish, rice, garlic and banana leaves) and processed som-fak (a Thai low-salt fermented fish product) were characterized by API 50-CH and other phenotypic criteria. Lactococcus lactis subsp. lactis and Leuconostoc citreum were specifically associated with fish fillet and minced fish, Lactobacillus paracasei subsp. paracasei with boiled rice and Weisella confusa with garlic mix and banana leaves. In addition, Lactobacillus plantarum, Lactobacillus pentosus and Pediococcus pentosaceus were isolated from raw materials. A succession of aciduric, homofermentative lactobacillus species, dominated by Lb. plantarum/pentosus, was found during fermentation. In total, 9% of the strains fermented starch and 19% fermented garlic, the two main carbohydrate components in som-fak. The ability to ferment garlic was paralleled by a capacity to ferment inulin. An increased percentage of garlic fermenting strains was found during fermentation of som-fak, from 8% at day 1 to 40% at day 5. No starch fermenting strains were isolated during fermentation. Three mixed LAB cultures, composed of either starch fermenting Lc. lactis subsp. lactis and Lb. paracasei subsp. paracasei, or garlic fermenting Lb. plantarum and Pd. pentosaceus, or a combination of these strains were inoculated into laboratory prepared som-fak with or without garlic. In som-fak without garlic, pH was above 4.8 after three days, irrespective of addition of mixed LAB cultures. The starch fermenting LAB were unable to ferment som-fak and sensory spoilage occurred after three days. Fermentation with the combined mix of starch and garlic fermenting strains led to production of 2.5% acid and a decrease in pH to 4.5 in two days. The fermentation was slightly slower with the garlic fermenting strains alone. This is the first report describing the role of garlic as carbohydrate source for LAB in fermented fish products.

Multiplex PCR to detect bacteriophages from natural whey cultures of buffalo milk and characterisation of two phages active against Lactococcus lactis, ΦApr-1 and ΦApr-2.

PubMed

Aprea, Giuseppe; Mullan, William Michael; Murru, Nicoletta; Fitzgerald, Gerald; Buonanno, Marialuisa; Cortesi, Maria Luisa; Prencipe, Vincenza Annunziata; Migliorati, Giacomo

2017-09-30

This work investigated bacteriophage induced starter failures in artisanal buffalo Mozzarella production plants in Southern Italy. Two hundred and ten samples of whey starter cultures were screened for bacteriophage infection. Multiplex polymerase chain reaction (PCR) revealed phage infection in 28.56% of samples, all showing acidification problems during cheese making. Based on DNA sequences, bacteriophages for Lactococcus lactis (L. lactis), Lactobacillus delbruekii (L. delbruekii) and Streptococcus thermophilus (S. thermophilus) were detected. Two phages active against L. lactis, ΦApr-1 and ΦApr-2, were isolated and characterised. The genomes, approximately 31.4 kb and 31 kb for ΦApr-1 and ΦApr-2 respectively, consisted of double-stranded linear DNA with pac-type system. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‑PAGE) showed one major structural protein of approximately 32.5 kDa and several minor proteins. This is the first report of phage isolation in buffalo milk and of the use of multiplex PCR to screen and study the diversity of phages against Lactic Acid Bacteria (LAB) strains in artisanal Water Buffalo Mozzarella starters.

Use of Taiwanese ropy fermented milk (TRFM) and Lactococcus lactis subsp. cremoris isolated from TRFM in manufacturing of functional low-fat cheeses.

PubMed

Chiang, Ming-Lun; Chen, Hsi-Chia; Wang, Sheng-Yao; Hsieh, Yueh-Ling; Chen, Ming-Ju

2011-09-01

The purpose of this study was to manufacture new functional low-fat cheeses using Taiwanese ropy fermented milk (TRFM) and Lactococcus lactis subsp. cremoris strains isolated from TRFM. After 28 d of ripening and storage, the viable populations of lactic acid bacteria (LAB) in the low-fat cheeses made with L. lactis subsp. cremoris TL1 (TL1), L. lactis subsp. cremoris TL4 (TL4), and TRFM still maintained above 10(8) CFU/g. The low-fat cheeses made with TL1 and TRFM showed higher moisture contents than the cheeses made with TL4, full-fat, and low-fat cheese controls. The low-fat cheeses made with TL1 and TL4 had higher customer preferential scores similar to full-fat cheese control in the sensory evaluation. Additionally, the low-fat cheeses fermented with TL1, TL4, and TRFM for 4 h had higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging and ferrous ion-chelating abilities than the cheeses fermented with the starters for 8 h, full-fat, and low-fat cheese controls. A better angiotensin-converting enzyme (ACE) inhibition activity was also observed in the low-fat cheeses made with TL1, TL4, and TRFM than that in the full-fat and low-fat cheese controls during ripening and storage period. As health-conscious consumers continue to seek low-fat alternatives in their diets, there remain strong interests for the dairy industry to develop low-fat cheeses to meet the demands. This study clearly demonstrated that the low-fat cheeses fermented with TL1 for 4 h showed a better overall acceptability and possessed antioxidative abilities and ACE inhibitory activities than other cheeses tested in this study. By improving its flavor and investigating the possible mechanisms of its functionalities in the future, this low-fat cheese might possibly be commercialized and give a positive impact on cheese consumption in the future. © 2011 Institute of Food Technologists®

Genetic Variation of Lactobacillus delbrueckii subsp. lactis Bacteriophages Isolated from Cheese Processing Plants in Finland

PubMed Central

Forsman, Päivi; Alatossava, Tapani

1991-01-01

The genomes of four Lactobacillus delbrueckii subsp. lactis bacteriophages were characterized by restriction endonuclease mapping, Southern hybridization, and heteroduplex analysis. The phages were isolated from different cheese processing plants in Finland between 1950 and 1972. All four phages had a small isometric head and a long noncontractile tail. Two different types of genome (double-stranded DNA) organization existed among the different phages, the pac type and the cos type, corresponding to alternative types of phage DNA packaging. Three phages belonged to the pac type, and a fourth was a cos-type phage. The pac-type phages were genetically closely related. In the genomes of the pac-type phages, three putative insertion/deletions (0.7 to 0.8 kb, 1.0 kb, and 1.5 kb) and one other region (0.9 kb) containing clustered base substitutions were discovered and localized. At the phenotype level, three main differences were observed among the pac-type phages. These concerned two minor structural proteins and the efficiency of phage DNA packaging. The genomes of the pac-type phages showed only weak homology with that of the cos-type phage. Phage-related DNA, probably a defective prophage, was located in the chromosome of the host strain sensitive to the cos-type phage. This DNA exhibited homology under stringent conditions to the pac-type phages. Images PMID:16348513

Use of murine models to detect the allergenicity of genetically modified Lactococcus lactis NZ9000/pNZPNK.

PubMed

Chiang, Shen-Shih; Liu, Chin-Feng; Ku, Ting-Wei; Mau, Jeng-Leun; Lin, Hsin-Tang; Pan, Tzu-Ming

2011-04-27

By introducing aprN into Lactococcus lactis NZ9000, the genetically modified L. lactis NZ9000/pNZPNK successfully expressed the nattokinase. The safety assessment of this novel strain was based on allergenicity of pepsin digestion stability and murine model serologic identity. Subjecting to the GM strain and host to pepsin digestion, the soluble fractions and cell debris were fast degraded completely. Feeding with ovalbumin resulted in significantly higher production of IgG1 and IgE as compared to that of L. lactis NZ9000/pNZPNK or L. lactis NZ9000. Further, the serum IgG2a level increased dose-dependently at week 2 and induced immune reaction toward Th1 pathway. Secretion of cytokines IL-4 and IL-10 fed with lactococci was significantly lower than that of the OVA group. L. lactis NZ9000/pNZPNK did not increase the proliferation of type 2 helper T cells in spleen or induce allergenicity in BALB/c mice. On the basis of the results, the new GM lactic acid bacterium is regarded as safe to use.

Dominant lactic acid bacteria and their technological properties isolated from the Himalayan ethnic fermented milk products.

PubMed

Dewan, Sailendra; Tamang, Jyoti Prakash

2007-10-01

Ethnic people of the Himalayan regions of India, Nepal, Bhutan and China consume a variety of indigenous fermented milk products made from cows milk as well as yaks milk. These lesser-known ethnic fermented foods are dahi, mohi, chhurpi, somar, philu and shyow. The population of lactic acid bacteria (LAB) ranged from 10(7) to 10(8) cfu/g in these Himalayan milk products. A total of 128 isolates of LAB were isolated from 58 samples of ethnic fermented milk products collected from different places of India, Nepal and Bhutan. Based on phenotypic characterization including API sugar test, the dominant lactic acid bacteria were identified as Lactobacillus bifermentans, Lactobacillus paracasei subsp. pseudoplantarum, Lactobacillus kefir, Lactobacillus hilgardii, Lactobacillus alimentarius, Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Enterococcus faecium. LAB produced a wide spectrum of enzymes and showed high galactosidase, leucine-arylamidase and phosphatase activities. They showed antagonistic properties against selected Gram-negative bacteria. None of the strains produced bacteriocin and biogenic amines under the test conditions used. Most strains of LAB coagulated skim milk with a moderate drop in pH. Some strains of LAB showed a high degree of hydrophobicity, suggesting these strains may have useful adhesive potential. This paper is the first report on functional lactic acid bacterial composition in some lesser-known ethnic fermented milk products of the Himalayas.

Classification of Lactococcus lactis cell envelope proteinase based on gene sequencing, peptides formed after hydrolysis of milk, and computer modeling.

PubMed

Børsting, M W; Qvist, K B; Brockmann, E; Vindeløv, J; Pedersen, T L; Vogensen, F K; Ardö, Y

2015-01-01

Lactococcus lactis strains depend on a proteolytic system for growth in milk to release essential AA from casein. The cleavage specificities of the cell envelope proteinase (CEP) can vary between strains and environments and whether the enzyme is released or bound to the cell wall. Thirty-eight Lc. lactis strains were grouped according to their CEP AA sequences and according to identified peptides after hydrolysis of milk. Finally, AA positions in the substrate binding region were suggested by the use of a new CEP template based on Streptococcus C5a CEP. Aligning the CEP AA sequences of 38 strains of Lc. lactis showed that 21 strains, which were previously classified as group d, could be subdivided into 3 groups. Independently, similar subgroupings were found based on comparison of the Lc. lactis CEP AA sequences and based on normalized quantity of identified peptides released from αS1-casein and β-casein. A model structure of Lc. lactis CEP based on the crystal structure of Streptococcus C5a CEP was used to investigate the AA positions in the substrate-binding region. New AA positions were suggested, which could be relevant for the cleavage specificity of CEP; however, these could only explain 2 out of 3 found subgroups. The third subgroup could be explained by 1 to 5 AA positions located opposite the substrate binding region. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Beneficial effect of Lactococcus lactis NCC 2287 in a murine model of eosinophilic esophagitis.

PubMed

Holvoet, S; Doucet-Ladevèze, R; Perrot, M; Barretto, C; Nutten, S; Blanchard, C

2016-12-01

Eosinophilic esophagitis (EoE) is a severe inflammatory disease of the esophagus which is characterized histologically by an eosinophilic infiltration into the esophageal tissue. The efficacy of probiotics in the context of atopic diseases has been well investigated but, to date, there has been no study which has evaluated probiotic effects on EoE inflammation. This study sought to identify a probiotic which improves esophageal inflammation in experimental EoE. Two candidate probiotics, Lactococcus lactis NCC 2287 and Bifidobacterium lactis NCC 2818, were tested in a murine model of EoE elicited by epicutaneous sensitization with Aspergillus fumigatus protein extract. Administration of bacterial strains in drinking water was used, respectively, as a preventive or treatment measure, or continuously throughout the study. Inflammatory parameters were assessed in the esophagus, skin, and lungs after allergen challenge. In this EoE model, supplementation with L. lactis NCC 2287 significantly decreased esophageal and bronchoalveolar eosinophilia but only when given as a therapeutic treatment. No significant effect on eosinophilia was observed when NCC 2287 was given as a preventive or a continuous intervention. NCC 2287 supplementation had no significant effect on immunoglobulin levels, skin symptom scores, or on transepidermal water loss. Supplementation with another probiotic, B. lactis NCC 2818, had no significant effect on esophageal eosinophilia. We identified a L. lactis strain, able to attenuate esophageal eosinophilic inflammation in a preclinical model of EoE. This effect is strain specific and depends on the timing and duration of bacterial supplementation. Confirmation of these observations in human clinical trials is warranted. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Expression of lycopene biosynthesis genes fused in line with Shine-Dalgarno sequences improves the stress-tolerance of Lactococcus lactis.

PubMed

Dong, Xiangrong; Wang, Yanping; Yang, Fengyuan; Zhao, Shanshan; Tian, Bing; Li, Tao

2017-01-01

Lycopene biosynthetic genes from Deinococcus radiodurans were co-expressed in Lactococcus lactis to produce lycopene and improve its tolerance to stress. Lycopene-related genes from D. radiodurans, DR1395 (crtE), DR0862 (crtB), and DR0861 (crtI), were fused in line with S hine-Dalgarno (SD) sequences and co-expressed in L. lactis. The recombinant strain produced 0.36 mg lycopene g -1  dry cell wt after 48 h fermentation. The survival rate to UV irradiation of the recombinant strain was higher than that of the non-transformed strain. The L. lactis with co-expressed genes responsible for lycopene biosynthesis from D. radiodurans produced lycopene and exhibited increased resistance to UV stress, suggesting that the recombinant strain has important application potential in food industry.

Versatile Cas9-Driven Subpopulation Selection Toolbox for Lactococcus lactis.

PubMed

van der Els, Simon; James, Jennelle K; Kleerebezem, Michiel; Bron, Peter A

2018-04-15

CRISPR-Cas9 technology has been exploited for the removal or replacement of genetic elements in a wide range of prokaryotes and eukaryotes. Here, we describe the extension of the Cas9 application toolbox to the industrially important dairy species Lactococcus lactis The Cas9 expression vector pLABTarget, encoding the Streptocccus pyogenes Cas9 under the control of a constitutive promoter, was constructed, allowing plug and play introduction of short guide RNA (sgRNA) sequences to target specific genetic loci. Introduction of a pepN -targeting derivative of pLABTarget into L. lactis strain MG1363 led to a strong reduction in the number of transformants obtained, which did not occur in a pepN deletion derivative of the same strain, demonstrating the specificity and lethality of the Cas9-mediated double-strand breaks in the lactococcal chromosome. Moreover, the same pLABTarget derivative allowed the selection of a pepN deletion subpopulation from its corresponding single-crossover plasmid integrant precursor, accelerating the construction and selection of gene-specific deletion derivatives in L. lactis Finally, pLABTarget, which contained sgRNAs designed to target mobile genetic elements, allowed the effective curing of plasmids, prophages, and integrative conjugative elements (ICEs). These results establish that pLABTarget enables the effective exploitation of Cas9 targeting in L. lactis , while the broad-host-range vector used suggests that this toolbox could readily be expanded to other Gram-positive bacteria. IMPORTANCE Mobile genetic elements in Lactococcus lactis and other lactic acid bacteria (LAB) play an important role in dairy fermentation, having both positive and detrimental effects during the production of fermented dairy products. The pLABTarget vector offers an efficient cloning platform for Cas9 application in lactic acid bacteria. Targeting Cas9 toward mobile genetic elements enabled their effective curing, which is of particular interest in the

A mouse mastitis model to study the effects of the intramammary infusion of a food-grade Lactococcus lactis strain.

PubMed

Camperio, Cristina; Armas, Federica; Biasibetti, Elena; Frassanito, Paolo; Giovannelli, Carlo; Spuria, Liliana; D'Agostino, Claudia; Tait, Sabrina; Capucchio, Maria Teresa; Marianelli, Cinzia

2017-01-01

Lactococcus lactis is one of the most important microorganisms in the dairy industry and has "generally recognized as safe" (GRAS) status. L. lactis belongs to the group of lactic acid bacteria (LAB) and is encountered in a wide range of environments. Recently, the use of the intramammary infusion of a live culture of LAB has been investigated as a new antibiotic alternative for treating mastitis in dairy ruminants. Controversial results are described in literature regarding its efficacy and safety. In this study we conducted in-depth investigation of the mammary gland immune response induced by intramammary inoculum of a live culture of L. lactis LMG 7930 using the mouse mastitis model. Overnight cultures either of L. lactis (≈ 107 CFU) or of the mastitis pathogens Staphylococcus chromogenes (≈ 105 CFU) or S. aureus (≈ 102 CFU/ml) were injected into the mouse inguinal glands. A double injection, consisting of S. chromogenes first and then L. lactis, was also investigated. Bacterial recovery from the gland and inflammatory cell infiltration were assessed. L. lactis-treated and control glands were analysed for proinflammatory cytokine production. Microbiological results showed that L. lactis was able to survive in the mammary gland 24 h post infection, as were the mastitis pathogens S. chromogenes and S. aureus. L. lactis reduced S. chromogenes survival in the glands and increased its own survival ability by coexisting with the pathogen. Histology showed that L. lactis-treated glands presented variable histological features, ranging from undamaged tissue with no inflammatory cell infiltrate to severe PMN infiltrate with focal areas of tissue damage. S. aureus-treated glands showed the most severe histological grade of inflammation despite the fact that the inoculum size was the smallest. In contrast, most S. chromogenes-treated glands showed normal structures with no infiltration or lesions. Significant increases in IL-1β and TNF-α levels were also found in

A mouse mastitis model to study the effects of the intramammary infusion of a food-grade Lactococcus lactis strain

PubMed Central

Biasibetti, Elena; Frassanito, Paolo; Giovannelli, Carlo; Spuria, Liliana; D’Agostino, Claudia; Tait, Sabrina; Capucchio, Maria Teresa

2017-01-01

Lactococcus lactis is one of the most important microorganisms in the dairy industry and has “generally recognized as safe” (GRAS) status. L. lactis belongs to the group of lactic acid bacteria (LAB) and is encountered in a wide range of environments. Recently, the use of the intramammary infusion of a live culture of LAB has been investigated as a new antibiotic alternative for treating mastitis in dairy ruminants. Controversial results are described in literature regarding its efficacy and safety. In this study we conducted in-depth investigation of the mammary gland immune response induced by intramammary inoculum of a live culture of L. lactis LMG 7930 using the mouse mastitis model. Overnight cultures either of L. lactis (≈ 107 CFU) or of the mastitis pathogens Staphylococcus chromogenes (≈ 105 CFU) or S. aureus (≈ 102 CFU/ml) were injected into the mouse inguinal glands. A double injection, consisting of S. chromogenes first and then L. lactis, was also investigated. Bacterial recovery from the gland and inflammatory cell infiltration were assessed. L. lactis-treated and control glands were analysed for proinflammatory cytokine production. Microbiological results showed that L. lactis was able to survive in the mammary gland 24 h post infection, as were the mastitis pathogens S. chromogenes and S. aureus. L. lactis reduced S. chromogenes survival in the glands and increased its own survival ability by coexisting with the pathogen. Histology showed that L. lactis-treated glands presented variable histological features, ranging from undamaged tissue with no inflammatory cell infiltrate to severe PMN infiltrate with focal areas of tissue damage. S. aureus-treated glands showed the most severe histological grade of inflammation despite the fact that the inoculum size was the smallest. In contrast, most S. chromogenes-treated glands showed normal structures with no infiltration or lesions. Significant increases in IL-1β and TNF-α levels were also found

Isolation and characterization of probiotics from dairies

PubMed Central

Haghshenas, Babak; Nami, Yousef; Almasi, Ali; Abdullah, Norhafizah; Radiah, Dayang; Rosli, Rozita; Barzegari, Abolfazl; Khosroushahi, Ahmad Yari

2017-01-01

Background and Objectives: Probiotics are live microorganisms, which show beneficial health effects on hosts once consumed in sufficient amounts. LAB group can be isolated and characterized from traditional dairy sources. This study aimed at isolating, identifying, and in vitro characterizing (low pH/high bile salt tolerance, antibacterial activity, and antibiotic susceptibility) LAB strains from traditional Iranian dairy products. Materials and Methods: Isolated strains were identified by Gram staining, catalase assay, and 3 molecular identification methods; namely, (GTG) 5-PCR fingerprinting, ARDRA, and 16S rDNA gene sequencing. Results: A total of 19 LAB strains belonging to 4 genera (Lactococcus, Leuconostoc, Lactobacillus and Enterococcus) were identified. Conclusion: The experiments revealed that L. plantarum 15HN, L. lactis subsp. cremoris 44L and E. mundtii 50H strains, which were isolated from shiraz, cheese and shiraz, respectively, displayed a desirable tolerance to low pH and high bile salts, favorable anti-pathogen activity, and acceptable antibiotic susceptibility; hence, they could be considered as novel probiotic candidates and applied in the food industry. PMID:29238459

Strain isolated ceramic coatings

NASA Technical Reports Server (NTRS)

Tolokan, R. P.; Brady, J. B.; Jarrabet, G. P.

1985-01-01

Plasma sprayed ceramic coatings are used in gas turbine engines to improve component temperature capability and cooling air efficiency. A compliant metal fiber strain isolator between a plasma sprayed ceramic coating and a metal substrate improves ceramic durability while allowing thicker coatings for better insulation. Development of strain isolated coatings has concentrated on design and fabrication of coatings and coating evaluation via thermal shock testing. In thermal shock testing, five types of failure are possible: buckling failure im compression on heat up, bimetal type failure, isothermal expansion mismatch failure, mudflat cracking during cool down, and long term fatigue. A primary failure mode for thermally cycled coatings is designated bimetal type failure. Bimetal failure is tensile failure in the ceramic near the ceramic-metal interface. One of the significant benefits of the strain isolator is an insulating layer protecting the metal substrate from heat deformation and thereby preventing bimetal type failure.

Heterologous expression of Streptococcus mutans Cnm in Lactococcus lactis promotes intracellular invasion, adhesion to human cardiac tissues and virulence.

PubMed

Freires, Irlan A; Avilés-Reyes, Alejandro; Kitten, Todd; Simpson-Haidaris, P J; Swartz, Michael; Knight, Peter A; Rosalen, Pedro L; Lemos, José A; Abranches, Jacqueline

2017-01-02

In S. mutans, the expression of the surface glycoprotein Cnm mediates binding to extracellular matrix proteins, endothelial cell invasion and virulence in the Galleria mellonella invertebrate model. To further characterize Cnm as a virulence factor, the cnm gene from S. mutans strain OMZ175 was expressed in the non-pathogenic Lactococcus lactis NZ9800 using a nisin-inducible system. Despite the absence of the machinery necessary for Cnm glycosylation, Western blot and immunofluorescence microscopy analyses demonstrated that Cnm was effectively expressed and translocated to the cell wall of L. lactis. Similar to S. mutans, expression of Cnm in L. lactis enabled robust binding to collagen and laminin, invasion of human coronary artery endothelial cells and increased virulence in G. mellonella. Using an ex vivo human heart tissue colonization model, we showed that Cnm-positive strains of either S. mutans or L. lactis outcompete their Cnm-negative counterparts for tissue colonization. Finally, Cnm expression facilitated L. lactis adhesion and colonization in a rabbit model of infective endocarditis. Collectively, our results provide unequivocal evidence that binding to extracellular matrices mediated by Cnm is an important virulence attribute of S. mutans and confirm the usefulness of the L. lactis heterologous system for further characterization of bacterial virulence factors.

Effects in the use of a genetically engineered strain of Lactococcus lactis delivering in situ IL-10 as a therapy to treat low-grade colon inflammation.

PubMed

Martín, Rebeca; Chain, Florian; Miquel, Sylvie; Natividad, Jane M; Sokol, Harry; Verdu, Elena F; Langella, Philippe; Bermúdez-Humarán, Luis G

2014-01-01

Irritable bowel syndrome (IBS) is a gastrointestinal disorder characterized by chronic abdominal pain, discomfort, and bloating. Interestingly, there is now evidence of the presence of a low-grade inflammatory status in many IBS patients, including histopathological and mucosal cytokine levels in the colon, as well as the presence of IBS-like symptoms in quiescent inflammatory bowel disease (IBD). The use of a genetically engineered food-grade bacterium, such as Lactococcus lactis, secreting the anti-inflammatory cytokine IL-10 has been proven by many pre-clinical studies to be a successful therapy to treat colon inflammation. In this study, we first reproduced the recovery-recurrence periods observed in IBS-patients in a new chronic model characterized by 2 episodes of DiNitro-BenzeneSulfonic-acid (DNBS)-challenge and we tested the effects of a recombinant strain of L. lactis secreting IL-10 under a Stress-Inducible Controlled Expression (SICE) system. In vivo gut permeability, colonic serotonin levels, cytokine profiles, and spleen cell populations were then measured as readouts of a low-grade inflammation. In addition, since there is increasing evidence that gut microbiota tightly regulates gut barrier function, tight junction proteins were also measured by qRT-PCR after administration of recombinant L. lactis in DNBS-treated mice. Strikingly, oral administration of L. lactis secreting active IL-10 in mice resulted in significant protective effects in terms of permeability, immune activation, and gut-function parameters. Although genetically engineered bacteria are, for now, used only as a "proof-of-concept," our study validates the interest in the use of the novel SICE system in L. lactis to express therapeutic molecules, such as IL-10, locally at mucosal surfaces.

Molecular cloning of the plasma membrane H(+)-ATPase from Kluyveromyces lactis: a single nucleotide substitution in the gene confers ethidium bromide resistance and deficiency in K+ uptake.

PubMed Central

Miranda, M; Ramírez, J; Peña, A; Coria, R

1995-01-01

A Kluyveromyces lactis strain resistant to ethidium bromide and deficient in potassium uptake was isolated. Studies on the proton-pumping activity of the mutant strain showed that a decreased H(+)-ATPase specific activity was responsible for the observed phenotypes. The putative K. lactis PMA1 gene encoding the plasma membrane H(+)-ATPase was cloned by its ability to relieve the potassium transport defect of this mutant and by reversing its resistance to ethidium bromide. Its deduced amino acid sequence predicts a protein 899 residues long that is structurally colinear in its full length to H(+)-ATPases cloned from different yeasts, except for the presence of a variable N-terminal domain. By PCR-mediated amplification, we identified a transition from G to A that rendered the substitution of the fully conserved methionine at position 699 by isoleucine. We attribute to this amino acid change the low capacity of the mutant H(+)-ATPase to pump out protons. PMID:7730265

Metabolic engineering of mannitol production in Lactococcus lactis: influence of overexpression of mannitol 1-phosphate dehydrogenase in different genetic backgrounds.

PubMed

Wisselink, H Wouter; Mars, Astrid E; van der Meer, Pieter; Eggink, Gerrit; Hugenholtz, Jeroen

2004-07-01

To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and (13)C nuclear magnetic resonance analysis revealed that small amounts (<1%) of mannitol were formed by growing cells of mtlD-overexpressing LDH-deficient and phosphofructokinase-reduced strains, whereas resting cells of the LDH-deficient transformant converted 25% of glucose into mannitol. Moreover, the formed mannitol was not reutilized upon glucose depletion. Of the metabolic-engineering strategies investigated in this work, mtlD-overexpressing LDH-deficient L. lactis seemed to be the most promising strain for mannitol production.

Metabolic characterization and transformation of the non-dairy Lactococcus lactis strain KF147, for production of ethanol from xylose.

PubMed

Petersen, Kia Vest; Liu, Jianming; Chen, Jun; Martinussen, Jan; Jensen, Peter Ruhdal; Solem, Christian

2017-08-01

The non-dairy lactic acid bacterium Lactococcus lactis KF147 can utilize xylose as the sole energy source. To assess whether KF147 could serve as a platform organism for converting second generation sugars into useful chemicals, the authors characterized growth and product formation for KF147 when grown on xylose. In a defined medium KF147 was found to co-metabolize xylose and arginine, resulting in bi-phasic growth. Especially at low xylose concentrations, arginine significantly improved growth rate. To facilitate further studies of the xylose metabolism, the authors eliminated arginine catabolism by deleting the arcA gene encoding the arginine deiminase. The fermentation product profile suggested two routes for xylose degradation, the phosphoketolase pathway and the pentose phosphate pathway. Inactivation of the phosphoketolase pathway redirected the entire flux through the pentose phosphate pathway whereas over-expression of phosphoketolase increased the flux through the phosphoketolase pathway. In general, significant amounts of the mixed-acid products, including lactate, formate, acetate and ethanol, were formed irrespective of xylose concentrations. To demonstrate the potential of KF147 for converting xylose into useful chemicals the authors chose to redirect metabolism towards ethanol production. A synthetic promoter library was used to drive the expression of codon-optimized versions of the Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase, and the outcome was a strain producing ethanol as the sole fermentation product with a high yield corresponding to 83% of the theoretical maximum. The results clearly indicate the great potential of using the more metabolically diverse non-dairy L. lactis strains for bio-production based on xylose containing feedstocks. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects in the use of a genetically engineered strain of Lactococcus lactis delivering in situ IL-10 as a therapy to treat low-grade colon inflammation

PubMed Central

Martín, Rebeca; Martín, Rebeca; Chain, Florian; Chain, Florian; Miquel, Sylvie; Miquel, Sylvie; Natividad, Jane M; Natividad, Jane M; Sokol, Harry; Sokol, Harry; Verdu, Elena F; Verdu, Elena F; Langella, Philippe; Langella, Philippe; Bermúdez-Humarán, Luis G; Bermúdez-Humarán, Luis G

2014-01-01

Irritable bowel syndrome (IBS) is a gastrointestinal disorder characterized by chronic abdominal pain, discomfort, and bloating. Interestingly, there is now evidence of the presence of a low-grade inflammatory status in many IBS patients, including histopathological and mucosal cytokine levels in the colon, as well as the presence of IBS-like symptoms in quiescent inflammatory bowel disease (IBD). The use of a genetically engineered food-grade bacterium, such as Lactococcus lactis, secreting the anti-inflammatory cytokine IL-10 has been proven by many pre-clinical studies to be a successful therapy to treat colon inflammation. In this study, we first reproduced the recovery-recurrence periods observed in IBS-patients in a new chronic model characterized by 2 episodes of DiNitro-BenzeneSulfonic-acid (DNBS)-challenge and we tested the effects of a recombinant strain of L. lactis secreting IL-10 under a Stress-Inducible Controlled Expression (SICE) system. In vivo gut permeability, colonic serotonin levels, cytokine profiles, and spleen cell populations were then measured as readouts of a low-grade inflammation. In addition, since there is increasing evidence that gut microbiota tightly regulates gut barrier function, tight junction proteins were also measured by qRT-PCR after administration of recombinant L. lactis in DNBS-treated mice. Strikingly, oral administration of L. lactis secreting active IL-10 in mice resulted in significant protective effects in terms of permeability, immune activation, and gut-function parameters. Although genetically engineered bacteria are, for now, used only as a “proof-of-concept,” our study validates the interest in the use of the novel SICE system in L. lactis to express therapeutic molecules, such as IL-10, locally at mucosal surfaces. PMID:24732667

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.

PubMed

Innocentin, Silvia; Guimarães, Valeria; Miyoshi, Anderson; Azevedo, Vasco; Langella, Philippe; Chatel, Jean-Marc; Lefèvre, François

2009-07-01

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.

NMR resonance assignments of the lantibiotic immunity protein NisI from Lactococcus lactis.

PubMed

Hacker, Carolin; Christ, Nina Alexandra; Duchardt-Ferner, Elke; Korn, Sophie; Berninger, Lucija; Kötter, Peter; Entian, Karl-Dieter; Wöhnert, Jens

2015-10-01

The lantibiotic nisin is a small antimicrobial peptide which acts against a wide range of Gram-positive bacteria. Nisin-producing Lactococcus lactis strains express four genes for self-protection against their own antimicrobial compound. This immunity system consists of the lipoprotein NisI and the ABC transporter NisFEG. NisI is attached to the outside of the cytoplasmic membrane via a covalently linked diacylglycerol anchor. Both the lipoprotein and the ABC transporter are needed for full immunity but the exact immunity mechanism is still unclear. To gain insights into the highly specific immunity mechanism of nisin producing strains on a structural level we present here the backbone resonance assignment of NisI (25.8 kDa) as well as the virtually complete (1)H,(15)N,(13)C chemical shift assignments for the isolated 12.7 kDa N-terminal and 14.6 kDa C-terminal domains of NisI.

Potential aquaculture probiont Lactococcus lactis TW34 produces nisin Z and inhibits the fish pathogen Lactococcus garvieae.

PubMed

Sequeiros, Cynthia; Garcés, Marisa E; Vallejo, Marisol; Marguet, Emilio R; Olivera, Nelda L

2015-04-01

Bacteriocin-producing Lactococcus lactis TW34 was isolated from marine fish. TW34 bacteriocin inhibited the growth of the fish pathogen Lactococcus garvieae at 5 AU/ml (minimum inhibitory concentration), whereas the minimum bactericidal concentration was 10 AU/ml. Addition of TW34 bacteriocin to L. garvieae cultures resulted in a decrease of six orders of magnitude of viable cells counts demonstrating a bactericidal mode of action. The direct detection of the bacteriocin activity by Tricine-SDS-PAGE showed an active peptide with a molecular mass ca. 4.5 kDa. The analysis by MALDI-TOF-MS detected a strong signal at m/z 2,351.2 that corresponded to the nisin leader peptide mass without the initiating methionine, whose sequence STKDFNLDLVSVSKKDSGASPR was confirmed by MS/MS. Sequence analysis of nisin structural gene confirmed that L. lactis TW34 was a nisin Z producer. This nisin Z-producing strain with probiotic properties might be considered as an alternative in the prevention of lactococcosis, a global disease in aquaculture systems.

Molecular identification and cluster analysis of homofermentative thermophilic lactobacilli isolated from dairy products.

PubMed

Andrighetto, C; De Dea, P; Lombardi, A; Neviani, E; Rossetti, L; Giraffa, G

1998-10-01

Twenty-five strains of thermophilic lactobacilli isolated from yoghurt and from semi-hard and hard cheeses (in parallel with nine type or reference strains) were identified and grouped according to their genetic relatedness. Strains were identified by sugar fermentation patterns using the "API 50 CHL" galleries, by species-specific DNA probes in dot-blot hybridization experiments, by amplification and restriction analysis of the 16S rRNA gene (ARDRA) and by polymerase chain reaction (PCR) using species-specific oligonucleotide primers. Strains were classified as Lactobacillus delbrueckii subsp. lactis and subsp. bulgaricus, L. helveticus, and L. acidophilus. Strains which were atypical by sugar fermentation patterns were also identified. Most of the strains could not be grouped using carbohydrate fermentation profiles. PCR fingerprinting was used to identify DNA profiles for the 25 lactobacilli. Experimentally obtained PCR profiles enabled discrimination of all strains, which were grouped according to the similarities in their combined patterns. In general, the clustering of the strains corresponded well with species delineation obtained by molecular identification. The dendrogram of genetic relatedness enabled the unambiguous identification of most of the strains which were shown to be atypical by the sugar fermentation profile, except for a discrepancy in one L. delbrueckii subsp. lactis strain and one atypical Lactobacillus sp. strain.

Influence of Technological Treatments on the Functionality of Bifidobacterium lactis INL1, a Breast Milk-Derived Probiotic.

PubMed

Zacarías, María Florencia; Souza, Tassia Costa; Zaburlín, Natalia; Carmona Cara, Denise; Reinheimer, Jorge; Nicoli, Jacques; Vinderola, Gabriel

2017-10-01

The aim of this study is to evaluate the influence of the technological processing on the functionality of the human breast milk probiotic strain Bifidobacterium lactis INL1. In vitro antagonistic activity of B. lactis INL1 was detected for Gram-positive and Gram-negative pathogens. B. lactis INL1 was administered to mice as fresh (F), frozen (Z), spray-dried (S), or lyophilized (L) culture. Immune parameters (IgA, IL-10, and IFN-γ) were determined and histological analysis was performed to assess functionality and protection capacity against Salmonella. In BALB/c mice, F and S cultures induced an increase in the number of IgA-producing cells in the small intestine and IL-10 levels were increased for L culture in the large intestine. In Swiss mice, B. lactis INL1 increased secretory-IgA levels in the small intestine before and after Salmonella infection, both as F or dehydrated culture. Also, an attenuation of damage in the intestinal epithelium and less inflammatory infiltrates were observed in animals that received F and S cultures, whereas in liver only F showed some effect. The anti-inflammatory effect was confirmed in both tissues by myeloperoxidase activity and by IFN-γ levels in the intestinal content. B. lactis INL1 showed inhibitory activity against pathogens and confirmed its probiotic potential in animal models. Technological processing of the probiotic strain affected its functionality. This work provides evidence about the influence of technology on the functionality of probiotics, which may help probiotics and functional food manufacturers to take processing into consideration when assessing the functionality of new strains. © 2017 Institute of Food Technologists®.

Bifidobacterium animalis ssp. lactis CNCM-I2494 Restores Gut Barrier Permeability in Chronically Low-Grade Inflamed Mice.

PubMed

Martín, Rebeca; Laval, Laure; Chain, Florian; Miquel, Sylvie; Natividad, Jane; Cherbuy, Claire; Sokol, Harry; Verdu, Elena F; van Hylckama Vlieg, Johan; Bermudez-Humaran, Luis G; Smokvina, Tamara; Langella, Philippe

2016-01-01

Growing evidence supports the efficacy of many probiotic strains in the management of gastrointestinal disorders associated with deregulated intestinal barrier function and/or structure. In particular, bifidobacteria have been studied for their efficacy to both prevent and treat a broad spectrum of animal and/or human gut disorders. The aim of the current work was thus to evaluate effects on intestinal barrier function of Bifidobacterium animalis ssp. lactis CNCM-I2494, a strain used in fermented dairy products. A chronic dinitrobenzene sulfonic acid (DNBS)-induced low-grade inflammation model causing gut dysfunction in mice was used in order to study markers of inflammation, intestinal permeability, and immune function in the presence of the bacterial strain. In this chronic low-grade inflammation mice model several parameters pointed out the absence of an over active inflammation process. However, gut permeability, lymphocyte populations, and colonic cytokines were found to be altered. B. animalis ssp. lactis CNCM-I2494 was able to protect barrier functions by restoring intestinal permeability, colonic goblet cell populations, and cytokine levels. Furthermore, tight junction (TJ) proteins levels were also measured by qRT-PCR showing the ability of this strain to specifically normalize the level of several TJ proteins, in particular for claudin-4. Finally, B. lactis strain counterbalanced CD4(+) lymphocyte alterations in both spleen and mesenteric lymphoid nodes. It restores the Th1/Th2 ratio altered by the DNBS challenge (which locally augments CD4(+) Th1 cells) by increasing the Th2 response as measured by the increase in the production of major representative Th2 cytokines (IL-4, IL-5, and IL-10). Altogether, these data suggest that B. animalis ssp. lactis CNCM-I2494 may efficiently prevent disorders associated with increased barrier permeability.

Development and evaluation of an efficient heterologous gene knock-in reporter system in Lactococcus lactis.

PubMed

Lu, Yifei; Yan, Hongxiang; Deng, Jiezhong; Huang, Zhigang; Jin, Xurui; Yu, Yanlan; Hu, Qiwen; Hu, Fuquan; Wang, Jing

2017-09-18

Lactococcus lactis is a food grade probiotics and widely used to express heterologous proteins. Generally, target genes are knocked into the L. lactis genome through double-crossover recombination to express heterologous proteins stably. However, creating marker-less heterologous genes knocked-in clones is laborious. In this study, an efficient heterologous gene knock-in reporter system was developed in L. lactis NZ9000. Our knock-in reporter system consists of a temperature-sensitive plasmid pJW and a recombinant L. lactis strain named NZB. The pJW contains homologous arms, and was constructed to knock-in heterologous genes at a fixed locus of NZ9000 genome. lacZ (β-galactosidase) gene was knocked into the chromosome of NZ9000 as a counter-selective marker through the plasmid pJW to generate NZB. The engineered NZB strain formed blue colonies on X-Gal plate. The desired double-crossover mutants formed white colonies distinctive from the predominantly blue colonies (parental and plasmid-integrated clones) when the embedded lacZ was replaced with the target heterologous genes carried by pJW in NZB. By using the system, the heterologous gene knocked-in clones are screened by colony phenotype change rather than by checking colonies individually. Our new knock-in reporter system provides an efficient method to create heterologous genes knocked-in clones.

Identification, Cloning, and Characterization of a Lactococcus lactis Branched-Chain α-Keto Acid Decarboxylase Involved in Flavor Formation

PubMed Central

Smit, Bart A.; van Hylckama Vlieg, Johan E. T.; Engels, Wim J. M.; Meijer, Laura; Wouters, Jan T. M.; Smit, Gerrit

2005-01-01

The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain α-keto acid decarboxylase (KdcA). The activity of the latter enzyme has been found only in a limited number of Lactococcus lactis strains. By using a random mutagenesis approach, the gene encoding KdcA in L. lactis B1157 was identified. The gene for this enzyme is highly homologous to the gene annotated ipd, which encodes a putative indole pyruvate decarboxylase, in L. lactis IL1403. Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3′ terminus of the ipd gene encoding a truncated nonfunctional decarboxylase. The kdcA gene was overexpressed in L. lactis for further characterization of the decarboxylase enzyme. Of all of the potential substrates tested, the highest activity was observed with branched-chain α-keto acids. Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 6.3, indicating that the enzyme might be active under cheese ripening conditions. PMID:15640202

Characterization of microbiota in Arapaima gigas intestine and isolation of potential probiotic bacteria.

PubMed

do Vale Pereira, G; da Cunha, D G; Pedreira Mourino, J L; Rodiles, A; Jaramillo-Torres, A; Merrifield, D L

2017-11-01

The aim of this study was to determine the intestinal microbiota of pirarucu (Arapaima gigas) in different growth stages (adult and fingerlings) and to isolate and identify potential probiotic bacteria. High-throughput sequencing analysis of the intestinal contents revealed that the majority of sequences belonged to the Proteobacteria, Fusobacteria and Firmicutes phyla. At the genus level, the greatest number of sequences belonged to Bradyrhizobium in adult fish, while Cetobacterium was the most abundant in juvenile fish. Twenty-three lactic-acid bacteria (LABs) were isolated on MRS agar from healthy juvenile fish. The isolates were tested in vitro for probiotic properties. Two isolates (identified as strains of Lactococcus lactis subsp. lactis and Enterococcus faecium) displayed antagonism against all 10 pathogens tested, were nonhaemolytic and maintained good viability for at least 3 weeks when supplemented to fish diets. The presence of a number of antibiotic resistance genes (ARGs), conferring resistance to erythromycin, tetracycline and chloramphenicol, was investigated by PCR. The absence of ARGs investigated the potential to antagonize pathogens, and favourable growth and survival characteristics indicate that these autochthonous isolates have the potential to be considered probiotics, which will be studied in future in vivo experiments. This study has demonstrated, for the first time, the normal microbiota in the A. gigas intestine during different life stages and the presence of LAB strains. It also demonstrated LAB antibiotic resistance and antagonistic behaviour against pathogens isolated from the same fish. © 2017 The Society for Applied Microbiology.

Transcriptome analysis shows activation of the arginine deiminase pathway in Lactococcus lactis as a response to ethanol stress.

PubMed

Díez, Lorena; Solopova, Ana; Fernández-Pérez, Rocío; González, Miriam; Tenorio, Carmen; Kuipers, Oscar P; Ruiz-Larrea, Fernanda

2017-09-18

This paper describes the molecular response of Lactococcus lactis NZ9700 to ethanol. This strain is a well-known nisin producer and a lactic acid bacteria (LAB) model strain. Global transcriptome profiling using DNA microarrays demonstrated a bacterial adaptive response to the presence of 2% ethanol in the culture broth and differential expression of 67 genes. The highest up-regulation was detected for those genes involved in arginine degradation through the arginine deiminase (ADI) pathway (20-40 fold up-regulation). The metabolic responses to ethanol of wild type L. lactis strains were studied and compared to those of regulator-deletion mutants MG∆argR and MG∆ahrC. The results showed that in the presence of 2% ethanol those strains with an active ADI pathway reached higher growth rates when arginine was available in the culture broth than in absence of arginine. In a chemically defined medium strains with an active ADI pathway consumed arginine and produced ornithine in the presence of 2% ethanol, hence corroborating that arginine catabolism is involved in the bacterial response to ethanol. This is the first study of the L. lactis response to ethanol stress to demonstrate the relevance of arginine catabolism for bacterial adaptation and survival in an ethanol containing medium. Copyright © 2017 Elsevier B.V. All rights reserved.

Absolute Enumeration of Probiotic Strains Lactobacillus acidophilus NCFM® and Bifidobacterium animalis subsp. lactis Bl-04 ® via Chip-Based Digital PCR.

PubMed

Hansen, Sarah J Z; Morovic, Wesley; DeMeules, Martha; Stahl, Buffy; Sindelar, Connie W

2018-01-01

The current standard for enumeration of probiotics to obtain colony forming units by plate counts has several drawbacks: long time to results, high variability and the inability to discern between bacterial strains. Accurate probiotic cell counts are important to confirm the delivery of a clinically documented dose for its associated health benefits. A method is described using chip-based digital PCR (cdPCR) to enumerate Bifidobacterium animalis subsp. lactis Bl-04 and Lactobacillus acidophilus NCFM both as single strains and in combination. Primers and probes were designed to differentiate the target strains against other strains of the same species using known single copy, genetic differences. The assay was optimized to include propidium monoazide pre-treatment to prevent amplification of DNA associated with dead probiotic cells as well as liberation of DNA from cells with intact membranes using bead beating. The resulting assay was able to successfully enumerate each strain whether alone or in multiplex. The cdPCR method had a 4 and 5% relative standard deviation (RSD) for Bl-04 and NCFM, respectively, making it more precise than plate counts with an industry accepted RSD of 15%. cdPCR has the potential to replace traditional plate counts because of its precision, strain specificity and the ability to obtain results in a matter of hours.

Cloning and Expression of Plantaricin W Produced by Lactobacillus plantarum U10 Isolate from "Tempoyak" Indonesian Fermented Food as Immunity Protein in Lactococcus lactis.

PubMed

Lages, Aksar Chair; Mustopa, Apon Zaenal; Sukmarini, Linda; Suharsono

2015-10-01

Plantaricins, one of bacteriocin produced by Lactobacillus plantarum, are already known to have activities against several pathogenic bacterium. L. plantarum U10 isolated from "tempoyak," an Indonesian fermented food, produced one kind of plantaricin designated as plantaricin W (plnW). The plnW is suggested as a putative membrane location of protein and has similar conserved motif which is important as immunity to bacteriocin itself. Thus, due to study about this plantaricin, several constructs have been cloned and protein was analyzed in Lactococcus lactis. In this study, plnW gene was successfully cloned into vector NICE system pNZ8148 and created the transformant named L. lactis NZ3900 pNZ8148-WU10. PlnW protein was 25.3 kDa in size. The concentration of expressed protein was significantly increased by 10 ng/mL nisin induction. Furthermore, PlnW exhibited protease activity with value of 2.22 ± 0.05 U/mL and specific activity about 1.65 ± 0.03 U/mg protein with 50 ng/mL nisin induction. Immunity study showed that the PlnW had immunity activity especially against plantaricin and rendered L. lactis recombinant an immunity broadly to other bacteriocins such as pediocin, fermentcin, and acidocin.

Leuconostoc strains isolated from dairy products: Response against food stress conditions.

PubMed

D'Angelo, Luisa; Cicotello, Joaquín; Zago, Miriam; Guglielmotti, Daniela; Quiberoni, Andrea; Suárez, Viviana

2017-09-01

A systematic study about the intrinsic resistance of 29 strains (26 autochthonous and 3 commercial ones), belonging to Leuconostoc genus, against diverse stress factors (thermal, acidic, alkaline, osmotic and oxidative) commonly present at industrial or conservation processes were evaluated. Exhaustive result processing was made by applying one-way ANOVA, Student's test (t), multivariate analysis by Principal Component Analysis (PCA) and Matrix Hierarchical Cluster Analysis. In addition, heat adaptation on 4 strains carefully selected based on previous data analysis was assayed. The strains revealed wide diversity of resistance to stress factors and, in general, a clear relationship between resistance and Leuconostoc species was established. In this sense, the highest resistance was shown by Leuconostoc lactis followed by Leuconostoc mesenteroides strains, while Leuconostoc pseudomesenteroides and Leuconostoc citreum strains revealed the lowest resistance to the stress factors applied. Heat adaptation improved thermal cell survival and resulted in a cross-resistance against the acidic factor. However, all adapted cells showed diminished their oxidative resistance. According to our knowledge, this is the first study regarding response of Leuconostoc strains against technological stress factors and could establish the basis for the selection of "more robust" strains and propose the possibility of improving their performance during industrial processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effect of dissolved oxygen on redox potential and milk acidification by lactic acid bacteria isolated from a DL-starter culture.

PubMed

Larsen, Nadja; Werner, Birgit Brøsted; Vogensen, Finn Kvist; Jespersen, Lene

2015-03-01

Milk acidification by DL-starter cultures [cultures containing Lactococcus lactis diacetylactis (D) and Leuconostoc (L) species] depends on the oxidation-reduction (redox) potential in milk; however, the mechanisms behind this effect are not completely clear. The objective of this study was to investigate the effect of dissolved oxygen on acidification kinetics and redox potential during milk fermentation by lactic acid bacteria (LAB). Fermentations were conducted by single strains isolated from mixed DL-starter culture, including Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris, and Leuconostoc mesenteroides ssp. cremoris, by the DL-starter culture, and by the type strains. High and low levels of oxygen were produced by flushing milk with oxygen or nitrogen, respectively. The kinetics of milk acidification was characterized by the maximum rate and time of acidification (Vamax and Tamax), the maximum rate and time of reduction (Vrmax and Trmax), the minimum redox potential (Eh7 final), and time of reaching Eh7 final (Trfinal). Variations in kinetic parameters were observed at both the species and strain levels. Two of the Lc. lactis ssp. lactis strains were not able to lower redox potential to negative values. Kinetic parameters of the DL-starter culture were comparable with the best acidifying and reducing strains, indicating their additive effects. Acidification curves were mostly diauxic at all oxygen levels, displaying 2 maxima of acidification rate: before (aerobic maximum) and after (anaerobic maximum) oxygen depletion. The redox potential decreased concurrently with oxygen consumption and continued to decrease at slower rate until reaching the final values, indicating involvement of both oxygen and microbiological activity in the redox state of milk. Oxygen flushing had a negative effect on reduction and acidification capacity of tested LAB. Reduction was significantly delayed at high initial oxygen, exhibiting longer Trmax, Trfinal, or both

DNA–DNA hybridization study of strains of Chryseobacterium, Elizabethkingia and Empedobacter and of other usually indole-producing non-fermenters of CDC groups IIc, IIe, IIh and IIi, mostly from human clinical sources, and proposals of Chryseobacterium bernardetii sp. nov., Chryseobacterium carnis sp. nov., Chryseobacterium lactis sp. nov., Chryseobacterium nakagawai sp. nov. and Chryseobacterium taklimakanense comb. nov

PubMed Central

Holmes, B.; Steigerwalt, A. G.; Nicholson, A. C.

2015-01-01

The taxonomic classification of 182 phenotypically similar isolates was evaluated using DNA–DNA hybridization and 16S rRNA gene sequence analysis. These bacterial isolates were mainly derived from clinical sources; all were Gram-negative non-fermenters and most were indoleproducing. Phenotypically, they resembled species from the genera Chryseobacterium, Elizabethkingia or Empedobacter or belonged to CDC groups IIc, IIe, IIh and IIi. Based on these analyses, four novel species are described: Chryseobacterium bernardetii sp. nov. (type strain NCTC 13530T=CCUG 60564T=CDC G229T), Chryseobacterium carnis sp. nov. (type strain NCTC 13525T=CCUG 60559T=CDC G81T), Chryseobacterium lactis sp. nov. (type strain NCTC 11390T=CCUG 60566T=CDC KC1864T) and Chryseobacterium nakagawai sp. nov. (type strain NCTC 13529T=CCUG 60563T=CDC G41T). The new combination Chryseobacterium taklimakanense comb. nov. (type strain NCTC 13490T=X-65T=CCTCC AB 208154T=NRRL B-51322T) is also proposed to accommodate the reclassified Planobacterium taklimakanense. PMID:23934253

16S-23S rDNA intergenic spacer region polymorphism of Lactococcus garvieae, Lactococcus raffinolactis and Lactococcus lactis as revealed by PCR and nucleotide sequence analysis.

PubMed

Blaiotta, Giuseppe; Pepe, Olimpia; Mauriello, Gianluigi; Villani, Francesco; Andolfi, Rosamaria; Moschetti, Giancarlo

2002-12-01

The intergenic spacer region (ISR) between the 16S and 23S rRNA genes was tested as a tool for differentiating lactococci commonly isolated in a dairy environment. 17 reference strains, representing 11 different species belonging to the genera Lactococcus, Streptococcus, Lactobacillus, Enterococcus and Leuconostoc, and 127 wild streptococcal strains isolated during the whole fermentation process of "Fior di Latte" cheese were analyzed. After 16S-23S rDNA ISR amplification by PCR, species or genus-specific patterns were obtained for most of the reference strains tested. Moreover, results obtained after nucleotide analysis show that the 16S-23S rDNA ISR sequences vary greatly, in size and sequence, among Lactococcus garvieae, Lactococcus raffinolactis, Lactococcus lactis as well as other streptococci from dairy environments. Because of the high degree of inter-specific polymorphism observed, 16S-23S rDNA ISR can be considered a good potential target for selecting species-specific molecular assays, such as PCR primer or probes, for a rapid and extremely reliable differentiation of dairy lactococcal isolates.

Mechanism of citrate metabolism by an oxaloacetate decarboxylase-deficient mutant of Lactococcus lactis IL1403.

PubMed

Pudlik, Agata M; Lolkema, Juke S

2011-08-01

Citrate metabolism in resting cells of Lactococcus lactis IL1403(pFL3) results in the formation of two end products from the intermediate pyruvate, acetoin and acetate (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:706-714, 2011). Pyruvate is formed from citrate following uptake by the transporter CitP through the subsequent actions of citrate lyase and oxaloacetate decarboxylase. The present study describes the metabolic response of L. lactis when oxaloacetate accumulates in the cytoplasm. The oxaloacetate decarboxylase-deficient mutant ILCitM(pFL3) showed nearly identical rates of citrate consumption, but the end product profile in the presence of glucose shifted from mainly acetoin to only acetate. In addition, in contrast to the parental strain, the mutant strain did not generate proton motive force. Citrate consumption by the mutant strain was coupled to the excretion of oxaloacetate, with a yield of 80 to 85%. Following citrate consumption, oxaloacetate was slowly taken up by the cells and converted to pyruvate by a cryptic decarboxylase and, subsequently, to acetate. The transport of oxaloacetate is catalyzed by CitP. The parental strain IL1403(pFL3) containing CitP consumed oxaloacetate, while the original strain, IL1403, not containing CitP, did not. Moreover, oxaloacetate consumption was enhanced in the presence of L-lactate, indicating exchange between oxaloacetate and L-lactate catalyzed by CitP. Hence, when oxaloacetate inadvertently accumulates in the cytoplasm, the physiological response of L. lactis is to excrete oxaloacetate in exchange with citrate by an electroneutral mechanism catalyzed by CitP. Subsequently, in a second step, oxaloacetate is taken up by CitP and metabolized to pyruvate and acetate.

Lactobacillus delbrueckii subsp lactis CIDCA 133 modulates response of human epithelial and dendritic cells infected with Bacillus cereus.

PubMed

Rolny, I S; Tiscornia, I; Racedo, S M; Pérez, P F; Bollati-Fogolín, M

2016-11-30

It is known that probiotic microorganisms are able to modulate pathogen virulence. This ability is strain dependent and involves multiple interactions between microorganisms and relevant host's cell populations. In the present work we focus on the effect of a potentially probiotic lactobacillus strain (Lactobacillus delbrueckii subsp. lactis CIDCA 133) in an in vitro model of Bacillus cereus infection. Our results showed that infection of intestinal epithelial HT-29 cells by B. cereus induces nuclear factor kappa B (NF-κB) pathway. Noteworthy, the presence of strain L. delbrueckii subsp.lactis CIDCA 133 increases stimulation. However, B. cereus-induced interleukin (IL)-8 production by epithelial cells is partially abrogated by L. delbrueckii subsp. lactis CIDCA 133. These findings suggest that signalling pathways other than that of NF-κB are involved. In a co-culture system (HT-29 and monocyte-derived dendritic cells), B. cereus was able to translocate from the epithelial (upper) to the dendritic cell compartment (lower). This translocation was partially abrogated by the presence of lactobacilli in the upper compartment. In addition, infection of epithelial cells in the co-culture model, led to an increase in the expression of CD86 by dendritic cells. This effect could not be modified in the presence of lactobacilli. Interestingly, infection of enterocytes with B. cereus triggers production of proinflammatory cytokines by dendritic cells (IL-8, IL-6 and tumour necrosis factor alpha (TNF-α)). The production of TNF-α (a protective cytokine in B. cereus infections) by dendritic cells was increased in the presence of lactobacilli. The present work demonstrates for the first time the effect of L. delbrueckii subsp. lactis CIDCA 133, a potentially probiotic strain, in an in vitro model of B. cereus infection. The presence of the probiotic strain modulates cell response both in infected epithelial and dendritic cells thus suggesting a possible beneficial effect of

Continuous D-lactic acid production by a novel thermotolerant Lactobacillus delbrueckii subsp. lactis QU 41.

PubMed

Tashiro, Yukihiro; Kaneko, Wataru; Sun, Yanqi; Shibata, Keisuke; Inokuma, Kentaro; Zendo, Takeshi; Sonomoto, Kenji

2011-03-01

We isolated and characterized a D-lactic acid-producing lactic acid bacterium (D-LAB), identified as Lactobacillus delbrueckii subsp. lactis QU 41. When compared to Lactobacillus coryniformis subsp. torquens JCM 1166 (T) and L. delbrueckii subsp. lactis JCM 1248 (T), which are also known as D-LAB, the QU 41 strain exhibited a high thermotolerance and produced D-lactic acid at temperatures of 50 °C and higher. In order to optimize the culture conditions of the QU 41 strain, we examined the effects of pH control, temperature, neutralizing reagent, and initial glucose concentration on D-lactic acid production in batch cultures. It was found that the optimal production of 20.1 g/l D-lactic acid was acquired with high optical purity (>99.9% of D-lactic acid) in a pH 6.0-controlled batch culture, by adding ammonium hydroxide as a neutralizing reagent, at 43 °C in MRS medium containing 20 g/l glucose. As a result of product inhibition and low cell density, continuous cultures were investigated using a microfiltration membrane module to recycle flow-through cells in order to improve D-lactic acid productivity. At a dilution rate of 0.87 h(-1), the high cell density continuous culture exhibited the highest D-lactic acid productivity of 18.0 g/l/h with a high yield (ca. 1.0 g/g consumed glucose) and a low residual glucose (<0.1 g/l) in comparison with systems published to date.

Isolation and identification of lactic acid bacteria from koumiss in Eastern Inner Mongolia of China

NASA Astrophysics Data System (ADS)

Bai, Lijuan; Ji, Shujuan

2017-01-01

Koumiss is a traditional fermented dairy product and known as its unique physiological actions. Isolation and identification of LAB in it will yield valuable knowledge. In total, 55 LAB strains were isolated and identified of 12 koumiss samples collected in limited regions of Eastern Inner Mongolia. 16S rRNA sequence analysis results showed that were Lactobacillus helveticus, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus delbrueckii, Enterococcus. durans, Leuconostoc lactis and Leuconostoc mesenteroides. It is benefit to further research on koumiss.

Antibiotic susceptibility of bifidobacterial strains distributed in the Japanese market.

PubMed

Xiao, Jin-Zhong; Takahashi, Sachiko; Odamaki, Toshitaka; Yaeshima, Tomoko; Iwatsuki, Keiji

2010-01-01

The aim of the present study was to analyze the antibiotic susceptibility of bifidobacterial strains distributed in the Japanese market. A total of 23 strains, including probiotic isolates from foods, supplements, pharmaceuticals and reference strains of each species (or subspecies), were tested for susceptibility to 15 antibiotics by the broth microdilution method and examined for the presence of possible resistant determinants. The strains were susceptible overall to chloramphenicol, ampicillin, vancomycin and linezolid, and were intrinsically resistant to aminoglycoside group agents. Susceptibility to erythromycin, clindamycin, rifampicin, tetracycline and trimethoprim varied among the strains. All strains of Bifidobacterium animalis subsp. lactis were resistant to tetracycline and appeared to harbor tet(W) genes. No risk factor for safety was found for bifidobacterial strains distributed in the Japanese market in respect of their antimicrobial resistance, although the presence of the tet(W) gene in some strains stresses the need for future evaluation.

Characterization, identification and application of lactic Acid bacteria isolated from forage paddy rice silage.

PubMed

Ni, Kuikui; Wang, Yanping; Li, Dongxia; Cai, Yimin; Pang, Huili

2015-01-01

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.

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

PubMed Central

Ni, Kuikui; Wang, Yanping; Li, Dongxia; Cai, Yimin; Pang, Huili

2015-01-01

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. PMID:25803578

Lactococcus lactis subsp. lactis infection in Bester sturgeon, a cultured hybrid of Huso huso × Acipenser ruthenus, in Taiwan.

PubMed

Chen, Ming-Hui; Hung, Shao-Wen; Shyu, Ching-Lin; Lin, Cheng-Chung; Liu, Pan-Chen; Chang, Chen-Hsuan; Shia, Wei-Yau; Cheng, Ching-Fu; Lin, Shiun-Long; Tu, Ching-Yu; Lin, Yu-Hsing; Wang, Way-Shyan

2012-10-01

Approximately 5300 hybrid sturgeons with an average body weight of 600-800 g were farmed in 3 round tankers measuring 3m in diameter each containing 28,000 L of aerated groundwater. According to the owner's description, the diseased fish had anorexia, pale body color, and reddish spots on the abdomen. The morbidity and lethality rates in this outbreak were about 70% (3706/5300) and 100% (3706/3706), respectively. The clinical examination revealed enteritis, enlarged abdomen, and rapid respiration rate. The gross findings revealed a volume of about 4 mL of ascites. The histopathological examination showed multiple massive, hemorrhagic or coagulative necrotic foci in the liver and spleen. Furthermore, there was diffuse infiltration of glycogen in hepatic cells, and a few polymorphonuclear and mononuclear leucocytes were observed surrounding the spleen. Some bacterial clumps were noted around the necrotic foci. We also observed that there was moderate to severe, acute, multifocal, coagulative necrosis in the renal parenchyma, with some necrotic foci present beneath the margin of the kidney. Additionally, multifocal, coagulative necrosis was found in the pancreas. Results of microbiologic examinations, including biochemical characteristics, PCR amplification of 16S rRNA gene, sequencing and comparison, and phylogenetic analysis, revealed the pathogen of this infection was Lactococcus lactis subsp. lactis, and based on the results of an antimicrobial agent sensitivity test the bacterium was only sensitive to ampicillin and florfenicol. Additionally, results of in vivo experimental infections in hybrid tilapia showed that 1×10(8) and 1×10(9) CFU/mL of our isolate caused death in all fish and LD(50) values ranged from 10(2) to 10(5) CFU/mL. To the best of the authors' knowledge, this is the first reported case of Lactococcus lactis subsp. lactis infection in hybrid sturgeon. Copyright © 2011 Elsevier Ltd. All rights reserved.

Engineering Trehalose Synthesis in Lactococcus lactis for Improved Stress Tolerance ▿ †

PubMed Central

Carvalho, Ana Lúcia; Cardoso, Filipa S.; Bohn, Andreas; Neves, Ana Rute; Santos, Helena

2011-01-01

Trehalose accumulation is a common cell defense strategy against a variety of stressful conditions. In particular, our team detected high levels of trehalose in Propionibacterium freudenreichii in response to acid stress, a result that led to the idea that endowing Lactococcus lactis with the capacity to synthesize trehalose could improve the acid tolerance of this organism. To this end, we took advantage of the endogenous genes involved in the trehalose catabolic pathway of L. lactis, i.e., trePP and pgmB, encoding trehalose 6-phosphate phosphorylase and β-phosphoglucomutase, respectively, which enabled the synthesis of trehalose 6-phosphate. Given that L. lactis lacks trehalose 6-phosphate phosphatase, the respective gene, otsB, from the food-grade organism P. freudenreichii was used to provide the required activity. The trehalose yield was approximately 15% in resting cells and in mid-exponential-phase cells grown without pH control. The intracellular concentration of trehalose reached maximal values of approximately 170 mM, but at least 67% of the trehalose produced was found in the growth medium. The viability of mutant and control strains was examined after exposure to heat, cold or acid shock, and freeze-drying. The trehalose-producing strains showed improved tolerance (5- to 10-fold-higher survivability) to acid (pH 3) and cold shock (4°C); there was also a strong improvement in cell survival in response to heat shock (45°C), and no protection was rendered against dehydration. The insight provided by this work may help the design of food-grade strains optimized for the dairy industry as well as for oral drug delivery. PMID:21515730

Use of the usp45 lactococcal secretion signal sequence to drive the secretion and functional expression of enterococcal bacteriocins in Lactococcus lactis.

PubMed

Borrero, Juan; Jiménez, Juan J; Gútiez, Loreto; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

2011-01-01

Replacement of the signal peptide (SP) of the bacteriocins enterocin P (EntP) and hiracin JM79 (HirJM79), produced by Enterococcus faecium P13 and Enterococcus hirae DCH5, respectively, by the signal peptide of Usp45 (SP(usp45)), the major Sec-dependent protein secreted by Lactococcus lactis, permits the production, secretion, and functional expression of EntP and HirJM79 by L. lactis. Chimeric genes encoding the SP(usp45) fused to either mature EntP (entP), with or without the immunity gene (entiP) or to mature HirJM79 (hirJM79), with or without the immunity gene (hiriJM79), were cloned into the expression vector pMG36c, carrying the P(32) constitutive promoter, and into pNZ8048 under control of the inducible PnisA promoter. The production of EntP and HirJM79 by most of the L. lactis recombinant strains was 1.5- to 3.7-fold higher and up to 3.6-fold higher than by the E. faecium P13 and E. hirae DCH5 control strains, respectively. However, the specific antimicrobial activity of the recombinant EntP was 1.1- to 6.2-fold higher than that produced by E. faecium P13, while that of the HirJM79 was a 40% to an 89% of that produced by E. hirae DCH5. Chimeras of SP(usp45) fused to mature EntP or HirJM79 drive the production and secretion of these bacteriocins in L. lactis in the absence of specific immunity and secretion proteins. The supernatants of the recombinant L. lactis NZ9000 strains, producers of EntP, showed a much higher antimicrobial activity against Listeria spp. than that of the recombinant L. lactis NZ9000 derivatives, producers of HirJM79.

The extracellular phage-host interactions involved in the bacteriophage LL-H infection of Lactobacillus delbrueckii ssp. lactis ATCC 15808

PubMed Central

Munsch-Alatossava, Patricia; Alatossava, Tapani

2013-01-01

The complete genome sequence of Lactobacillus bacteriophage LL-H was determined in 1996. Accordingly, LL-H has been used as a model phage for the infection of dairy Lactobacillus, specifically for thermophilic Lactobacillus delbrueckii ssp. lactis host strains, such as ATCC 15808. One of the major goals of phage LL-H research consisted of the characterization of the first phage-host interactions at the level of phage adsorption and phage DNA injection steps to determine effective and practical methods to minimize the risks associated with the appearance and attack of phages in the manufacture of yogurt, and Swiss or Italian hard type cheeses, which typically use thermophilic lactic acid bacteria starter cultures containing L. delbrueckii strains among others. This mini review article summarizes the present data concerning (i) the special features, particle structure, and components of phage LL-H and (ii) the structure and properties of lipoteichoic acids (LTAs), which are the phage LL-H receptor components of L. delbrueckii ssp. lactis host strains. Moreover, a model of the first, extracellular, phage-host interactions for the infection of L. delbrueckii ssp. lactis ATCC 15808 by phage LL-H is presented and further discussed. PMID:24400001

The extracellular phage-host interactions involved in the bacteriophage LL-H infection of Lactobacillus delbrueckii ssp. lactis ATCC 15808.

PubMed

Munsch-Alatossava, Patricia; Alatossava, Tapani

2013-12-24

The complete genome sequence of Lactobacillus bacteriophage LL-H was determined in 1996. Accordingly, LL-H has been used as a model phage for the infection of dairy Lactobacillus, specifically for thermophilic Lactobacillus delbrueckii ssp. lactis host strains, such as ATCC 15808. One of the major goals of phage LL-H research consisted of the characterization of the first phage-host interactions at the level of phage adsorption and phage DNA injection steps to determine effective and practical methods to minimize the risks associated with the appearance and attack of phages in the manufacture of yogurt, and Swiss or Italian hard type cheeses, which typically use thermophilic lactic acid bacteria starter cultures containing L. delbrueckii strains among others. This mini review article summarizes the present data concerning (i) the special features, particle structure, and components of phage LL-H and (ii) the structure and properties of lipoteichoic acids (LTAs), which are the phage LL-H receptor components of L. delbrueckii ssp. lactis host strains. Moreover, a model of the first, extracellular, phage-host interactions for the infection of L. delbrueckii ssp. lactis ATCC 15808 by phage LL-H is presented and further discussed.

Mutation of the oxaloacetate decarboxylase gene of Lactococcus lactis subsp. lactis impairs the growth during citrate metabolism.

PubMed

Augagneur, Y; Garmyn, D; Guzzo, J

2008-01-01

Citrate metabolism generates metabolic energy through the generation of a membrane potential and a pH gradient. The purpose of this work was to study the influence of oxaloacetate decarboxylase in citrate metabolism and intracellular pH maintenance in relation to acidic conditions. A Lactococcus lactis oxaloacetate decarboxylase mutant [ILCitM (pFL3)] was constructed by double homologous recombination. During culture with citrate, and whatever the initial pH, the growth rate of the mutant was lower. In addition, the production of diacetyl and acetoin was altered in the mutant strain. However, our results indicated no relationship with a change in the maintenance of intracellular pH. Experiments performed on resting cells clearly showed that oxaloacetate accumulated temporarily in the supernatant of the mutant. This accumulation could be involved in the perturbations observed during citrate metabolism, as the addition of oxaloacetate in M17 medium inhibited the growth of L. lactis. The mutation of oxaloacetate decarboxylase perturbed citrate metabolism and reduced the benefits of its utilization during growth under acidic conditions. This study allows a better understanding of citrate metabolism and the role of oxaloacetate decarboxylase in the tolerance of lactic acid bacteria to acidic conditions.

Extraction of the same novel homoglycan mixture from two different strains of Bifidobacterium animalis and three strains of Bifidobacterium breve.

PubMed

Alhudhud, M; Sadiq, S; Ngo, H N; Hidalgo-Cantabrana, C; Ruas-Madiedo, P; van Sinderen, D; Humphreys, P N; Laws, A P

2018-06-15

Three strains of Bifidobacterium breve (JCM 7017, JCM 7019 and JCM 2258) and two strains of Bifidobacterium animalis subsp. lactis (AD011 and A1dOxR) were grown in broth cultures or on plates, and a standard exopolysaccharide extraction method was used in an attempt to recover exocellular polysaccharides. When the extracted materials were analysed by NMR it was clear that mixtures of polysaccharides were being isolated including exopolysaccharides (EPS) cell wall polysaccharides and intracellular polysaccharides. Treatment of the cell biomass from the B. breve strains, or the B. animalis subsp. lactis AD011 strain, with aqueous sodium hydroxide provided a very similar mixture of polysaccharides but without the EPS. The different polysaccharides were partially fractionated by selective precipitation from an aqueous solution upon the addition of increasing percentages of ethanol. The polysaccharides extracted from B. breve JCM 7017 grown in HBM media supplemented with glucose (or isotopically labelled D-glucose-1- 13 C) were characterised using 1D and 2D-NMR spectroscopy. Addition of one volume of ethanol generated a medium molecular weight glycogen (Mw=1×10 5 Da, yield 200 mg/l). The addition of two volumes of ethanol precipitated an intimate mixture of a low molecular weight β-(1→6)-glucan and a low molecular weight β-(1→6)-galactofuranan which could not be separated (combined yield 46 mg/l). When labelled D-glucose-1- 13 C was used as a carbon supplement, the label was incorporated into >95% of the anomeric carbons of each polysaccharide confirming they were being synthesised in situ. Similar 1 H NMR profiles were obtained for polysaccharides recovered from the cells of B. animalis subsp. lactis AD011and A1dOxR (in combination with an EPS), B. breve JCM 7017, B. breve JCM 7019, B. breve JCM 2258 and from an EPS (-ve) mutant of B. breve 7017 (a non-EPS producer).

Display of the Viral Epitopes on Lactococcus lactis: A Model for Food Grade Vaccine against EV71

PubMed Central

Varma, Nadimpalli Ravi S.; Toosa, Haryanti; Foo, Hooi Ling; Alitheen, Noorjahan Banu Mohamed; Nor Shamsudin, Mariana; Arbab, Ali S.; Yusoff, Khatijah; Abdul Rahim, Raha

2013-01-01

In this study, we have developed a system for display of antigens of Enterovirus type 71 (EV71) on the cell surface of L. lactis. The viral capsid protein (VP1) gene from a local viral isolate was utilized as the candidate vaccine for the development of oral live vaccines against EV71 using L. lactis as a carrier. We expressed fusion proteins in E. coli and purified fusion proteins were incubated with L. lactis. We confirmed that mice orally fed with L. lactis displaying these fusion proteins on its surface were able to mount an immune response against the epitopes of EV71. This is the first example of an EV71 antigen displayed on the surface of a food grade organism and opens a new perspective for alternative vaccine strategies against the EV71. We believe that the method of protein docking utilized in this study will allow for more flexible presentations of short peptides and proteins on the surface of L. lactis to be useful as a delivery vehicle. PMID:23476790

From field to fermentation: the origins of Lactococcus lactis and its domestication to the dairy environment.

PubMed

Cavanagh, Daniel; Fitzgerald, Gerald F; McAuliffe, Olivia

2015-05-01

Lactococcus lactis is an organism of substantial economic importance, used extensively in the production of fermented foods and widely held to have evolved from plant strains. The domestication of this organism to the milk environment is associated with genome reduction and gene decay, and the acquisition of specific genes involved in protein and lactose utilisation by horizontal gene transfer. In recent years, numerous studies have focused on uncovering the physiology and molecular biology of lactococcal strains from the wider environment for exploitation in the dairy industry. This in turn has facilitated comparative genome analysis of lactococci from different environments and provided insight into the natural phenotypic and genetic diversity of L. lactis. This diversity may be exploited in dairy fermentations to develop products with improved quality and sensory attributes. In this review, we discuss the classification of L. lactis and the problems that arise with phenotype/genotype designation. We also discuss the adaptation of non-dairy lactococci to milk, the traits associated with this adaptation and the potential application of non-dairy lactococci to dairy fermentations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Use of PCR-Based Methods for Rapid Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis

PubMed Central

Torriani, Sandra; Zapparoli, Giacomo; Dellaglio, Franco

1999-01-01

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412T, which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains. PMID:10508059

Use of PCR-based methods for rapid differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis.

PubMed

Torriani, S; Zapparoli, G; Dellaglio, F

1999-10-01

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412(T), which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains.

Lactococcus lactis expressing food-grade β-galactosidase alleviates lactose intolerance symptoms in post-weaning Balb/c mice.

PubMed

Li, Jingjie; Zhang, Wen; Wang, Chuan; Yu, Qian; Dai, Ruirui; Pei, Xiaofang

2012-12-01

The endogenous β-galactosidase expressed in intestinal microbes is demonstrated to help humans in lactose usage, and treatment associated with the promotion of beneficial microorganism in the gut is correlated with lactose tolerance. From this point, a kind of recombinant live β-galactosidase delivery system using food-grade protein expression techniques and selected probiotics as vehicle was promoted by us for the purpose of application in lactose intolerance subjects. Previously, a recombinant Lactococcus lactis MG1363 strain expressing food-grade β-galactosidase, the L. lactis MG1363/FGZW, was successfully constructed and evaluated in vitro. This study was conducted to in vivo evaluate its efficacy on alleviating lactose intolerance symptoms in post-weaning Balb/c mice, which were orally administered with 1 × 10⁶ CFU or 1 × 10⁸ CFU of L. lactis MG1363/FGZW daily for 4 weeks before lactose challenge. In comparison with naïve mice, the mice administered with L. lactis MG1363/FGZW showed significant alleviation of diarrhea symptoms in less total feces weight within 6 h post-challenge and suppressed intestinal motility after lactose challenge, although there was no significant increase of β-galactosidase activity in small intestine. The alleviation also correlated with higher species abundance, more Bifidobacterium colonization, and stronger colonization resistance in mice intestinal microflora. Therefore, this recombinant L. lactis strain effectively alleviated diarrhea symptom induced by lactose uptake in lactose intolerance model mice with the probable mechanism of promotion of lactic acid bacteria to differentiate and predominantly colonize in gut microbial community, thus making it a promising probiotic for lactose intolerance subjects.

Phenotypic and Genotypic Characterization of Some Lactic Acid Bacteria Isolated from Bee Pollen: A Preliminary Study

PubMed Central

BELHADJ, Hani; HARZALLAH, Daoud; BOUAMRA, Dalila; KHENNOUF, Seddik; Dahamna, Saliha; GHADBANE, Mouloud

2014-01-01

In the present work, five hundred and sixty-seven isolates of lactic acid bacteria were recovered from raw bee pollen grains. All isolates were screened for their antagonistic activity against both Gram-positive and Gram-negative pathogenic bacteria. Neutralized supernatants of 54 lactic acid bacteria (LAB) cultures from 216 active isolates inhibited the growth of indicator bacteria. They were phenotypically characterized, based on the fermentation of 39 carbohydrates. Using the simple matching coefficient and unweighted pair group algorithm with arithmetic averages (UPGMA), seven clusters with other two members were defined at the 79% similarity level. The following species were characterized: Lactobacillus plantarum, Lactobacillus fermentum, Lactococcus lactis, Pediococcus acidilactici, Pediococcus pentosaceus, and unidentified lactobacilli. Phenotypic characteristics of major and minor clusters were also identified. Partial sequencing of the 16S rRNA gene of representative isolates from each cluster was performed, and ten strains were assigned to seven species: Lactobacillus plantarum, Lactobacillus fermentum, Lactococcus lactis, Lactobacillus ingluviei, Pediococcus pentosaceus, Lactobacillus acidipiscis and Weissella cibaria. The molecular method used failed to determine the exact taxonomic status of BH0900 and AH3133. PMID:24936378

Phenotypic and genotypic characterization of some lactic Acid bacteria isolated from bee pollen: a preliminary study.

PubMed

Belhadj, Hani; Harzallah, Daoud; Bouamra, Dalila; Khennouf, Seddik; Dahamna, Saliha; Ghadbane, Mouloud

2014-01-01

In the present work, five hundred and sixty-seven isolates of lactic acid bacteria were recovered from raw bee pollen grains. All isolates were screened for their antagonistic activity against both Gram-positive and Gram-negative pathogenic bacteria. Neutralized supernatants of 54 lactic acid bacteria (LAB) cultures from 216 active isolates inhibited the growth of indicator bacteria. They were phenotypically characterized, based on the fermentation of 39 carbohydrates. Using the simple matching coefficient and unweighted pair group algorithm with arithmetic averages (UPGMA), seven clusters with other two members were defined at the 79% similarity level. The following species were characterized: Lactobacillus plantarum, Lactobacillus fermentum, Lactococcus lactis, Pediococcus acidilactici, Pediococcus pentosaceus, and unidentified lactobacilli. Phenotypic characteristics of major and minor clusters were also identified. Partial sequencing of the 16S rRNA gene of representative isolates from each cluster was performed, and ten strains were assigned to seven species: Lactobacillus plantarum, Lactobacillus fermentum, Lactococcus lactis, Lactobacillus ingluviei, Pediococcus pentosaceus, Lactobacillus acidipiscis and Weissella cibaria. The molecular method used failed to determine the exact taxonomic status of BH0900 and AH3133.

Identification and characterization of lactic acid bacteria isolated from mixed pasture of timothy and orchardgrass, and its badly preserved silage.

PubMed

Tohno, Masanori; Kobayashi, Hisami; Nomura, Masaru; Uegaki, Ryuichi; Cai, Yimin

2012-04-01

In order to understand the relationship between lactic acid bacteria (LAB) species and silage fermentation, a total of 65 LAB strains isolated from mixed pasture of timothy (Phleum pratense L.) and orchardgrass (Dactylis glomerata L.), and its badly preserved silages were subjected to phenotypic and genetic analysis. According to these analyses, the isolates were divided into 13 groups, including Enterococcus gallinarum, Lactobacillus acidipiscis, L. coryniformis subsp. coryniformis, L. coryniformis subsp. torquens, L. curvatus, L. paraplantarum, L. plantarum subsp. argentoratensis, L. plantarum subsp. plantarum, L. sakei subsp. carnosus, Lactococcus garvieae, Lactococcus lactis subsp. cremoris, Leuconostoc pseudomesenteroides, Pediococcus acidilactici, Pediococcus pentosaceus, Weissella hellenica, Weissella paramesenteroides and Carnobacterium divergens. This is the first report to document that C. divergens, L. acidipiscis, L. sakei subsp. carnosus, L. garvieae, phenotypically novel L. lactis subsp. cremoris, E. gallinarum and W. hellenica are present in vegetative forage crops. L. plantarum group strains were most frequently isolated from the badly preserved silages. Some isolates showed a wide range of growth preferences for carbohydrate utilization, optimal growth pH and temperature in vitro, indicating that they have a high growth potential. These results are useful in understanding the diversity of LAB associated with decayed silage of timothy and orchardgrass. © 2011 The Authors. Animal Science Journal © 2011 Japanese Society of Animal Science.

DNA-DNA hybridization study of strains of Chryseobacterium, Elizabethkingia and Empedobacter and of other usually indole-producing non-fermenters of CDC groups IIc, IIe, IIh and IIi, mostly from human clinical sources, and proposals of Chryseobacterium bernardetii sp. nov., Chryseobacterium carnis sp. nov., Chryseobacterium lactis sp. nov., Chryseobacterium nakagawai sp. nov. and Chryseobacterium taklimakanense comb. nov.

PubMed

Holmes, B; Steigerwalt, A G; Nicholson, A C

2013-12-01

The taxonomic classification of 182 phenotypically similar isolates was evaluated using DNA-DNA hybridization and 16S rRNA gene sequence analysis. These bacterial isolates were mainly derived from clinical sources; all were Gram-negative non-fermenters and most were indole-producing. Phenotypically, they resembled species from the genera Chryseobacterium, Elizabethkingia or Empedobacter or belonged to CDC groups IIc, IIe, IIh and IIi. Based on these analyses, four novel species are described: Chryseobacterium bernardetii sp. nov. (type strain NCTC 13530(T) = CCUG 60564(T) = CDC G229(T)), Chryseobacterium carnis sp. nov. (type strain NCTC 13525(T) = CCUG 60559(T) = CDC G81(T)), Chryseobacterium lactis sp. nov. (type strain NCTC 11390(T) = CCUG 60566(T) = CDC KC1864(T)) and Chryseobacterium nakagawai sp. nov. (type strain NCTC 13529(T) = CCUG 60563(T) = CDC G41(T)). The new combination Chryseobacterium taklimakanense comb. nov. (type strain NCTC 13490(T) = X-65(T) = CCTCC AB 208154(T) = NRRL B-51322(T)) is also proposed to accommodate the reclassified Planobacterium taklimakanense.

Antifungal activity of strains of lactic acid bacteria isolated from a semolina ecosystem against Penicillium roqueforti, Aspergillus niger and Endomyces fibuliger contaminating bakery products.

PubMed

Valerio, Francesca; Favilla, Mara; De Bellis, Palmira; Sisto, Angelo; de Candia, Silvia; Lavermicocca, Paola

2009-09-01

Thirty samples of Italian durum wheat semolina and whole durum wheat semolina, generally used for the production of Southern Italy's traditional breads, were subjected to microbiological analysis in order to explore their lactic acid bacteria (LAB) diversity and to find strains with antifungal activity. A total of 125 presumptive LAB isolates (Gram-positive and catalase-negative) were characterized by repetitive extragenic palindromic-PCR (REP-PCR) and sequence analysis of the 16S rRNA gene, leading to the identification of the following species: Weissella confusa, Weissella cibaria, Leuconostoc citreum, Leuconostoc mesenteroides, Lactococcus lactis, Lactobacillus rossiae and Lactobacillus plantarum. The REP-PCR results delineated 17 different patterns whose cluster analysis clearly differentiated W. cibaria from W. confusa isolates. Seventeen strains, each characterized by a different REP-PCR pattern, were screened for their antifungal properties. They were grown in a flour-based medium, comparable to a real food system, and the resulting fermentation products (FPs) were tested against fungal species generally contaminating bakery products, Aspergillus niger, Penicillium roqueforti and Endomyces fibuliger. The results of the study indicated a strong inhibitory activity - comparable to that obtained with the common preservative calcium propionate (0.3% w/v) - of ten LAB strains against the most widespread contaminant of bakery products, P. roqueforti. The screening also highlighted the unexplored antifungal activity of L. citreum, L. rossiae and W. cibaria (1 strain), which inhibited all fungal strains to the same or a higher extent compared with calcium propionate. The fermentation products of these three strains were characterized by low pH values, and a high content of lactic and acetic acids.

Immunopathological evaluation of recombinant mycobacterial antigen Hsp65 expressed in Lactococcus lactis as a novel vaccine candidate

PubMed Central

Herrera Ramírez, J. C.; De la Mora, A. Ch.; De la Mora Valle, A.; Lopez-Valencia, G.; Hurtado, R. M. B.; Rentería Evangelista, T. B.; Rodríguez Castillo, J. L.; Rodríguez Gardea, A.; Gómez Gómez, S. D.; Medina-Basulto, G. E.

2017-01-01

Bovine tuberculosis (TBB) is a zoonotic disease distributed worldwide and is of great importance for public health and the livestock industry. Several experimental vaccines against this disease have been evaluated in recent years, yielding varying results. An example is the Bacillus Calmette-Guérin (BCG) vaccine, which has been used extensively in humans and tested in cattle showing mixed results related to protection (0-80%) against Mycobacterium bovis. In this study, we used the food-grade bacterium Lactococcus lactis as an expression system for production of mycobacterial protein Hsp65. For this purpose, the construction of a replicable plasmid in strain NZ9000 L. lactis (pVElepr) was conducted, which expressed the Mycobacterium leprae Hsp65 antigen, and was recognized by traded anti-Hsp65 antibodies. The strain NZ9000-pVElepr was applied to calves that were negative to tuberculin test and the immune response was monitored. The results showed that immune response was not significantly increased in calves with NZ9000-pVElepr with respect to control groups, and no injury was observed in any lung or lymph of the calves. Finally, this study suggest that the recombinant NZ9000 strain of L. lactis may protect against the development of M. bovis infection, although studies with longer exposure to this pathogen are necessary to conclude the matter. PMID:29163649

Isolating Escherichia coli strains for recombinant protein production.

PubMed

Schlegel, Susan; Genevaux, Pierre; de Gier, Jan-Willem

2017-03-01

Escherichia coli has been widely used for the production of recombinant proteins. To improve protein production yields in E. coli, directed engineering approaches have been commonly used. However, there are only few reported examples of the isolation of E. coli protein production strains using evolutionary approaches. Here, we first give an introduction to bacterial evolution and mutagenesis to set the stage for discussing how so far selection- and screening-based approaches have been used to isolate E. coli protein production strains. Finally, we discuss how evolutionary approaches may be used in the future to isolate E. coli strains with improved protein production characteristics.

Strain variation in Mycobacterium marinum fish isolates.

PubMed

Ucko, M; Colorni, A; Kvitt, H; Diamant, A; Zlotkin, A; Knibb, W R

2002-11-01

A molecular characterization of two Mycobacterium marinum genes, 16S rRNA and hsp65, was carried out with a total of 21 isolates from various species of fish from both marine and freshwater environments of Israel, Europe, and the Far East. The nucleotide sequences of both genes revealed that all M. marinum isolates from fish in Israel belonged to two different strains, one infecting marine (cultured and wild) fish and the other infecting freshwater (cultured) fish. A restriction enzyme map based on the nucleotide sequences of both genes confirmed the divergence of the Israeli marine isolates from the freshwater isolates and differentiated the Israeli isolates from the foreign isolates, with the exception of one of three Greek isolates from marine fish which was identical to the Israeli marine isolates. The second isolate from Greece exhibited a single base alteration in the 16S rRNA sequence, whereas the third isolate was most likely a new Mycobacterium species. Isolates from Denmark and Thailand shared high sequence homology to complete identity with reference strain ATCC 927. Combined analysis of the two gene sequences increased the detection of intraspecific variations and was thus of importance in studying the taxonomy and epidemiology of this aquatic pathogen. Whether the Israeli M. marinum strain infecting marine fish is endemic to the Red Sea and found extremely susceptible hosts in the exotic species imported for aquaculture or rather was accidentally introduced with occasional imports of fingerlings from the Mediterranean Sea could not be determined.

Early adaptation to oxygen is key to the industrially important traits of Lactococcus lactis ssp. cremoris during milk fermentation.

PubMed

Cretenet, Marina; Le Gall, Gwenaëlle; Wegmann, Udo; Even, Sergine; Shearman, Claire; Stentz, Régis; Jeanson, Sophie

2014-12-03

Lactococcus lactis is the most used species in the dairy industry. Its ability to adapt to technological stresses, such as oxidative stress encountered during stirring in the first stages of the cheese-making process, is a key factor to measure its technological performance. This study aimed to understand the response to oxidative stress of Lactococcus lactis subsp. cremoris MG1363 at the transcriptional and metabolic levels in relation to acidification kinetics and growth conditions, especially at an early stage of growth. For those purposes, conditions of hyper-oxygenation were initially fixed for the fermentation. Kinetics of growth and acidification were not affected by the presence of oxygen, indicating a high resistance to oxygen of the L. lactis MG1363 strain. Its resistance was explained by an efficient consumption of oxygen within the first 4 hours of culture, leading to a drop of the redox potential. The efficient consumption of oxygen by the L. lactis MG1363 strain was supported by a coherent and early adaptation to oxygen after 1 hour of culture at both gene expression and metabolic levels. In oxygen metabolism, the over-expression of all the genes of the nrd (ribonucleotide reductases) operon or fhu (ferrichrome ABC transports) genes was particularly significant. In carbon metabolism, the presence of oxygen led to an early shift at the gene level in the pyruvate pathway towards the acetate/2,3-butanediol pathway confirmed by the kinetics of metabolite production. Finally, the MG1363 strain was no longer able to consume oxygen in the stationary growth phase, leading to a drastic loss of culturability as a consequence of cumulative stresses and the absence of gene adaptation at this stage. Combining metabolic and transcriptomic profiling, together with oxygen consumption kinetics, yielded new insights into the whole genome adaptation of L. lactis to initial oxidative stress. An early and transitional adaptation to oxidative stress was revealed for L

Isolation and characterization of ethanol tolerant yeast strains

PubMed Central

Tikka, Chiranjeevi; Osuru, Hari Prasad; Atluri, Navya; Raghavulu, Praveen Chakravarthi Veera; yellapu, Nanda Kumar; Mannur, Ismail Shaik; Prasad, Uppu Venkateswara; Aluru, Sudheer; K, Narasimha Varma; Bhaskar, Matcha

2013-01-01

Yeast strains are commonly associated with sugar rich environments. Various fruit samples were selected as source for isolating yeast cells. The isolated cultures were identified at Genus level by colony morphology, biochemical characteristics and cell morphological characters. An attempt has been made to check the viability of yeast cells under different concentrations of ethanol. Ethanol tolerance of each strain was studied by allowing the yeast to grow in liquid YEPD (Yeast Extract Peptone Dextrose) medium having different concentrations of ethanol. A total of fifteen yeast strains isolated from different samples were used for the study. Seven strains of Saccharomyces cerevisiae obtained from different fruit sources were screened for ethanol tolerance. The results obtained in this study show a range of tolerance levels between 7%-12% in all the stains. Further, the cluster analysis based on 22 RAPD (Random Amplified polymorphic DNA) bands revealed polymorphisms in these seven Saccharomyces strains. PMID:23750092

Mechanism of Citrate Metabolism by an Oxaloacetate Decarboxylase-Deficient Mutant of Lactococcus lactis IL1403 ▿

PubMed Central

Pudlik, Agata M.; Lolkema, Juke S.

2011-01-01

Citrate metabolism in resting cells of Lactococcus lactis IL1403(pFL3) results in the formation of two end products from the intermediate pyruvate, acetoin and acetate (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:706–714, 2011). Pyruvate is formed from citrate following uptake by the transporter CitP through the subsequent actions of citrate lyase and oxaloacetate decarboxylase. The present study describes the metabolic response of L. lactis when oxaloacetate accumulates in the cytoplasm. The oxaloacetate decarboxylase-deficient mutant ILCitM(pFL3) showed nearly identical rates of citrate consumption, but the end product profile in the presence of glucose shifted from mainly acetoin to only acetate. In addition, in contrast to the parental strain, the mutant strain did not generate proton motive force. Citrate consumption by the mutant strain was coupled to the excretion of oxaloacetate, with a yield of 80 to 85%. Following citrate consumption, oxaloacetate was slowly taken up by the cells and converted to pyruvate by a cryptic decarboxylase and, subsequently, to acetate. The transport of oxaloacetate is catalyzed by CitP. The parental strain IL1403(pFL3) containing CitP consumed oxaloacetate, while the original strain, IL1403, not containing CitP, did not. Moreover, oxaloacetate consumption was enhanced in the presence of l-lactate, indicating exchange between oxaloacetate and l-lactate catalyzed by CitP. Hence, when oxaloacetate inadvertently accumulates in the cytoplasm, the physiological response of L. lactis is to excrete oxaloacetate in exchange with citrate by an electroneutral mechanism catalyzed by CitP. Subsequently, in a second step, oxaloacetate is taken up by CitP and metabolized to pyruvate and acetate. PMID:21665973

Antibiotic resistance of lactic acid bacteria and Bifidobacterium spp. isolated from dairy and pharmaceutical products.

PubMed

D'Aimmo, Maria Rosaria; Modesto, Monica; Biavati, Bruno

2007-04-01

The outlines of antibiotic resistance of some probiotic microorganisms were studied. This study was conducted with the double purpose of verifying their ability to survive if they are taken simultaneously with an antibiotic therapy and to increase the selective properties of suitable media for the isolation of samples containing mixed bacterial populations. We isolated from commercial dairy and pharmaceutical products, 34 strains declared as probiotics, belonging to the genera Bifidobacterium and Lactobacillus, and 21 strains of starter culture bacteria. All the microorganisms have been compared by electrophoresis of the soluble proteins for the purpose of identifying them. A Multiplex-PCR with genus- and species-specific primers was used to detect for Bifidobacterium animalis subsp. lactis presence. All bifidobacteria were B. animalis subsp. lactis except one Bifidobacterium longum. Sometimes the identification showed that the used strain was not the one indicated on the label. The lactobacilli were Lactobacillus acidophilus, Lactobacillus casei, and Lactobacillus delbrueckii subsp. bulgaricus. The streptococci were all Streptococcus thermophilus. The minimal inhibitory concentration (MIC) of 24 common antibiotic substances has been valued by the broth microdilution method. All tested strains were susceptible to ampicillin, bacitracin, clindamycin, dicloxacillin, erytromycin, novobiocin, penicillin G, rifampicin (MIC(90) ranging from 0.01 to 4 microg/ml); resistant to aztreonam, cycloserin, kanamycin, nalidixic acid, polymyxin B and spectinomycin (MIC(90) ranging from 64 to >1000 microg/ml). The susceptibility to cephalothin, chloramphenicol, gentamicin, lincomycin, metronidazole, neomycin, paromomycin, streptomycin, tetracycline and vancomycin was variable and depending on the species.

Improvement of LysM-Mediated Surface Display of Designed Ankyrin Repeat Proteins (DARPins) in Recombinant and Nonrecombinant Strains of Lactococcus lactis and Lactobacillus Species

PubMed Central

Zadravec, Petra; Štrukelj, Borut

2015-01-01

Safety and probiotic properties make lactic acid bacteria (LAB) attractive hosts for surface display of heterologous proteins. Protein display on nonrecombinant microorganisms is preferred for therapeutic and food applications due to regulatory requirements. We displayed two designed ankyrin repeat proteins (DARPins), each possessing affinity for the Fc region of human IgG, on the surface of Lactococcus lactis by fusing them to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA, containing lysine motif (LysM) repeats. Growth medium containing a secreted fusion protein was used to test its heterologous binding to 10 strains of species of the genus Lactobacillus, using flow cytometry, whole-cell enzyme-linked immunosorbent assay (ELISA), and fluorescence microscopy. The fusion proteins bound to the surfaces of all lactobacilli; however, binding to the majority of bacteria was only 2- to 5-fold stronger than that of the control. Lactobacillus salivarius ATCC 11741 demonstrated exceptionally strong binding (32- to 55-fold higher than that of the control) and may therefore be an attractive host for nonrecombinant surface display. Genomic comparison of the species indicated the exopolysaccharides of Lb. salivarius as a possible reason for the difference. Additionally, a 15-fold concentration-dependent increase in nonrecombinant surface display on L. lactis was demonstrated by growing bacteria with sublethal concentrations of the antibiotics chloramphenicol and erythromycin. Nonrecombinant surface display on LAB, based on LysM repeats, was optimized by selecting Lactobacillus salivarius ATCC 11741 as the optimal host and by introducing antibiotics as additives for increasing surface display on L. lactis. Additionally, effective display of DARPins on the surfaces of nonrecombinant LAB has opened up several new therapeutic possibilities. PMID:25576617

Some Technological Properties of Lactic Acid Bacteria Isolated from Dahi and Datshi, Naturally Fermented Milk Products of Bhutan

PubMed Central

Shangpliang, H. N. J.; Sharma, Sharmila; Rai, Ranjita; Tamang, Jyoti P.

2017-01-01

Dahi and datshi are common naturally fermented milk (NFM) products of Bhutan. Population of lactic acid bacteria (LAB) in dahi (pH 3.7) and datshi (pH 5.2) was 1.4 × 107 and 3.9 × 108 cfu/ml, respectively. Based on 16S rRNA gene sequencing isolates of LAB from dahi and datshi were identified as Enterococcus faecalis, E. faecium, Lactococcus lactis subsp. lactis. LAB strains were tested for some technological properties. All LAB strains except E. faecalis CH2:17 caused coagulation of milk at both 30°C for 48 h. Only E. faecium DH4:05 strain was resistant to pH 3. No significant difference (P > 0.05) of viable counts was observed in MRS broth with and without lysozyme. All LAB strains grew well in 0.3% bile showing their ability to tolerate bile salt. None of the LAB strains showed >70% hydrophobicity. This study, being the first of its microbiological analysis of the NFM of Bhutan, has opened up to an extent of research work that gives a new insight to the products. PMID:28203227

Ultraviolet light-induced mutants of Streptococcus lactis subspecies diacetylactis with enhanced acid- or flavor-producing abilities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuila, R.K.; Ranganathan, B.

1978-04-01

A strain of Streptococcus lactis subspecies diacetylactis S/sub 1/ isolated from fresh milk was exposed to 7200 ergs/mm/sup 2/ of ultraviolet radiation. Over 8100 colonies surviving from 7.4 x 10/sup 6/ cells exposed to radiation were screened on citrate agar for detection and isolation of mutants with increased flavor and/or acid production. Of the survivors, 960 were type-I mutants that exhibited clear zone on citrate agar after 18 h (presumed to be high diacetyl producers), and 288 were type-II mutants which did not exhibit clear zones on citrate agar for up to 72 h (high acid producers). Type-II mutants producedmore » an average .93 percent titratable acidity which was 34 percent more than the .69 percent of the parent. Reduction in titratable acidity (56 percent less) was considerable in type-I mutants, compared with the parent culture. Diacetyl + acetoin production by type-I mutants was 137.9 ppM which has 4.5 times more than that of the parental strain. Acetaldehyde production in the mutants varied from 1.5 to 34.5 ppM (parent culture 3.0 ppM). The mutants with increased acid and high acetoin plus diacetyl production were stable after 50 subcultures in milk.« less

Transformation of Saccharomyces cerevisiae with linear DNA killer plasmids from Kluyveromyces lactis.

PubMed Central

Gunge, N; Murata, K; Sakaguchi, K

1982-01-01

Protoplasts of Saccharomyces cerevisiae were mixed with linear DNA plasmids, pGKl1 and pGKl2, isolated from a Kluyveromyces lactis killer strain and treated with polyethylene glycol. Out of 2,000 colonies regenerated on a nonselective medium, two killer transformants were obtained. The pGKl plasmids and the killer character were stably maintained in one (Pdh-1) of them. Another transformant, Pdl-1, was a weak killer, and the subclones consisted of a mixture of weak and nonkiller cells. The weak killers were characterized by the presence of pGKl1 in a decreased amount, and nonkillers were characterized by the absence of pGKl1. The occurrence of two new plasmids which migrated faster than pGKl1 in an agarose gel was observed in Pdl-1 and its subclones, whether weak or nonkillers. Staining with 4',6-diamidino-2-phenylindole revealed that the pGKl plasmids exist in the cytosol of transformant cells with numerous copy numbers. Images PMID:7045080

Greek Goat Encephalitis Virus Strain Isolated from Ixodes ricinus, Greece

PubMed Central

Pavlidou, Vasiliki; Antoniadis, Antonis

2008-01-01

A strain of Greek goat encephaltitis virus was isolated from engorged Ixodes ricinus ticks that had fed on goats in northern Greece. The strain was almost identical to the prototype strain isolated 35 years ago. PMID:18258134

Subcutaneous or oral immunization of mice with Lactococcus lactis expressing F4 fimbrial adhesin FaeG.

PubMed

Liu, Shujie; Li, Yongming; Xu, Ziwei; Wang, Yicheng

2013-01-01

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrhea in neonatal and postweaning piglets. Fimbrial adhesion of ETEC has been considered an important colonization factor with antigenicity. To safely and effectively deliver the F4 (K88) fimbrial adhesin FaeG to the immune system, we have previously constructed the secretory expression vector pNZ8112-faeG, and FaeG was produced in cytoplasmic form in Lactococcus lactis. In this work, BALB/c mice were immunized with recombinant L. lactis to further determine the immunogenicity of recombinant FaeG (rFaeG) via the subcutaneous or oral route. Subcutaneous immunization in mice with recombinant L. lactis induced a significant increase in the F4-specific serum IgG titer and the number of antibody-secreting cells (ASCs) in the spleen. Oral immunization of mice with recombinant L. lactis induced mucosal and systemic F4-specific immune responses and increased the number of ASCs in the spleen, mesenteric lymph nodes and Peyer's patches. High-dose (2.8 × 10(11) CFU) recombinant strains and adjuvant cholera toxin B subunit enhanced specific mucosal immune responses. The results suggest the feasibility of delivering rFaeG expressed in L. lactis to the immune system in order to induce an F4-specific immune response.

Isolation and purification of Gallid herpesvirus 2 strains currently distributed in Japan.

PubMed

Machida, Yuka; Murata, Shiro; Matsuyama-Kato, Ayumi; Isezaki, Masayoshi; Taneno, Akira; Sakai, Eishi; Konnai, Satoru; Ohashi, Kazuhiko

2017-01-20

Gallid herpesvirus 2 (GaHV-2) causes malignant lymphomas in chickens (Marek's disease, MD). Although MD is controlled through vaccination efforts, field isolates of GaHV-2 have increased in virulence worldwide and even cause MD in vaccinated chickens. GaHV-2 strains are classified into four categories (mild, virulent, very virulent and very virulent +) based on the virulence exhibited in experimental infection in unvaccinated or MD-vaccinated susceptible chickens. Although MD cases are sporadically reported in Japan, the recent field strains of GaHV-2 in Japan have not been characterized. During isolation of recent field strains by using primary chicken kidney cell cultures, a method classically used for GaHV-2 isolation, vaccine strains were simultaneously isolated. Therefore, it is necessary to separate vaccine strains to characterize the virulence and pathogenicity of the GaHV-2 strains currently distributed in Japan. In this study, we prepared cell suspensions from the spleens of MD-symptomatic chickens, inoculated day-old-chicks and isolated GaHV-2 strains by primary chicken kidney cell cultures at 2-3 weeks post inoculation. The isolated strains were passaged several times on chicken embryo fibroblast cells, and PCR analysis revealed that the isolated strains were not contaminated with vaccine strains. Moreover, the contaminant vaccine strains were completely removed by the purification of plaques observed in chicken kidney cells. These procedures are necessary to isolate GaHV-2 field strains from vaccine strains in order to carry out future studies to characterize these strains and glean insights into GaHV-2 virulence and pathogenicity.

Fermentation and aerobic metabolism of cellodextrins by yeasts. [Candida wickerhamii; C. guiliermondii; C. molischiana; Debaryomyces polymorphus; Pichia guilliermondii; Clavispora lusitaniae; Kluyveromyces lactis; Brettanomyces claussenii; Rhodotorula minuta; Dekkera intermedia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Freer, S.N.

1991-03-01

The fermentation and aerobic metabolism of cellodextrins by 14 yeast species or strains was monitored. When grown aerobically, Candida wickerhamii, C. guilliermondii, and C. molischiana metabolized cellodextrins of degree of polymerization 3 to 6. C. wicherhamii and C. molischiana also fermented these substrates, while C. guilliermondii fermented only cellodextrins of degree of polymerization {<=} 3. Debaryomyces polymorphus, Pichia guilliermondii, Clavispora lusitaniae, and one of two strains of Kluyveromyces lactis metabolized glucose, cellobiose, and cellotriose when grown aerobically. These yeasts also fermented these substrates, except for K. lactis, which fermented only glucose and cellobiose. The remaining species/strains tested, K. lactis, Brettanomycesmore » claussenii, Brettanomyces anomalus, Kluyveromyces dobzhanskii, Rhodotorula minuta, and Dekkera intermedia, both fermented and aerobically metabolized glucose and cellobiose. Crude enzyme preparations from all 14 yeast species or strains were tested for ability to hydrolyze cellotriose and cellotretose. Most of the yeasts produced an enzyme(s) capable of hydrolyzing cellotriose. However, with two exceptions, R. minuta and P. guilliermondii, only the yeasts that metabolized cellodextrins of degree of polymerization >3 produced an enzyme(s) that hydrolyzed cellotretose.« less

Anticandidal activity of cell extracts from 13 probiotic Lactobacillus strains and characterisation of lactic acid and a novel fatty acid derivative from one strain.

PubMed

Nyanzi, Richard; Awouafack, Maurice D; Steenkamp, Paul; Jooste, Piet J; Eloff, Jacobus N

2014-12-01

This study investigated the anti-Candida activity of methanol extracts from freeze-dried probiotic cells and the isolation of some constituents in the extracts. The MIC values of the probiotic methanol cell extracts against Candida albicans ranged between 1.25 and 5mg/ml after 48 h of incubation. However, Lactococcus latics subsp. lactis strain X and Lactobacillus casei strain B extracts had an MIC of 10mg/ml after 48 h of incubation. The extracts had fungistatic rather than fungicidal activity. These extracts had a much higher antifungal activity than antifungal compounds isolated from the growth medium by many other authors. This indicates that probiotics may also release antifungal compounds in their cells that could contribute to a therapeutic effect. Lactic acid (1) and 6-O-(α-D-glucopyranosyl)-1,6-di-O-pentadecanoyl-α-D-glucopyranose a novel fatty acid derivative (2) were isolated from methanol probiotic extracts and the structure of these compounds were elucidated using NMR (1 and 2D) and mass spectrometry (MS). Copyright © 2014 Elsevier Ltd. All rights reserved.

Lactococcus lactis carrying the pValac eukaryotic expression vector coding for IL-4 reduces chemically-induced intestinal inflammation by increasing the levels of IL-10-producing regulatory cells.

PubMed

Souza, Bianca Mendes; Preisser, Tatiane Melo; Pereira, Vanessa Bastos; Zurita-Turk, Meritxell; de Castro, Camila Prósperi; da Cunha, Vanessa Pecini; de Oliveira, Rafael Pires; Gomes-Santos, Ana Cristina; de Faria, Ana Maria Caetano; Machado, Denise Carmona Cara; Chatel, Jean-Marc; Azevedo, Vasco Ariston de Carvalho; Langella, Philippe; Miyoshi, Anderson

2016-08-30

Inflammatory bowel diseases are characterized by chronic intestinal inflammation that leads to severe destruction of the intestinal mucosa. Therefore, the understanding of their aetiology as well as the development of new medicines is an important step for the treatment of such diseases. Consequently, the development of Lactococcus lactis strains capable of delivering a eukaryotic expression vector encoding the interleukin 4 (IL-4) of Mus musculus would represent a new strategy for the elaboration of a more effective alternative therapy against Crohn's disease. The murine IL-4 ORF was cloned into the eukaryotic expression vector pValac::dts. The resulting plasmid-pValac::dts::IL-4-was transfected into CHO cells so that its functionality could be evaluated in vitro. With fluorescent confocal microscopy, flow cytometry and ELISA, it was observed that pValac::dts::IL-4-transfected cells produced IL-4, while non-transfected cells and cells transfected with the empty vector did not. Then, pValac::dts::IL-4 was inserted into L. lactis MG1363 FnBPA(+) in order to evaluate the therapeutic potential of the recombinant strain against TNBS-induced colitis. Intragastric administration of L. lactis MG1363 FnBPA(+) (pValac::dts::IL-4) was able to decrease the severity of colitis, with animals showing decreased levels of IL-12, IL-6 and MPO activity; and increased levels of IL-4 and IL-10. Finally, LP-isolated cells from mice administered TNBS were immunophenotyped so that the main IL-4 and IL-10 producers were identified. Mice administered the recombinant strain presented significantly higher percentages of F4/80(+)MHCII(+)Ly6C(-)IL-4(+), F4/80(+)MHCII(+)Ly6C(-)IL-10(+), F4/80(+)MHCII(+)Ly6C(-)CD206(+)CD124(+)IL-10(+) and CD4(+)Foxp3(+)IL10(+) cells compared to the other groups. This study shows that L. lactis MG1363 FnBPA(+) (pValac::dts::IL-4) is a good candidate to maintain the anti-inflammatory and proinflammatory balance in the gastrointestinal tract, increasing the levels

Protective immunity induced by an intranasal multivalent vaccine comprising 10 Lactococcus lactis strains expressing highly prevalent M-protein antigens derived from Group A Streptococcus.

PubMed

Wozniak, Aniela; Scioscia, Natalia; García, Patricia C; Dale, James B; Paillavil, Braulio A; Legarraga, Paulette; Salazar-Echegarai, Francisco J; Bueno, Susan M; Kalergis, Alexis M

2018-04-28

Streptococcus pyogenes (group A Streptococcus) causes diseases ranging from mild pharyngitis to severe invasive infections. The N-terminal fragment of Streptococcal M protein elicits protective antibodies and is an attractive vaccine target. However, this N- terminal fragment is hypervariable and there are more than 200 different M types. We are developing an intranasal live bacterial vaccine comprised of 10 strains of Lactococcus lactis, each expressing one N-terminal fagment of M protein. Live bacterial-vectored vaccines have lower associated costs because of its less complex manufacturing processes compared to protein subunit vaccines. Moreover, intranasal administration does not require syringe or specilized personnel. The evaluation of individual vaccine types (M1, M2, M3, M4, M6, M9, M12, M22, M28 and M77) showed that most of them protected mice against challenge with virulent S. pyogenes. All of the 10 strains combined in a 10-valent vaccine (Mx10) induced serum and bronchoalveolar lavages IgG titers that ranged from 3 to 10-fold those of unimmunized mice. Survival of Mx10-immunized mice after intranasal challenge with M28 streptococci is significantly higher than unimmunized mice. In contrast, when mice were challenged with M75 streptococci, survival of Mx10-immunized mice was not significantly different from unimmunized mice. Mx-10 immunized mice were significantly less colonized with S. pyogenes in oropharyngeal washes and developed less severe disease symptoms after challenge compared to unimmunized mice. Our L. lactis-based vaccine may provide an alternative solution to the development of broadly protective group A streptococcal vaccines. © 2018 The Societies and John Wiley & Sons Australia, Ltd.

Antagonistic lactic acid bacteria isolated from goat milk and identification of a novel nisin variant Lactococcus lactis

PubMed Central

2014-01-01

Background The raw goat milk microbiota is considered a good source of novel bacteriocinogenic lactic acid bacteria (LAB) strains that can be exploited as an alternative for use as biopreservatives in foods. The constant demand for such alternative tools justifies studies that investigate the antimicrobial potential of such strains. Results The obtained data identified a predominance of Lactococcus and Enterococcus strains in raw goat milk microbiota with antimicrobial activity against Listeria monocytogenes ATCC 7644. Enzymatic assays confirmed the bacteriocinogenic nature of the antimicrobial substances produced by the isolated strains, and PCR reactions detected a variety of bacteriocin-related genes in their genomes. Rep-PCR identified broad genetic variability among the Enterococcus isolates, and close relations between the Lactococcus strains. The sequencing of PCR products from nis-positive Lactococcus allowed the identification of a predicted nisin variant not previously described and possessing a wide inhibitory spectrum. Conclusions Raw goat milk was confirmed as a good source of novel bacteriocinogenic LAB strains, having identified Lactococcus isolates possessing variations in their genomes that suggest the production of a nisin variant not yet described and with potential for use as biopreservatives in food due to its broad spectrum of action. PMID:24521354

Poly(dimethyl siloxane) surface modification with biosurfactants isolated from probiotic strains.

PubMed

Pinto, S; Alves, P; Santos, A C; Matos, C M; Oliveiros, B; Gonçalves, S; Gudiña, E; Rodrigues, L R; Teixeira, J A; Gil, M H

2011-09-15

Depending on the final application envisaged for a given biomaterial, many surfaces must be modified before use. The material performance in a biological environment is mainly mediated by its surface properties that can be improved using suitable modification methods. The aim of this work was to coat poly(dimethyl siloxane) (PDMS) surfaces with biosurfactants (BSs) and to evaluate how these compounds affect the PDMS surface properties. BSs isolated from four probiotic strains (Lactococcus lactis, Lactobacillus paracasei, Streptococcus thermophilus A, and Streptococcus thermophilus B) were used. Bare PDMS and PDMS coated with BSs were characterized by contact angle measurements, infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). The influence of the surface modifications on the materials blood compatibility was studied through thrombosis and hemolysis assays. The cytotoxicity of these materials was tested against rat peritoneal macrophages. AFM results demonstrated the successful coating of the surfaces. Also, by contact angle measurements, an increase of the coated surfaces hydrophilicity was seen. Furthermore, XPS analysis indicated a decrease of the silicon content at the surface, and ATR-FTIR results showed the presence of BS characteristic groups as a consequence of the modification. All the studied materials revealed no toxicity and were found to be nonhemolytic. The proposed approach for the modification of PDMS surfaces was found to be effective and opens new possibilities for the application of these surfaces in the biomedical field. Copyright © 2011 Wiley Periodicals, Inc.

[Enhanced nisin production by overexpression of nisin immunity gene nisI in the nisin-producing strain].

PubMed

Hu, Hongmei; Jiang, Like; Lin, Yuheng; Huan, Liandong; Zhong, Jin

2010-10-01

Our aim was to enhance nisin production by overexpression of nisin immunity gene nisI in nisin-producing strains. Nisin immunity gene nisI with a strong promoter P59 was cloned into vector pHJ201 and introduced into Lacotococcus lactis NZ9800, resulting in a recombinant strain L. lactis NZ9800/pHMI. Then the differences between the recombinant strain and the control strain L. lactis NZ9800/pHJ201 were analyzed in several aspects, including their growth curves, nisin resistance level and antibacterial activity against indicator strain Microccus flavus NCIB 8166. The overexpression of nisI had no significant difference in growth rate between recombinant strain and contrast strain. However, it promoted recombinant strain tolerance 25% higer nisin resistance level and stronger antibacterial activity against M. flavus NCIB 8166, which was increased by 32% and 25% when fermented for 6 and 8 hours, respectively. These results indicated that overexpression of nisI gene in the nisin producing strain can effectively enhance nisin resistence level and thus improve nisin production.

[Isolation and identification of Cronobacter (Enterobacter sakazakii) strains from food].

PubMed

Dong, Xiaohui; Li, Chengsi; Wu, Qingping; Zhang, Jumei; Mo, Shuping; Guo, Weipeng; Yang, Xiaojuan; Xu, Xiaoke

2013-05-04

This study aimed to detect and quantify Cronobacter in 300 powdered milk samples and 50 non-powdered milk samples. Totally, 24 Cronobacter (formerly Enterobacter sakazakii) strains isolated from powdered milk and other foods were identified and confirmed. Cronobacter strains were detected quantitatively using most probable number (MPN) method and molecular detection method. We identified 24 Cronobacter strains using biochemical patterns, including indole production and dulcitol, malonate, melezitose, turanose, and myo-Inositol utilization. Of the 24 strains, their 16S rRNA genes were sequenced, and constructed phylogenetic tree by N-J (Neighbour-Joining) with the 16S rRNA gene sequences of 17 identified Cronobacter strains and 10 non-Cronobacter strains. Quantitative detection showed that Cronobacter strains were detected in 23 out of 350 samples yielding 6.6% detection rate. Twenty-four Cronobacter strains were isolated from 23 samples and the Cronobacter was more than 100 MPN/100g in 4 samples out of 23 samples. The 24 Cronobacter spp. isolates strains were identified and confirmed, including 19 Cronobacter sakazakii strains, 2 C. malonaticus strains, 2 C. dubliensis subsp. lactaridi strains, and 1 C. muytjensii strain. The combination of molecular detection method and most probable number (MPN) method could be suitable for the detection of Cronobacter in powdered milk, with low rate of contamination and high demand of quantitative detection. 24 isolated strains were confirmed and identified by biochemical patterns and molecular technology, and C. sakazakii could be the dominant species. The problem of Cronobacter in powdered milk should be a hidden danger to nurseling, and should catch the government and consumer's attention.

Phylogenetic analysis of Hungarian goose parvovirus isolates and vaccine strains.

PubMed

Tatár-Kis, Tímea; Mató, Tamás; Markos, Béla; Palya, Vilmos

2004-08-01

Polymerase chain reaction and sequencing were used to analyse goose parvovirus field isolates and vaccine strains. Two fragments of the genome were amplified. Fragment "A" represents a region of VP3 gene, while fragment "B" represents a region upstream of the VP3 gene, encompassing part of the VP1 gene. In the region of fragment "A" the deduced amino acid sequence of the strains was identical, therefore differentiation among strains could be done only at the nucleotide level, which resulted in the formation of three groups: Hungarian, West-European and Asian strains. In the region of fragment "B", separation of groups could be done by both nucleotide and deduced amino acid sequence level. The nucleotide sequences resulted in the same groups as for fragment "A" but with a different clustering pattern among the Hungarian strains. Within the "Hungarian" group most of the recent field isolates fell into one cluster, very closely related or identical to each other, indicating a very slow evolutionary change. The attenuated strains and field isolates from 1979/80 formed a separate cluster. When vaccine strains and field isolates were compared, two specific amino acid differences were found that can be considered as possible markers for vaccinal strains. Sequence analysis of fragment "B" seems to be a suitable method for differentiation of attenuated vaccine strains from virulent strains. Copyright 2004 Houghton Trust Ltd

Manipulation for plasmid elimination by transforming synthetic competitors diversifies lactococcus lactis starters applicable to food products.

PubMed

Kobayashi, Miho; Nomura, Masaru; Kimoto, Hiromi

2007-11-01

This study was designed selectively to eliminate a theta-plasmid from Lactococcus lactis strains by transforming synthetic competitors. A shuttle vector for Escherichia coli and L. lactis, pDB1, was constructed by ligating a partial replicon of pDR1-1B, which is a 7.3 kb theta-plasmid in L. lactis DRC1, with an erythromycin resistance gene into pBluescript II KS(+). This versatile vector was used to construct competitors to common lactococcal theta-plasmids. pDB1 contains the 5' half of the replication origin and the 3' region of repB of pDR1-1B, but lacks the 1.1-kb region normally found between these two segments. A set of primers, Pv3 and Pv4, was designed to amplify the 1.1-kb middle parts of the general theta-replicons of lactococcal plasmids. When the PCR products were cloned into the Nru I and Xho I sites of pDB1, synthetic replicons were constructed and replication activity was restored. A number of theta-plasmids in L. lactis ssp. lactis and cremoris were eliminated selectively by transforming the synthetic competitors. These competitors were easily eliminated by subculture for a short time in the absence of selection. The resulting variants contained no exogenous DNA and are suitable for food products, since part of the phenotype was altered without altering other plasmids indispensable for fermentation.

Cloning and functional expression of the mitochondrial alternative oxidase gene (aox1) of Aspergillus niger in Lactococcus lactis and its induction by oxidizing conditions.

PubMed

Papagianni, Maria; Avramidis, Nicholaos

2012-01-05

Lactococcus lactis is a widely used food bacterium mainly known for its fermentation metabolism. An important, and for long time overlooked, trait of this species is its ability to perform respiratory metabolism in the presence of heme and under aerobic conditions. There is no evidence however for the presence of an alternative respiration pathway and AOX activity. In this study, a cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for alternative respiration, from a citric acid producing Aspergillus niger strain was cloned and expressed in L. lactis as a host strain. Expression of aox1 conferred on this organism cyanide-resistant and salicylhydroxamate-sensitive growth. Bioreactor cultures under fully aerobic conditions of the transformed L. lactis showed that the alternative respiratory pathway operates and improves significantly the microorganism's response to oxidizing stress conditions as it enhances biomass production, suppresses lactate formation, and leads to accumulation of large amounts of nisin. Copyright © 2011 Elsevier Inc. All rights reserved.

Endosomal recognition of Lactococcus lactis G121 and its RNA by dendritic cells is key to its allergy-protective effects.

PubMed

Stein, Karina; Brand, Stephanie; Jenckel, André; Sigmund, Anna; Chen, Zhijian James; Kirschning, Carsten J; Kauth, Marion; Heine, Holger

2017-02-01

Bacterial cowshed isolates are allergy protective in mice; however, the underlying mechanisms are largely unknown. We examined the ability of Lactococcus lactis G121 to prevent allergic inflammatory reactions. We sought to identify the ligands and pattern recognition receptors through which L lactis G121 confers allergy protection. L lactis G121-induced cytokine release and surface expression of costimulatory molecules by untreated or inhibitor-treated (bafilomycin and cytochalasin D) human monocyte-derived dendritic cells (moDCs), bone marrow-derived mouse dendritic cells (BMDCs), and moDC/naive CD4 + T-cell cocultures were analyzed by using ELISA and flow cytometry. The pathology of ovalbumin-induced acute allergic airway inflammation after adoptive transfer of BMDCs was examined by means of microscopy. L lactis G121-treated murine BMDCs and human moDCs released T H 1-polarizing cytokines and induced T H 1 T cells. Inhibiting phagocytosis and endosomal acidification in BMDCs or moDCs impaired the release of T H 1-polarizing cytokines, costimulatory molecule expression, and T-cell activation on L lactis G121 challenge. In vivo allergy protection mediated by L lactis G121 was dependent on endosomal acidification in dendritic cells (DCs). Toll-like receptor (Tlr) 13 -/- BMDCs showed a weak response to L lactis G121 and were unresponsive to its RNA. The T H 1-polarizing activity of L lactis G121-treated human DCs was blocked by TLR8-specific inhibitors, mediated by L lactis G121 RNA, and synergistically enhanced by activation of nucleotide-binding oligomerization domain-containing protein (NOD) 2. Bacterial RNA is the main driver of L lactis G121-mediated protection against experimentally induced allergy and requires both bacterial uptake by DCs and endosomal acidification. In mice L lactis G121 RNA signals through TLR13; however, the most likely intracellular receptor in human subjects is TLR8. Copyright © 2016 American Academy of Allergy, Asthma & Immunology

Safety evaluation of HOWARU® Restore (Lactobacillus acidophilus NCFM, Lactobacillus paracasei Lpc-37, Bifidobacterium animalis subsp. lactis Bl-04 and B. lactis Bi-07) for antibiotic resistance, genomic risk factors, and acute toxicity.

PubMed

Morovic, Wesley; Roper, Jason M; Smith, Amy B; Mukerji, Pushkor; Stahl, Buffy; Rae, Jessica Caverly; Ouwehand, Arthur C

2017-12-01

Although probiotic lactobacilli and bifidobacteria are generally considered safe by various regulatory agencies, safety properties, such as absence of transferable antibiotic resistance, must still be determined for each strain prior to market introduction as a probiotic. Safety requirements for probiotics vary regionally and evaluation methods are not standardized, therefore methodologies are often adopted from food ingredients or chemicals to assess microbial safety. Four individual probiotic strains, Lactobacillus acidophilus NCFM ® , Lactobacillus paracasei Lpc-37 ® , Bifidobacterium animalis subsp. lactis strains Bl-04 ® , and Bi-07 ® , and their combination (HOWARU ® Restore) were examined for antibiotic resistance by broth microdilution culture, toxin genes by PCR and genome mining, and acute oral toxicity in rats. Only B. lactis Bl-04 exhibited antibiotic resistance above a regulated threshold due to a tetW gene previously demonstrated to be non-transferable. Genomic mining did not reveal any bacterial toxin genes known to harm mammalian hosts in any of the strains. The rodent studies did not indicate any evidence of acute toxicity following a dose of 1.7-4.1 × 10 12  CFU/kg body weight. Considering a 100-fold safety margin, this corresponds to 1.2-2.8 × 10 12  CFU for a 70 kg human. Our findings demonstrate a comprehensive approach of in vitro, in silico, and in vivo safety testing for probiotics. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Biodiversity of Lactobacillus sanfranciscensis strains isolated from five sourdoughs.

PubMed

Kitahara, M; Sakata, S; Benno, Y

2005-01-01

Five different sourdoughs were investigated for the composition of lactic acid bacteria (LAB) and the biodiversity of Lactobacillus sanfranciscensis strains. A total of 57 strains were isolated from five sourdoughs. Isolated strains were all identified by the 16S rDNA sequence and species-specific primers for L. sanfranciscensis. Results of identification showed that LAB strains were L. sanfranciscensis, Lactobacillus plantarum, Lactobacillus paralimentarius, Lactobacillus fermentum, Lactobacillus pontis, Lactobacillus casei, Weisella confusa and Pediococcus pentosaceus. A total of 21 strains were identified as L. sanfranciscensis and these isolates were detected in all five sourdoughs. Ribotyping was applied to investigate the relationship between intraspecies diversity of L. sanfranciscensis and sourdough. A total of 22 strains of L. sanfranciscensis including L. sanfranciscensis JCM 5668T were compared by ribotyping. The dendrogram of 21 ribotyping patterns showed four clusters, and L. sanfranciscensis JCM 5668T was independent of the others. The different biotypes of L. sanfranciscensis were present in two sourdoughs compared with other three sourdoughs. The LAB compositions of five sourdoughs were different and the relationship between intraspecies diversity of L. sanfranciscensis strains and five sourdoughs was shown by ribotyping. This study demonstrated that ribotyping was useful for distinguishing L. sanfranciscensis strains. A further important result is that the intra-species diversity of L. sanfranciscensis strains seems to be related to the sourdough preparation.

Cloning of the citrate permease gene of Lactococcus lactis subsp. lactis biovar diacetylactis and expression in Escherichia coli.

PubMed Central

Sesma, F; Gardiol, D; de Ruiz Holgado, A P; de Mendoza, D

1990-01-01

The citrate plasmid (Cit+ plasmid) from Lactococcus lactis subsp. lactis biovar diacetylactis was cloned into the EcoRI site of plasmid pUC18. This recombinant plasmid enabled Escherichia coli K-12 to transport and utilize citrate as a source of energy, indicating expression of the citrate permease from L. lactis biovar diacetylactis. The citrate permease was under the control of the lac promoter of pUC18. Genetic expression of the Cit+ plasmid in maxicells revealed that the plasmid encoded two polypeptides of 47 and 32 kilodaltons, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Images PMID:2117878

Inhibition of Listeria monocytogenes biofilms by bacteriocin-producing bacteria isolated from mushroom substrate.

PubMed

Bolocan, A S; Pennone, V; O'Connor, P M; Coffey, A; Nicolau, A I; McAuliffe, O; Jordan, K

2017-01-01

This study was designed to investigate the ability of naturally occurring bacteria isolated from mushroom substrate to prevent biofilm formation by Listeria monocytogenes or to remove existing biofilms in mushroom production facilities. It is generally recognized that L. monocytogenes forms biofilms that can facilitate its survival in food-processing environments. Eleven bacteriocin-producing isolates were identified and the bacteriocins characterized based on heat and enzyme inactivation studies. Further characterization was undertaken by MALDI-TOF mass spectrometry, PCR and sequencing. Production of nisin Z (by Lactococcus lactis isolates), subtilomycin (by Bacillus subtilis isolates) and lichenicidin (by Bacillus licheniformis and Bacillus sonorensis isolates) was detected. In co-culture with L. monocytogenes, the bacteriocin-producing strains could prevent biofilm formation and reduce pre-formed biofilms. Mushroom substrate can be a source of bacteriocin-producing bacteria that can antagonize L. monocytogenes. The results highlight the potential of bacteriocin-producing strains from mushroom substrate to reduce L. monocytogenes biofilm in food production environments, contributing to a reduction in the risk of food contamination from the environment. © 2016 The Society for Applied Microbiology.

Response of Leuconostoc strains against technological stress factors: Growth performance and volatile profiles.

PubMed

Cicotello, Joaquín; Wolf, Irma V; D'Angelo, Luisa; Guglielmotti, Daniela M; Quiberoni, Andrea; Suárez, Viviana B

2018-08-01

The ability of twelve strains belonging to three Leuconostoc species (Leuconostoc mesenteroides, Leuconostoc lactis and Leuconostoc pseudomesenteroides) to grow under diverse sub-lethal technological stress conditions (cold, acidic, alkaline and osmotic) was evaluated in MRS broth. Two strains, Leuconostoc lactis Ln N6 and Leuconostoc mesenteroides Ln MB7, were selected based on their growth under sub-lethal conditions, and volatile profiles in RSM (reconstituted skim milk) at optimal and under stress conditions were analyzed. Growth rates under sub-lethal conditions were strain- and not species-dependent. Volatilomes obtained from the two strains studied were rather diverse. Particularly, Ln N6 (Ln. lactis) produced more ethanol and acetic acid than Ln MB7 (Ln. mesenteroides) and higher amounts and diversity of the rest of volatile compounds as well, at all times of incubation. For the two strains studied, most of stress conditions applied diminished the amounts of ethanol and acetic acid produced and the diversity and levels of the rest of volatile compounds. These results were consequence of the different capacity of the strains to grow under each stress condition tested. Copyright © 2018 Elsevier Ltd. All rights reserved.

Listeria monocytogenes and Salmonella enterica affect the expression of nisin gene and its production by Lactococcus lactis.

PubMed

Abdollahi, Soosan; Ghahremani, Mohammad Hossein; Setayesh, Neda; Samadi, Nasrin

2018-06-13

The Lactococcus lactis is known as a probiotic bacterium and also as a producer of nisin. Nisin has been approved by related legal agencies to be used as an antimicrobial peptide in food preservation. In fact, the L. lactis is present in different food products along with other micro-organisms especially pathogenic bacteria. So, it is important to predict the behavior of nisin-producer strain in contact with other pathogens. In this regard, nisin gene expression and the level of secreted biologically active form of nisin by L. lactis subsp. lactis in modified MRS broth and whey solution in co-culture with Listeria monocytogenes or Salmonella enterica were studied. The nisin concentration was determined by microbiological assay method and the transcription level of nisin gene was assayed through quantitative reverse transcription PCR (RT-qPCR). According to our results, the highest concentration of nisin and its gene transcription level were detected in mono- and co-cultures after 16 h of incubation, concurrent with the end of L. lactis exponential phase of growth. The nisin mRNA copies in co-cultures were higher than mono-cultures only at 16 h of incubation. But, differences between nisin concentrations in mono- and co-cultures were significant at 16, 24 h and at 12, 16, 24 h of incubation in the modified MRS medium and whey solution, respectively. This incompatibility could be related to the low availability of components required for nisin precursor modification, transportation and processing in mono-cultures. Overall, the L. lactis produced more mature and active nisin when it was in contact with pathogenic bacteria. Copyright © 2018. Published by Elsevier Ltd.

A Deficiency in Aspartate Biosynthesis in Lactococcus lactis subsp. lactis C2 Causes Slow Milk Coagulation†

PubMed Central

Wang, Hua; Yu, Weizhu; Coolbear, Tim; O’Sullivan, Dan; McKay, Larry L.

1998-01-01

A mutant of fast milk-coagulating (Fmc+) Lactococcus lactis subsp. lactis C2, designated L. lactis KB4, was identified. Although possessing the known components essential for utilizing casein as a nitrogen source, which include functional proteinase (PrtP) activity and oligopeptide, di- and tripeptide, and amino acid transport systems, KB4 exhibited a slow milk coagulation (Fmc−) phenotype. When the amino acid requirements of L. lactis C2 were compared with those of KB4 by use of a chemically defined medium, it was found that KB4 was unable to grow in the absence of aspartic acid. This aspartic acid requirement could also be met by aspartate-containing peptides. The addition of aspartic acid to milk restored the Fmc+ phenotype of KB4. KB4 was found to be defective in pyruvate carboxylase and thus was deficient in the ability to form oxaloacetate and hence aspartic acid from pyruvate and carbon dioxide. The results suggest that when lactococci are propagated in milk, aspartate derived from casein is unable to meet fully the nutritional demands of the lactococci, and they become dependent upon aspartate biosynthesis. PMID:9572935

Isolation of noninhibitory strains of Zymomonas mobilis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haffie, T.L.; Louie, P.W.; Khachatourians, G.G.

1985-04-01

Wild-type Zymomonas mobilis strains inhibit the growth of Escherichia coli. The authors report the first isolation of noninhibitory strains, called Zymomonas inhibition negative (Zin/sup -/), after treatment with N-methyl-N'-nitro-N-nitrosoguanidine. A standardized soft-agar overlay procedure for detecting E. coli growth inhibition was also developed.

Epilithonimonas psychrotolerans sp. nov., isolated from alpine permafrost.

PubMed

Ge, Liang; Zhao, Qi; Sheng, Hongmei; Wu, Jianmin; An, Lizhe

2015-11-01

A bacterial strain, designated TSBY 57T, was isolated during a study on the phylogenetic diversity of culturable bacteria from alpine permafrost in Tianshan Mountains, China, and was classified by means of a polyphasic taxonomic approach. The novel strain was found to belong to the genus Epilithonimonas and was distinguished from recognized species of this genus. Strain TSBY 57T grew aerobically, at 0-30 °C, with 0-1.5% (w/v) NaCl and at pH 6-8.Cells were Gram-stain-negative, non-motile, non-spore-forming rods. Compared with the reference strains, the novel strain was psychrotolerant. The predominant fatty acids were summed feature 3 (consisting of C16:1ω7c and/or C16:1ω6c), anteiso-C15:0 and iso-C15:0.The sole respiratory quinone was MK-6.Phosphatidylethanolamine was predominant in the polar lipid profile of strain TSBY 57T. These chemotaxonomic traits were in good agreement with the characteristics of the genus Epilithonimonas. On the basis of 16S rRNA gene sequence similarity, strain TSBY 57T was a member of the genus Epilithonimonas and was closely related to Epilithonimonas tenax DSM 16811T (99.0%), Epilithonimonas ginsengisoli DCY78T (98.6%) and Epilithonimonas lactis H1T (98.5%). However, DNA-DNA reassociation values between strain TSBY 57T and E. tenax DSM 16811T, E. ginsengisoli DCY78T and E. lactis H1T were 39.5 ± 2.6, 37.7 ± 1.0 and 37.3 ± 1.1%, respectively. The G+C content of the DNA was 34.4 ± 0.2  mol%. Based on data from this polyphasic taxonomic study, strain TSBY 57T represents a novel species of the genus Epilithonimonas, for which the name Epilithonimonas psychrotolerans sp. nov. is proposed. The type strain is TSBY 57T ( = NRRL B-51307T=CCTCC AB 207182T).

In vitro evaluation of the probiotic and functional potential of Lactobacillus strains isolated from fermented food and human intestine.

PubMed

Ren, Dayong; Li, Chang; Qin, Yanqing; Yin, Ronglan; Du, Shouwen; Ye, Fei; Liu, Cunxia; Liu, Hongfeng; Wang, Maopeng; Li, Yi; Sun, Yang; Li, Xiao; Tian, Mingyao; Jin, Ningyi

2014-12-01

This study aims to evaluate the functional and probiotic characteristics of eight indigenous Lactobacillus strains in vitro. The selected lactobacilli include strains of Lactobacillus casei subsp. casei, Lactobacillus salivarius subsp. salicinius, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus delbrueckii subsp. lactis, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus rhamnosus. All strains tolerated both pH 2 for 3 h and 1% bile salt for 24 h. The strains CICC 23174 and CGMCC 1.557 were the most adhesive strains producing the highest quantity of EPS. Although a wide variation in the ability of the eight strains to deplete cholesterol and nitrite, antagonize pathogens, scavenge free radical, and stimulate innate immune response were observed, the strains CICC 23174 and CGMCC 1.557 showed the widest range of these useful traits. Taken together, the strains CICC 23174 and CGMCC 1.557 exhibited the best probiotic properties with the potential for use in the production of probiotic fermented foods. Copyright © 2014 Elsevier Ltd. All rights reserved.

Isolation of five Rubrobacter strains from biodeteriorated monuments

NASA Astrophysics Data System (ADS)

Laiz, L.; Miller, A. Z.; Jurado, V.; Akatova, E.; Sanchez-Moral, S.; Gonzalez, J. M.; Dionísio, A.; Macedo, M. F.; Saiz-Jimenez, C.

2009-01-01

In the last few years, the microbial colonisation of mural paintings in ancient monuments has been attracting the attention of microbiologists and conservators. The genus Rubrobacter is commonly found in biodeteriorated monuments, where it has been reported to cause rosy discolouration. However, to date, only three species of this genus have been isolated, all from thermophilic environments. In this paper, we studied three monuments: the Servilia and Postumio tombs in the Roman Necropolis of Carmona (Spain), and Vilar de Frades church (Portugal), in search of Rubrobacter strains. In all cases, biodeterioration and the formation of efflorescences were observed, and five Rubrobacter strains were isolated. These isolates showed different physiology and migration in denaturing gradient gel electrophoresis, suggesting they might represent new species within this genus. The isolates reproduced some biodeterioration processes in the laboratory and revealed their biomediation in crystal formation.

Characterization of some bacteriocins produced by lactic acid bacteria isolated from fermented foods.

PubMed

Grosu-Tudor, Silvia-Simona; Stancu, Mihaela-Marilena; Pelinescu, Diana; Zamfir, Medana

2014-09-01

Lactic acid bacteria (LAB) isolated from different sources (dairy products, fruits, fresh and fermented vegetables, fermented cereals) were screened for antimicrobial activity against other bacteria, including potential pathogens and food spoiling bacteria. Six strains have been shown to produce bacteriocins: Lactococcus lactis 19.3, Lactobacillus plantarum 26.1, Enterococcus durans 41.2, isolated from dairy products and Lactobacillus amylolyticus P40 and P50, and Lactobacillus oris P49, isolated from bors. Among the six bacteriocins, there were both heat stable, low molecular mass polypeptides, with a broad inhibitory spectrum, probably belonging to class II bacteriocins, and heat labile, high molecular mass proteins, with a very narrow inhibitory spectrum, most probably belonging to class III bacteriocins. A synergistic effect of some bacteriocins mixtures was observed. We can conclude that fermented foods are still important sources of new functional LAB. Among the six characterized bacteriocins, there might be some novel compounds with interesting features. Moreover, the bacteriocin-producing strains isolated in our study may find applications as protective cultures.

Trypanosoma cruzi: clones isolated from the Colombian strain, reproduce the parental strain characteristics, with ubiquitous histotropism

PubMed Central

Camandaroba, Edson; Thé, Torriceli S; Pessina, Daniel Huber; Andrade, Sonia G

2006-01-01

Clonal histotropism and biological characters of five clones isolated during the early acute phase of the infection of Swiss mice with the Colombian strain of Trypanosoma cruzi (T. cruzi I), Biodeme Type III, were investigated. Clones were isolated from mice at the 10th and the 30th day of infection with the Colombian strain. Isolation was performed by micromanipulation and injection of one trypomatigote blood form into newborn mice, followed by passages into suckling mice for obtaining the inocula for the experimental groups. Mice infected with parental strain were also studied. All the clones have shown the basic characteristics of Biodeme Type III, with the same patterns of parasitemia, tissue tropism, morphological characters and isoenzymic profiles, such as the parental strain. Histotropism was most intense to myocardium and skeletal muscles, with intense lesions found in the advanced phase (20th to 30th day of infection). Both parental strain and the clones were seen to parasitize several organs and tissues; amastigote nests were identified in the cytoplasm of macrophages, adipose cells, smooth muscle of intestinal wall and Auerbach's neuronal plexus. The findings of the present study confirm the homology of the clones isolated from the Colombian strain, with predominance of a ‘principal clone’ and an ubiquitous distribution of parasites belonging to a same clone. PMID:16709229

Prevalence of toxoplasmosis and genetic characterization of Toxoplasma gondii strains isolated in wild birds of prey and their relation with previously isolated strains from Turkey.

PubMed

Karakavuk, Muhammet; Aldemir, Duygu; Mercier, Aurélien; Atalay Şahar, Esra; Can, Hüseyin; Murat, Jean-Benjamin; Döndüren, Ömer; Can, Şengül; Özdemir, Hüseyin Gökhan; Değirmenci Döşkaya, Aysu; Pektaş, Bayram; Dardé, Marie-Laure; Gürüz, Adnan Yüksel; Döşkaya, Mert

2018-01-01

Toxoplasma gondii is a protozoon parasite that causes congenital toxoplasmosis, as well as other serious clinical presentations, in immune compromised humans. Analyses of the prevalence and genotyping of strains from the definitive host and intermediate hosts will help to understanding the circulation of the different strains and elucidating the role of the genotype(s) in human toxoplasmosis. Turkey has a specific geographic location bridging Africa, Europe, and Asia. We hypothesized that T. gondii strains may have been transferred to Turkey from these continents via migratory birds or vice versa. The present study aimed to assess the prevalence of toxoplasmosis in wild birds of prey of İzmir and Manisa provinces as well as genetically characterize T. gondii strains from these wild birds to show the relation between bird strains and neighboring stray cats as well as human strains previously isolated in Turkey. Tissues obtained from 48 wild birds were investigated for the presence of T. gondii DNA and then bioassayed in mouse. Isolated strains were genotyped using 15 microsatellite markers. The prevalence of T. gondii DNA was found to be 89.6% (n: 43/48) in wild birds. Out of 43 positive samples, a total of 14 strains were genotyped by 15 microsatellite markers. Among them, eight were type II, three were type III and three were mixture of genotypes (two type II/II and one was II/III). These are the first data that showed the presence of T. gondii and types II and III genotypes in wild birds of Turkey. Moreover, Africa 1 was not detected. In addition, cluster analysis showed that T. gondii strains within type II and III lineage have close relation with strains previously isolated from stray cats in İzmir. Further studies are required to isolate more strains from human cases, other intermediate hosts, and water sources to reveal this relation.

Prevalence of toxoplasmosis and genetic characterization of Toxoplasma gondii strains isolated in wild birds of prey and their relation with previously isolated strains from Turkey

PubMed Central

Karakavuk, Muhammet; Aldemir, Duygu; Mercier, Aurélien; Atalay Şahar, Esra; Can, Hüseyin; Murat, Jean-Benjamin; Döndüren, Ömer; Can, Şengül; Özdemir, Hüseyin Gökhan; Değirmenci Döşkaya, Aysu; Pektaş, Bayram; Dardé, Marie-Laure; Gürüz, Adnan Yüksel

2018-01-01

Toxoplasma gondii is a protozoon parasite that causes congenital toxoplasmosis, as well as other serious clinical presentations, in immune compromised humans. Analyses of the prevalence and genotyping of strains from the definitive host and intermediate hosts will help to understanding the circulation of the different strains and elucidating the role of the genotype(s) in human toxoplasmosis. Turkey has a specific geographic location bridging Africa, Europe, and Asia. We hypothesized that T. gondii strains may have been transferred to Turkey from these continents via migratory birds or vice versa. The present study aimed to assess the prevalence of toxoplasmosis in wild birds of prey of İzmir and Manisa provinces as well as genetically characterize T. gondii strains from these wild birds to show the relation between bird strains and neighboring stray cats as well as human strains previously isolated in Turkey. Tissues obtained from 48 wild birds were investigated for the presence of T. gondii DNA and then bioassayed in mouse. Isolated strains were genotyped using 15 microsatellite markers. The prevalence of T. gondii DNA was found to be 89.6% (n: 43/48) in wild birds. Out of 43 positive samples, a total of 14 strains were genotyped by 15 microsatellite markers. Among them, eight were type II, three were type III and three were mixture of genotypes (two type II/II and one was II/III). These are the first data that showed the presence of T. gondii and types II and III genotypes in wild birds of Turkey. Moreover, Africa 1 was not detected. In addition, cluster analysis showed that T. gondii strains within type II and III lineage have close relation with strains previously isolated from stray cats in İzmir. Further studies are required to isolate more strains from human cases, other intermediate hosts, and water sources to reveal this relation. PMID:29668747

Acid or erythromycin stress significantly improves transformation efficiency through regulating expression of DNA binding proteins in Lactococcus lactis F44.

PubMed

Wang, Binbin; Zhang, Huawei; Liang, Dongmei; Hao, Panlong; Li, Yanni; Qiao, Jianjun

2017-12-01

Lactococcus lactis is a gram-positive bacterium used extensively in the dairy industry and food fermentation, and its biological characteristics are usually improved through genetic manipulation. However, poor transformation efficiency was the main restriction factor for the construction of engineered strains. In this study, the transformation efficiency of L. lactis F44 showed a 56.1-fold increase in acid condition (pH 5.0); meanwhile, erythromycin stress (0.04 μg/mL) promoted the transformation efficiency more significantly (76.9-fold). Notably, the transformation efficiency of F44e (L. lactis F44 harboring empty pLEB124) increased up to 149.1-fold under the synergistic stresses of acid and erythromycin. In addition, the gene expression of some DNA binding proteins (DprA, RadA, RadC, RecA, RecQ, and SsbA) changed correspondingly. Especially for radA, 25.1-fold improvement was detected when F44e was exposed to pH 5.0. Overexpression of some DNA binding proteins could improve the transformation efficiency. The results suggested that acid or erythromycin stress could improve the transformation efficiency of L. lactis through regulating gene expression of DNA binding proteins. We have proposed a simple but promising strategy for improving the transformation efficiency of L. lactis and other hard-transformed microorganisms. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

High Yields of 2,3-Butanediol and Mannitol in Lactococcus lactis through Engineering of NAD+ Cofactor Recycling ▿ †

PubMed Central

Gaspar, Paula; Neves, Ana Rute; Gasson, Michael J.; Shearman, Claire A.; Santos, Helena

2011-01-01

Manipulation of NADH-dependent steps, and particularly disruption of the las-located lactate dehydrogenase (ldh) gene in Lactococcus lactis, is common to engineering strategies envisaging the accumulation of reduced end products other than lactate. Reverse transcription-PCR experiments revealed that three out of the four genes assigned to lactate dehydrogenase in the genome of L. lactis, i.e., the ldh, ldhB, and ldhX genes, were expressed in the parental strain MG1363. Given that genetic redundancy is often a major cause of metabolic instability in engineered strains, we set out to develop a genetically stable lactococcal host tuned for the production of reduced compounds. Therefore, the ldhB and ldhX genes were sequentially deleted in L. lactis FI10089, a strain with a deletion of the ldh gene. The single, double, and triple mutants, FI10089, FI10089ΔldhB, and FI10089ΔldhBΔldhX, showed similar growth profiles and displayed mixed-acid fermentation, ethanol being the main reduced end product. Hence, the alcohol dehydrogenase-encoding gene, the adhE gene, was inactivated in FI10089, but the resulting strain reverted to homolactic fermentation due to induction of the ldhB gene. The three lactate dehydrogenase-deficient mutants were selected as a background for the production of mannitol and 2,3-butanediol. Pathways for the biosynthesis of these compounds were overexpressed under the control of a nisin promoter, and the constructs were analyzed with respect to growth parameters and product yields under anaerobiosis. Glucose was efficiently channeled to mannitol (maximal yield, 42%) or to 2,3-butanediol (maximal yield, 67%). The theoretical yield for 2,3-butanediol was achieved. We show that FI10089ΔldhB is a valuable basis for engineering strategies aiming at the production of reduced compounds. PMID:21841021

Functional Expression of an Orchid Fragrance Gene in Lactococcus lactis

PubMed Central

Song, Adelene Ai Lian; Abdullah, Janna O.; Abdullah, Mohd Puad; Shafee, Norazizah; Rahim, Raha A.

2012-01-01

Vanda Mimi Palmer (VMP), an orchid hybrid of Vanda tesselata and Vanda Tan Chay Yan is a highly scented tropical orchid which blooms all year round. Previous studies revealed that VMP produces a variety of isoprenoid volatiles during daylight. Isoprenoids are well known to contribute significantly to the scent of most fragrant plants. They are a large group of secondary metabolites which may possess valuable characteristics such as flavor, fragrance and toxicity and are produced via two pathways, the mevalonate (MVA) pathway or/and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. In this study, a sesquiterpene synthase gene denoted VMPSTS, previously isolated from a floral cDNA library of VMP was cloned and expressed in Lactococcus lactis to characterize the functionality of the protein. L. lactis, a food grade bacterium which utilizes the mevalonate pathway for isoprenoid production was found to be a suitable host for the characterization of plant terpene synthases. Through recombinant expression of VMPSTS, it was revealed that VMPSTS produced multiple sesquiterpenes and germacrene D dominates its profile. PMID:22408409

Lactose-induced cell death of beta-galactosidase mutants in Kluyveromyces lactis.

PubMed

Lodi, Tiziana; Donnini, Claudia

2005-05-01

The Kluyveromyces lactis lac4 mutants, lacking the beta-galactosidase gene, cannot assimilate lactose, but grow normally on many other carbon sources. However, when these carbon sources and lactose were simultaneously present in the growth media, the mutants were unable to grow. The effect of lactose was cytotoxic since the addition of lactose to an exponentially-growing culture resulted in 90% loss of viability of the lac4 cells. An osmotic stabilizing agent prevented cells killing, supporting the hypothesis that the lactose toxicity could be mainly due to intracellular osmotic pressure. Deletion of the lactose permease gene, LAC12, abolished the inhibitory effect of lactose and allowed the cell to assimilate other carbon substrates. The lac4 strains gave rise, with unusually high frequency, to spontaneous mutants tolerant to lactose (lar1 mutation: lactose resistant). These mutants were unable to take up lactose. Indeed, lar1 mutation turned out to be allelic to LAC12. The high mutability of the LAC12 locus may be an advantage for survival of K. lactis whose main habitat is lactose-containing niches.

Influence of Cofermentation by Amylolytic Lactobacillus plantarum and Lactococcus lactis Strains on the Fermentation Process and Rheology of Sorghum Porridge

PubMed Central

Byaruhanga, Yusuf B.; Muyanja, Charles M. B. K.; Aijuka, Matthew; Schüller, Reidar B.; Sahlstrøm, Stefan; Langsrud, Thor; Narvhus, Judith A.

2012-01-01

Amylolytic lactic acid bacteria (ALAB) can potentially replace malt in reducing the viscosity of starchy porridges. However, the drawback of using ALAB is their low and delayed amylolytic activity. This necessitates searching for efficient ALAB and strategies to improve their amylolytic activity. Two ALAB, Lactobacillus plantarum MNC 21 and Lactococcus lactis MNC 24, isolated from Obushera, were used to ferment starches in MRS broth: sorghum, millet, sweet potato, and commercial soluble starch. The amylolytic activity of MNC 21 was comparable to that of the ALAB collection strain Lb. plantarum A6, while that of MNC 24 was extremely low. MNC 21, MNC 24, and their coculture were compared to A6 and sorghum malt for ability to ferment and reduce the viscosity of sorghum porridge (11.6% dry matter). ALAB and the coculture lowered the pH from 6.2 to <4.5 within 12 h, while malt as a carrier of wild starter took about 20 h. Coculturing increased lactic acid yield by 46% and 76.8% compared to the yields of MNC 21 and MNC 24 monocultures, respectively. The coculture accumulated significantly larger (P < 0.05) amounts of maltose and diacetyl than the monocultures. Sorghum malt control and the coculture hydrolyzed more starch in sorghum porridge than the monocultures. The coculture initiated changes in the rheological parameters storage modulus (G′), loss modulus (G″), phase angle (δ), and complex viscosity (η*) earlier than its constituent monocultures. The shear viscosity of sorghum porridge was reduced significantly (P < 0.05) from 1950 cP to 110 cP (malt), 281 cP (coculture), 382 cP (MNC 21), 713 cP (MNC 24), and 722 cP (A6). Coculturing strong ALAB with weak ALAB or non-ALAB can be exploited for preparation of nutrient-dense weaning foods and increasing lactic acid yield from starchy materials. PMID:22610432

Influence of cofermentation by amylolytic Lactobacillus plantarum and Lactococcus lactis strains on the fermentation process and rheology of sorghum porridge.

PubMed

Mukisa, Ivan M; Byaruhanga, Yusuf B; Muyanja, Charles M B K; Aijuka, Matthew; Schüller, Reidar B; Sahlstrøm, Stefan; Langsrud, Thor; Narvhus, Judith A

2012-08-01

Amylolytic lactic acid bacteria (ALAB) can potentially replace malt in reducing the viscosity of starchy porridges. However, the drawback of using ALAB is their low and delayed amylolytic activity. This necessitates searching for efficient ALAB and strategies to improve their amylolytic activity. Two ALAB, Lactobacillus plantarum MNC 21 and Lactococcus lactis MNC 24, isolated from Obushera, were used to ferment starches in MRS broth: sorghum, millet, sweet potato, and commercial soluble starch. The amylolytic activity of MNC 21 was comparable to that of the ALAB collection strain Lb. plantarum A6, while that of MNC 24 was extremely low. MNC 21, MNC 24, and their coculture were compared to A6 and sorghum malt for ability to ferment and reduce the viscosity of sorghum porridge (11.6% dry matter). ALAB and the coculture lowered the pH from 6.2 to <4.5 within 12 h, while malt as a carrier of wild starter took about 20 h. Coculturing increased lactic acid yield by 46% and 76.8% compared to the yields of MNC 21 and MNC 24 monocultures, respectively. The coculture accumulated significantly larger (P < 0.05) amounts of maltose and diacetyl than the monocultures. Sorghum malt control and the coculture hydrolyzed more starch in sorghum porridge than the monocultures. The coculture initiated changes in the rheological parameters storage modulus (G'), loss modulus (G″), phase angle (δ), and complex viscosity (η*) earlier than its constituent monocultures. The shear viscosity of sorghum porridge was reduced significantly (P < 0.05) from 1950 cP to 110 cP (malt), 281 cP (coculture), 382 cP (MNC 21), 713 cP (MNC 24), and 722 cP (A6). Coculturing strong ALAB with weak ALAB or non-ALAB can be exploited for preparation of nutrient-dense weaning foods and increasing lactic acid yield from starchy materials.

The Nanomechanical Properties of Lactococcus lactis Pili Are Conditioned by the Polymerized Backbone Pilin

PubMed Central

Castelain, Mickaël; Duviau, Marie-Pierre; Canette, Alexis; Schmitz, Philippe; Loubière, Pascal; Cocaign-Bousquet, Muriel; Piard, Jean-Christophe; Mercier-Bonin, Muriel

2016-01-01

Pili produced by Lactococcus lactis subsp. lactis are putative linear structures consisting of repetitive subunits of the major pilin PilB that forms the backbone, pilin PilA situated at the distal end of the pilus, and an anchoring pilin PilC that tethers the pilus to the peptidoglycan. We determined the nanomechanical properties of pili using optical-tweezers force spectroscopy. Single pili were exposed to optical forces that yielded force-versus-extension spectra fitted using the Worm-Like Chain model. Native pili subjected to a force of 0–200 pN exhibit an inextensible, but highly flexible ultrastructure, reflected by their short persistence length. We tested a panel of derived strains to understand the functional role of the different pilins. First, we found that both the major pilin PilB and sortase C organize the backbone into a full-length organelle and dictate the nanomechanical properties of the pili. Second, we found that both PilA tip pilin and PilC anchoring pilin were not essential for the nanomechanical properties of pili. However, PilC maintains the pilus on the bacterial surface and may play a crucial role in the adhesion- and biofilm-forming properties of L. lactis. PMID:27010408

Proteogenomic Investigation of Strain Variation in Clinical Mycobacterium tuberculosis Isolates.

PubMed

Heunis, Tiaan; Dippenaar, Anzaan; Warren, Robin M; van Helden, Paul D; van der Merwe, Ruben G; Gey van Pittius, Nicolaas C; Pain, Arnab; Sampson, Samantha L; Tabb, David L

2017-10-06

Mycobacterium tuberculosis consists of a large number of different strains that display unique virulence characteristics. Whole-genome sequencing has revealed substantial genetic diversity among clinical M. tuberculosis isolates, and elucidating the phenotypic variation encoded by this genetic diversity will be of the utmost importance to fully understand M. tuberculosis biology and pathogenicity. In this study, we integrated whole-genome sequencing and mass spectrometry (GeLC-MS/MS) to reveal strain-specific characteristics in the proteomes of two clinical M. tuberculosis Latin American-Mediterranean isolates. Using this approach, we identified 59 peptides containing single amino acid variants, which covered ∼9% of all coding nonsynonymous single nucleotide variants detected by whole-genome sequencing. Furthermore, we identified 29 distinct peptides that mapped to a hypothetical protein not present in the M. tuberculosis H37Rv reference proteome. Here, we provide evidence for the expression of this protein in the clinical M. tuberculosis SAWC3651 isolate. The strain-specific databases enabled confirmation of genomic differences (i.e., large genomic regions of difference and nonsynonymous single nucleotide variants) in these two clinical M. tuberculosis isolates and allowed strain differentiation at the proteome level. Our results contribute to the growing field of clinical microbial proteogenomics and can improve our understanding of phenotypic variation in clinical M. tuberculosis isolates.

Bioactivity characterization of Lactobacillus strains isolated from dairy products

PubMed Central

Haghshenas, Babak; Nami, Yousef; Haghshenas, Minoo; Abdullah, Norhafizah; Rosli, Rozita; Radiah, Dayang; Yari Khosroushahi, Ahmad

2015-01-01

This study aimed to find candidate strains of Lactobacillus isolated from sheep dairy products (yogurt and ewe colostrum) with probiotic and anticancer activity. A total of 100 samples were randomly collected from yogurt and colostrum and 125 lactic acid bacteria were isolated. Of these, 17 Lactobacillus strains belonging to five species (L. delbrueckii, L. plantarum, L. rhamnosus, L. paracasei, and L. casei) were identified. L. plantarum 17C and 13C, which isolated from colostrums, demonstrated remarkable results such as resistant to low pH and high concentrations of bile salts, susceptible to some antibiotics and good antimicrobial activity that candidate them as potential probiotics. Seven strains (1C, 5C, 12C, 13C, 17C, 7M, and 40M), the most resistant to simulated digestion, were further investigated to evaluate their capability to adhere to human intestinal Caco-2 cells. L. plantarum 17C was the most adherent strain. The bioactivity assessment of L. plantarum 17C showed anticancer effects via the induction of apoptosis on HT-29 human cancer cells and negligible side effects on one human epithelial normal cell line (FHs 74). The metabolites produced by this strain can be used as alternative pharmaceutical compounds with promising therapeutic indices because they are not cytotoxic to normal mammalian cells. PMID:26219634

Growth, nisA Gene Expression, and In Situ Activity of Novel Lactococcus lactis subsp. cremoris Costarter Culture in Commercial Hard Cheese Production.

PubMed

Noutsopoulos, Dimitrios; Kakouri, Athanasia; Kartezini, Eleftheria; Pappas, Dimitrios; Hatziloukas, Efstathios; Samelis, John

2017-12-01

This study evaluated in situ expression of the nisA gene by an indigenous, nisin A-producing (NisA+) Lactococcus lactis subsp. cremoris raw milk genotype, represented by strain M78, in traditional Greek Graviera cheeses under real factory-scale manufacturing and ripening conditions. Cheeses were produced with added a mixed thermophilic and mesophilic commercial starter culture (CSC) or with the CSC plus strain M78 (CSC+M78). Cheeses were sampled after curd cooking (day 0), fermentation of the unsalted molds for 24 h (day 1), brining (day 7), and ripening of the brined molds (14 to 15 kg each) for 30 days in a fully controlled industrial room (16.5°C; 91% relative humidity; day 37). Total RNA was directly extracted from the cheese samples, and the expression of nisA gene was evaluated by real-time reverse transcription PCR (qRT-PCR). Agar overlay and well diffusion bioassays were correspondingly used for in situ detection of the M78 NisA+ colonies in the cheese agar plates and antilisterial activity in whole-cheese slurry samples, respectively. Agar overlay assays showed good growth (>8 log CFU/g of cheese) of the NisA+ strain M78 in coculture with the CSC and vice versa. The nisA expression was detected in CSC+M78 cheese samples only, with its expression levels being the highest (16-fold increase compared with those of the control gene) on day 1, followed by significant reduction on day 7 and almost negligible expression on day 37. Based on the results, certain intrinsic and mainly implicit hurdle factors appeared to reduce growth prevalence rates and decrease nisA gene expression, as well as the nisin A-mediated antilisterial activities of the NisA+ strain M78 postfermentation. To our knowledge, this is the first report on quantitative expression of the nisA gene in a Greek cooked hard cheese during commercial manufacturing and ripening conditions by using a novel, rarely isolated, indigenous NisA+ L. lactis subsp. cremoris genotype as costarter culture.

Optimization of nisin production by Lactococcus lactis UQ2 using supplemented whey as alternative culture medium.

PubMed

González-Toledo, S Y; Domínguez-Domínguez, J; García-Almendárez, B E; Prado-Barragán, L A; Regalado-González, C

2010-08-01

Lactococcus lactis UQ2 is a nisin A-producing native strain. In the present study, the production of nisin by L. lactis UQ2 in a bioreactor using supplemented sweet whey (SW) was optimized by a statistical design of experiments and response surface methodology (RSM). In a 1st approach, a fractional factorial design (FFD) of the order 2(5-1) with 3 central points was used. The effect on nisin production of air flow, SW, soybean peptone (SP), MgSO(4)/MnSO(4) mixture, and Tween 80 was evaluated. From FFD, the most significant factors affecting nisin production were SP (P = 0.011), and SW (P = 0.037). To find optimum conditions, a central composite design (CCD) with 2 central points was used. Three factors were considered, SW (7 to 10 g/L), SP (7 to10 g/L), and small amounts of added nisin as self-inducer (NI 34.4 to 74.4 IU/L). Nisin production was expressed as international units (IU). From RSM, an optimum nisin activity of 180 IU/mL was predicted at 74.4 IU/L NI, 13.8 g/L SP, and 14.9 or 5.11 g/L SW, while confirmatory experiments showed a maximum activity of 178 +/- 5.2 IU/mL, verifying the validity of the model. The 2nd-order model showed a coefficient of determination (R(2)) of 0.828. Optimized conditions were used for constant pH fermentations, where a maximum activity of 575 +/- 17 IU/mL was achieved at pH 6.5 after 12 h. The adsorption-desorption technique was used to partially purify nisin, followed by drying. The resulting powder showed an activity of 102150 IU/g. Practical Application: Nisin production was optimized using supplemented whey as alternative culture medium, using a native L. lactis UQ2 strain. Soybean peptone, SW, and subinhibitory amounts of nisin were successfully employed to optimize nisin production by L. lactis UQ2. Dried semipurified nisin showed an activity of 102150 IU/g.

Another Brick in the Wall: a Rhamnan Polysaccharide Trapped inside Peptidoglycan of Lactococcus lactis.

PubMed

Sadovskaya, Irina; Vinogradov, Evgeny; Courtin, Pascal; Armalyte, Julija; Meyrand, Mickael; Giaouris, Efstathios; Palussière, Simon; Furlan, Sylviane; Péchoux, Christine; Ainsworth, Stuart; Mahony, Jennifer; van Sinderen, Douwe; Kulakauskas, Saulius; Guérardel, Yann; Chapot-Chartier, Marie-Pierre

2017-09-12

Polysaccharides are ubiquitous components of the Gram-positive bacterial cell wall. In Lactococcus lactis , a polysaccharide pellicle (PSP) forms a layer at the cell surface. The PSP structure varies among lactococcal strains; in L. lactis MG1363, the PSP is composed of repeating hexasaccharide phosphate units. Here, we report the presence of an additional neutral polysaccharide in L. lactis MG1363 that is a rhamnan composed of α-l-Rha trisaccharide repeating units. This rhamnan is still present in mutants devoid of the PSP, indicating that its synthesis can occur independently of PSP synthesis. High-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) analysis of whole bacterial cells identified a PSP at the surface of wild-type cells. In contrast, rhamnan was detected only at the surface of PSP-negative mutant cells, indicating that rhamnan is located underneath the surface-exposed PSP and is trapped inside peptidoglycan. The genetic determinants of rhamnan biosynthesis appear to be within the same genetic locus that encodes the PSP biosynthetic machinery, except the gene tagO encoding the initiating glycosyltransferase. We present a model of rhamnan biosynthesis based on an ABC transporter-dependent pathway. Conditional mutants producing reduced amounts of rhamnan exhibit strong morphological defects and impaired division, indicating that rhamnan is essential for normal growth and division. Finally, a mutation leading to reduced expression of lcpA , encoding a protein of the LytR-CpsA-Psr (LCP) family, was shown to severely affect cell wall structure. In lcpA mutant cells, in contrast to wild-type cells, rhamnan was detected by HR-MAS NMR, suggesting that LcpA participates in the attachment of rhamnan to peptidoglycan. IMPORTANCE In the cell wall of Gram-positive bacteria, the peptidoglycan sacculus is considered the major structural component, maintaining cell shape and integrity. It is decorated with other glycopolymers, including

Identification of novel anti-inflammatory probiotic strains isolated from pulque.

PubMed

Torres-Maravilla, Edgar; Lenoir, Marion; Mayorga-Reyes, Lino; Allain, Thibault; Sokol, Harry; Langella, Philippe; Sánchez-Pardo, María E; Bermúdez-Humarán, Luis G

2016-01-01

Probiotics are live microorganisms which when administered in adequate amounts, confer health benefits on the host. Their use is more and more widespread for both prevention and treatment of diseases, including traveler’s diarrhea and inflammatory bowel diseases (IBDs). In this work, we isolated and characterized novel candidate probiotic strains from pulque (xaxtle), a traditional Mexican alcoholic fermented beverage. A total of 14 strains were obtained from xaxtle samples isolated from three different Mexican regions. Species identification was performed by biochemical methods and 16S rRNA gene targeted PCR. The isolates belonged to the Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus brevis, and Lactobacillus composti phylogenetic groups, with L. brevis being the most dominant group. Bacteria were tested for lysozyme, low pH, and bile acid resistance. Moreover, the strains were tested for adherence to human intestinal epithelial cells and screened for their immunomodulatory properties using a cellular model. Selected bacterial strains with anti-inflammatory properties were then tested in vivo in a dinitro-benzene sulfonic acid (DNBS)-induced chronic colitis mouse model, and weight loss, gut permeability, and cytokine profiles were measured as readouts of inflammation. One of the selected strains, Lactobacillus sanfranciscensis LBH1068, improved mice health as observed by a reduction of weight loss, significant decreases in gut permeability, and cytokine modulation. Altogether, our results highlighted the potential of lactobacilli isolated from pulque and in particular the strain L. sanfranciscensis LBH1068 as a novel probiotic to treat IBD.

Characterization of lactic acid bacteria isolated from artisanal Travnik young cheeses, sweet creams and sweet kajmaks over four seasons.

PubMed

Terzic-Vidojevic, Amarela; Mihajlovic, Sanja; Uzelac, Gordana; Veljovic, Katarina; Tolinacki, Maja; Nikolic, Milica; Topisirovic, Ljubisa; Kojic, Milan

2014-05-01

The aim of this study was to investigate the composition of lactic acid bacteria (LAB) in autochthonous young cheeses, sweet creams and sweet kajmaks produced in the Vlašić mountain region of central Bosnia and Herzegovina near the town of Travnik over a four season period. These three products were made from cow's milk by a traditional method without the addition of a starter culture. Preliminary characterization with phenotype-based assays and identification using rep-PCR with a (GTG)5 primer and 16S rDNA sequence analysis were undertaken for 460 LAB isolates obtained from all the examined samples. Fifteen species were identified as follows: Lactococcus lactis, Lactococcus raffinolactis, Lactococcus garviae, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus helveticus, Enterococcus faecium, Enterococcus durans, Enterococcus faecalis, Enterococcus italicus, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Leuconostoc lactis, Streptococcus thermophilus and Streptococcus mitis. A wide genotypic and phenotypic heterogeneity of the species was observed, particularly within the Lc. lactis strains. In all of the tested dairy products across four seasons, a significantly positive correlation (r = 0.690) between the presence of lactococci and enterococci and a negative correlation (r = 0.722) between the presence of lactococci and leuconostocs were recorded. Forty-five percent of the lactobacilli and 54.4% of the lactococci exhibited proteolytic activity, whereas 18.7% of the total LAB isolates exhibited antimicrobial activity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Exploring optimization parameters to increase ssDNA recombineering in Lactococcus lactis and Lactobacillus reuteri

PubMed Central

van Pijkeren, Jan-Peter; Neoh, Kar Mun; Sirias, Denise; Findley, Anthony S.; Britton, Robert A.

2012-01-01

Single-stranded DNA (ssDNA) recombineering is a technology which is used to make subtle changes in the chromosome of several bacterial genera. Cells which express a single-stranded DNA binding protein (RecT or Bet) are transformed with an oligonucleotide which is incorporated via an annealing and replication-dependent mechanism. By in silico analysis we identified ssDNA binding protein homologs in the genus Lactobacillus and Lactococcus lactis. To assess whether we could further improve the recombineering efficiency in Lactobacillus reuteri ATCC PTA 6475 we expressed several RecT homologs in this strain. RecT derived from Enterococcus faecalis CRMEN 19 yielded comparable efficiencies compared with a native RecT protein, but none of the other proteins further increased the recombineering efficiency. We successfully improved recombineering efficiency 10-fold in L. lactis by increasing oligonucleotide concentration combined with the use of oligonucleotides containing phosphorothioate-linkages (PTOs). Surprisingly, neither increased oligonucleotide concentration nor PTO linkages enhanced recombineering in L. reuteri 6475. To emphasize the utility of this technology in improving probiotic features we modified six bases in a transcriptional regulatory element region of the pdu-operon of L. reuteri 6475, yielding a 3-fold increase in the production of the antimicrobial compound reuterin. Directed genetic modification of lactic acid bacteria through ssDNA recombineering will simplify strain improvement in a way that, when mutating a single base, is genetically indistinguishable from strains obtained through directed evolution. PMID:22750793

Suppression of oral tolerance by Lactococcus lactis in mice.

PubMed

Sakai, Tohru; Hirota, Yuko; Nakamoto, Mariko; Shuto, Emi; Hosaka, Toshio; Makino, Seiya; Ikegami, Shuji

2011-01-01

Although oral ovabumin (OVA) administration suppressed the antibody (Ab) response in OVA-immunized mice, Lactococcus lactis increased OVA-specific IgG2a in these mice. L. lactis increased the casein-specific IgG level in NC/Nga mice fed on a casein diet. The percentage of CD4(+)CD25(+) cells was increased in DO11.10 mice orally given OVA, but this increase of CD4(+)CD25(+) cells were suppressed in L. lactis-fed DO11.10 mice.

Expression of Curli by Escherichia coli O157:H7 Strains Isolated from Patients during Outbreaks Is Different from Similar Strains Isolated from Leafy Green Production Environments.

PubMed

Ravva, Subbarao V; Sarreal, Chester Z; Cooley, Michael B

2016-01-01

We previously reported that the strains of Escherichia coli O157:H7 (EcO157) that survived longer in austere soil environment lacked expression of curli, a fitness trait linked with intestinal colonization. In addition, the proportion of curli-positive variants of EcO157 decreased with repeated soil exposure. Here we evaluated 84 and 176 clinical strains from outbreaks and sporadic infections in the US, plus 211 animal fecal and environmental strains for curli expression. These shiga-toxigenic strains were from 328 different genotypes, as characterized by multi-locus variable-number tandem-repeat analysis (MLVA). More than half of the fecal strains (human and animal) and a significant proportion of environmental isolates (82%) were found to lack curli expression. EcO157 strains from several outbreaks linked with the consumption of contaminated apple juice, produce, hamburgers, steak, and beef were also found to lack curli expression. Phylogenetic analysis of fecal strains indicates curli expression is distributed throughout the population. However, a significant proportion of animal fecal isolates (84%) gave no curli expression compared to human fecal isolates (58%). In addition, analysis of environmental isolates indicated nearly exclusive clustering of curli expression to a single branch of the minimal spanning tree. This indicates that curli expression depends primarily upon the type of environmental exposure and the isolation source, although genotypic differences also contribute to clonal variation in curli. Furthermore, curli-deficient phenotype appears to be a selective trait for survival of EcO157 in agricultural environments.

Genome Sequence of Lactobacillus delbrueckii subsp. lactis CNRZ327, a Dairy Bacterium with Anti-Inflammatory Properties.

PubMed

El Kafsi, Hela; Binesse, Johan; Loux, Valentin; Buratti, Julien; Boudebbouze, Samira; Dervyn, Rozenn; Hammani, Amal; Maguin, Emmanuelle; van de Guchte, Maarten

2014-07-17

Lactobacillus delbrueckii subsp. lactis CNRZ327 is a dairy bacterium with anti-inflammatory properties both in vitro and in vivo. Here, we report the genome sequence of this bacterium, which appears to contain no less than 215 insertion sequence (IS) elements, an exceptionally high number regarding the small genome size of the strain. Copyright © 2014 El Kafsi et al.

Multilocus Sequence Typing of Cronobacter Strains Isolated from Retail Foods and Environmental Samples.

PubMed

Killer, Jiří; Skřivanová, Eva; Hochel, Igor; Marounek, Milan

2015-06-01

Cronobacter spp. are bacterial pathogens that affect children and immunocompromised adults. In this study, we used multilocus sequence typing (MLST) to determine sequence types (STs) in 11 Cronobacter spp. strains isolated from retail foods, 29 strains from dust samples obtained from vacuum cleaners, and 4 clinical isolates. Using biochemical tests, species-specific polymerase chain reaction, and MLST analysis, 36 strains were identified as Cronobacter sakazakii, and 6 were identified as Cronobacter malonaticus. In addition, one strain that originated from retail food and one from a dust sample from a vacuum cleaner were identified on the basis of MLST analysis as Cronobacter dublinensis and Cronobacter turicensis, respectively. Cronobacter spp. strains isolated from the retail foods were assigned to eight different MLST sequence types, seven of which were newly identified. The strains isolated from the dust samples were assigned to 7 known STs and 14 unknown STs. Three clinical isolates and one household dust isolate were assigned to ST4, which is the predominant ST associated with neonatal meningitis. One clinical isolate was classified based on MLST analysis as Cronobacter malonaticus and belonged to an as-yet-unknown ST. Three strains isolated from the household dust samples were assigned to ST1, which is another clinically significant ST. It can be concluded that Cronobacter spp. strains of different origin are genetically quite variable. The recovery of C. sakazakii strains belonging to ST1 and ST4 from the dust samples suggests the possibility that contamination could occur during food preparation. All of the novel STs and alleles for C. sakazakii, C. malonaticus, C. dublinensis, and C. turicensis determined in this study were deposited in the Cronobacter MLST database available online ( http://pubmlst.org/cronobacter/).

Postmating Reproductive isolation between strains of Drosophila willistoni.

PubMed

Mardiros, Xian B; Park, Ronni; Clifton, Bryan; Grewal, Gurman; Khizar, Amina K; Markow, Therese A; Ranz, José M; Civetta, Alberto

2016-10-01

Speciation can occur through the presence of reproductive isolation barriers that impede mating, restrict cross-fertilization, or render inviable/sterile hybrid progeny. The D. willistoni subgroup is ideally suited for studies of speciation, with examples of both allopatry and sympatry, a range of isolation barriers, and the availability of one species complete genome sequence to facilitate genetic studies of divergence. D. w. willistoni has the largest geographic distribution among members of the Drosophila willistoni subgroup, spanning from Argentina to the southern United States, including the Caribbean islands. A subspecies of D. w. willistoni, D. w. quechua, is geographically separated by the Andes mountain range and has evolved unidirectional sterility, in that only male offspring of D. w. quechua females × D. w. willistoni males are sterile. Whether D. w. willistoni flies residing east of the Andes belong to one or more D. willistoni subspecies remains unresolved. Here we perform fecundity assays and show that F1 hybrid males produced from crosses between different strains found in Central America, North America, and northern Caribbean islands are reproductively isolated from South American and southern Caribbean island strains as a result of unidirectional hybrid male sterility. Our results show the existence of a reproductive isolation barrier between the northern and southern strains and suggest a subdivision of the previously identified D. willistoni willistoni species into 2 new subspecies.

Genetic relatedness of ciprofloxacin-resistant Shigella dysenteriae type 1 strains isolated in south Asia.

PubMed

Talukder, Kaisar A; Khajanchi, Bijay K; Islam, M Aminul; Dutta, Dilip K; Islam, Zhahirul; Safa, Ashrafus; Khan, G Y; Alam, Khorshed; Hossain, M A; Malla, Sarala; Niyogi, S K; Rahman, Mustafizur; Watanabe, Haruo; Nair, G Balakrish; Sack, David A

2004-10-01

The aim of the present study was to determine the clonal relationships of ciprofloxacin-resistant Shigella dysenteriae type 1 strains isolated from south Asia, and S. dysenteriae 1 strains associated with epidemics in 1978, 1984 and 1994. The antimicrobial susceptibilities were examined by NCCLS methods. Molecular epidemiological characterization was performed by plasmid profiling, pulsed-field gel electrophoresis (PFGE) and mutation analysis of the quinolone resistance-determining region (QRDR) of gyrA by sequencing. Plasmid patterns of the current ciprofloxacin-resistant strains from India, Nepal and Bangladesh were very similar to those of the 1978, 1984 and 1994 epidemic isolates of S. dysenteriae 1, except for the presence of a new plasmid of approximately 2.6 MDa, which was found in one recent ciprofloxacin-resistant strain isolated in Bangladesh. PFGE analysis showed that the ciprofloxacin-resistant strains isolated in Bangladesh, India and Nepal belonged to a PFGE type (type A), which was possibly related to that of the 1984 and 1994 clone of S. dysenteriae 1, but different from 1978 epidemic strains. The current ciprofloxacin-resistant strains belong to five subtypes (A3-A7), all of which were found in India, but in Bangladesh and Nepal, only A3 existed. Mutation analysis of the QRDR of gyrA revealed that amino acid substitutions at positions 83 and 87 of ciprofloxacin-resistant strains isolated in Bangladesh were similar to those of the strains isolated in Nepal, but different (at position 87) from ciprofloxacin-resistant strains isolated in India. PFGE and mutation analysis of gyrA showed differences between the current ciprofloxacin-resistant S. dysenteriae 1 strains isolated in south Asia and those associated with epidemics in 1978, 1984 and 1994.

Safety Evaluation of Enterocin Producer Enterococcus sp. Strains Isolated from Traditional Turkish Cheeses.

PubMed

Avcı, Mine; Özden Tuncer, Banu

2017-07-06

The purpose of this study was to determine the antimicrobial activity and occurrence of bacteriocin structural genes in Enterococcus spp. isolated from different cheeses and also investigate some of their virulence factors. Enterococcus strains were isolated from 33 different cheeses. Enterococcus faecium (6 strains) and Enterococcus faecalis (5 strains) enterocin-producing strains were identified by 16S rDNA analyses. Structural genes entA, entB, entP and entX were detected in some isolates. Multiple enterocin structural genes were found in 7 strains. None of the tested enterococci demonstrated anyβ-haemolytic activity and only one strain had gelatinase activity. Six strains showed multiple antibiotic resistance patterns and in addition, vanA and several virulence genes were detected in many strains. Only E. faecalis MBE1-9 showed tyrosine decarboxylase activity and tdc gene was detected only in this strain.

Plasmid linkage of the D-tagatose 6-phosphate pathway in Streptococcus lactis: effect on lactose and galactose metabolism.

PubMed Central

Crow, V L; Davey, G P; Pearce, L E; Thomas, T D

1983-01-01

The three enzymes of the D-tagatose 6-phosphate pathway (galactose 6-phosphate isomerase, D-tagatose 6-phosphate kinase, and tagatose 1,6-diphosphate aldolase) were absent in lactose-negative (Lac-) derivatives of Streptococcus lactis C10, H1, and 133 grown on galactose. The lactose phosphoenolpyruvate-dependent phosphotransferase system and phospho-beta-galactosidase activities were also absent in Lac- derivatives of strains H1 and 133 and were low (possibly absent) in C10 Lac-. In all three Lac- derivatives, low galactose phosphotransferase system activity was found. On galactose, Lac- derivatives grew more slowly (presumably using the Leloir pathway) than the wild-type strains and accumulated high intracellular concentrations of galactose 6-phosphate (up to 49 mM); no intracellular tagatose 1,6-diphosphate was detected. The data suggest that the Lac phenotype is plasmid linked in the three strains studied, with the evidence being more substantial for strain H1. A Lac- derivative of H1 contained a single plasmid (33 megadaltons) which was absent from the Lac- mutant. We suggest that the genes linked to the lactose plasmid in S. lactis are more numerous than previously envisaged, coding for all of the enzymes involved in lactose metabolism from initial transport to the formation of triose phosphates via the D-tagatose 6-phosphate pathway. Images PMID:6294064

Construction of a lactose-assimilating strain of baker's yeast.

PubMed

Adam, A C; Prieto, J A; Rubio-Texeira, M; Polaina, J

1999-09-30

A recombinant strain of baker's yeast has been constructed which can assimilate lactose efficiently. This strain has been designed to allow its propagation in whey, the byproduct resulting from cheese-making. The ability to metabolize lactose is conferred by the functional expression of two genes from Kluyveromyces lactis, LAC12 and LAC4, which encode a lactose permease and a beta-galactosidase, respectively. To make the recombinant strain more acceptable for its use in bread-making, the genetic transformation of the host baker's yeast was carried out with linear fragments of DNA of defined sequence, carrying as the only heterologous material the coding regions of the two K. lactis genes. Growth of the new strain on cheese whey affected neither the quality of bread nor the yeast gassing power. The significance of the newly developed strain is two-fold: it affords a cheap alternative to the procedure generally used for the propagation of baker's yeast, and it offers a profitable use for cheese whey. Copyright 1999 John Wiley & Sons, Ltd.

Characterization of functional properties of Enterococcus faecium strains isolated from human gut.

PubMed

İspirli, Hümeyra; Demirbaş, Fatmanur; Dertli, Enes

2015-11-01

The aim of this work was to characterize the functional properties of Enterococcus faecium strains identified after isolation from human faeces. Of these isolates, strain R13 showed the best resistance to low pH, bile salts, and survival in the simulated in vitro digestion assay, and demonstrated an important level of adhesion to hexadecane as a potential probiotic candidate. Analysis of the antibiotic resistance of E. faecium strains indicated that in general these isolates were sensitive to the tested antibiotics and no strain appeared to be resistant to vancomycin. Examination of the virulence determinants for E. faecium strains demonstrated that all strains contained the virulence genes common in gut- and food-originated enterococci, and strain R13 harboured the lowest number of virulence genes. Additionally, no strain contained the genes related to cytolysin metabolism and showed hemolytic activity. The antimicrobial role of E. faecium strains was tested against several pathogens, in which different levels of inhibitory effects were observed, and strain R13 was inhibitory to all tested pathogens. PCR screening of genes encoding enterocin A and B indicated the presence of these genes in E. faecium strains. Preliminary characterization of bacteriocins revealed that their activity was lost after proteolytic enzyme treatments, but no alteration in antimicrobial activity was observed at different pHs (3.5 to 9.5) and after heat treatments. In conclusion, this study revealed the functional characteristics of E. faecium R13 as a gut isolate, and this strain could be developed as a new probiotic after further tests.

Developing a xylanase XYNZG from Plectosphaerella cucumerina for baking by heterologously expressed in Kluyveromyces lactis.

PubMed

Zhan, Fei Xiang; Wang, Qin Hong; Jiang, Si Jing; Zhou, Yu Ling; Zhang, Gui Min; Ma, Yan He

2014-12-16

Xylanase can replace chemical additives to improve the volume and sensory properties of bread in the baking. Suitable baking xylanase with improved yield will promote the application of xylanase in baking industry. The xylanase XYNZG from the Plectosphaerella cucumerina has been previously characterized by heterologous expression in Pichia pastoris. However, P. pastoris is not a suitable host for xylanase to be used in the baking process since P. pastoris does not have GRAS (Generally Regarded As Safe) status and requires large methanol supplement during the fermentation in most conditions, which is not allowed to be used in the food industry. Kluyveromyces lactis, as another yeast expression host, has a GRAS status, which has been successfully used in food and feed applications. No previous work has been reported concerning the heterologous expression of xylanase gene xynZG in K. lactis with an aim for application in baking. The xylanase gene xynZG from the P. cucumerina was heterologously expressed in K. lactis. The recombinant protein XYNZG in K. lactis presented an approximately 19 kDa band on SDS-PAGE and zymograms analysis. Transformant with the highest halo on the plate containing the RBB-xylan (Remazol Brilliant Blue-xylan) was selected for the flask fermentation in different media. The results indicated that the highest activity of 115 U/ml at 72 h was obtained with the YLPU medium. The mass spectrometry analysis suggested that the hydrolytic products of xylan by XYNZG were mainly xylobiose and xylotriose. The results of baking trials indicated that the addition of XYNZG could reduce the kneading time of dough, increase the volume of bread, improve the texture, and have more positive effects on the sensory properties of bread. Xylanase XYNZG is successfully expressed in K. lactis, which exhibits the highest activity among the published reports of the xylanase expression in K. lactis. The recombinant XYNZG can be used to improve the volume and sensory

Use of waste materials for Lactococcus lactis development.

PubMed

Rodríguez, Noelia; Torrado, Ana; Cortés, Sandra; Domínguez, José Manuel

2010-08-15

Lactococcus lactis is an interesting microorganism with several industrial applications, particularly in the food industry. As well as being a probiotic species, L. lactis produces several metabolites with interesting properties, such as lactic acid (LA) and biosurfactants. Nevertheless, L. lactis is an especially demanding species since it has strong nutritional requirements, implying the use of complex and expensive culture media. The results showed the potential of L. lactis CECT-4434 as a LA and biosurfactant producer. The economical cost of L. lactis cultures can be reduced by replacing the MRS medium by the use of two waste materials: trimming vine shoots as C source, and 20 g L(-1) distilled wine lees (vinasses) as N, P and micronutrient sources. From the hemicellulosic fraction, 14.3 g L(-1) LA and 1.7 mg L(-1) surfactin equivalent were achieved after 74 h (surface tension reduction of 14.4 mN m(-1)); meanwhile, a simultaneous saccharification and fermentation process allowed the generation of 10.8 g L(-1) LA and 1.5 mg L(-1) surfactin equivalent after 72 h, reducing the surface tension by 12.1 units at the end of fermentation. Trimming vine shoots and vinasses can be used as alternative economical media for LA and cell-bound biosurfactant production. Copyright (c) 2010 Society of Chemical Industry.

Pathogenic Potential of Saccharomyces Strains Isolated from Dietary Supplements

PubMed Central

Monteoliva, Lucía; Querol, Amparo; Molina, María; Fernández-Espinar, María T.

2014-01-01

Saccharomyces cerevisiae plays a beneficial role in health because of its intrinsic nutritional value and bio-functional properties, which is why it is also used as a dietary supplement. However, the perception that S. cerevisiae is harmless has changed due to an increasing number of infections caused by this yeast. Given this scenario, we have tested whether viable strains contained in dietary supplements displayed virulence-associated phenotypic traits that could contribute to virulence in humans. We have also performed an in vivo study of the pathogenic potential of these strains using a murine model of systemic infection by intravenous inoculation. A total of 5 strains were isolated from 22 commercial products and tested. Results highlight one strain (D14) in terms of burden levels in brains and kidneys and ability to cause death, whereas the other two strains (D2 and D4) were considered of low virulence. Our results suggest a strong relationship between some of the virulence-associated phenotypic traits (ability to grow at 39°C and pseudohyphal growth) and the in vivo virulence in a mouse model of intravenous inoculation for isolates under study. The isolate displaying greatest virulence (D14) was evaluated in an experimental murine model of gastrointestinal infection with immunosuppression and disruption of mucosal integrity, which are common risk factors for developing infection in humans, and results were compared with an avirulent strain (D23). We showed that D14 was able to spread to mesenteric nodes and distant organs under these conditions. Given the widespread consumption of dietary supplements, we recommend only safe strains be used. PMID:24879417

[Production of pertussis toxin by Bordetella pertussis strains isolated from patients with whooping cough].

PubMed

Zaĭtsev, E M; Mertsalova, N U; Shinkarev, A S; Mazurova, I K; Zakharova, N S

2011-01-01

To assess level of pertussin toxin (PT) production by vaccine strains of Bordetella pertussis and strains isolated from patients with whooping cough. Concentration of PT in supernatants of microbial cultures of 3 vaccine strains and 25 strains of B. pertussis isolated from patients with pertussis in 2001 - 2005 was measured with enzyme immunoassay using gamma-globulin fractions of rabbit antiserum to PT as immunosorbent or included in peroxidase conjugates. Level of PT production by strains isolated from infected persons varied from 3 +/- 0.5 to 64.8 +/- 12.2 ng/MFU/ml: in 9 strains--from 3 +/- 0.5 to 9.4 +/- 2.1 ng/MFU/ml, in 7--10.5 +/- 1.8 to 18.4 +/- 2.6 ng/MFU/ml, and in 9--23.6 +/- 4.5 to 64.8 +/- 12.2 ng/MFU/ml. B. pertussis strains isolated from patients were heterogeneous on level of PT production. Difference in expression of PT between strains were as high as 20-fold. Conditionally low, moderate and high levels of PT production had 9 (36%), 7 (28%), and 9 (36%) of 25 studied strains. Three vaccine strains had levels of toxin production similar to recently isolated strains with moderate level of its production.

Thermal inactivation kinetics of Lactococcus lactis subsp. lactis bacteriophage pll98-22.

PubMed

Sanlibaba, Pinar; Buzrul, S; Akkoç, Nefise; Alpas, H; Akçelik, M

2009-03-01

Survival curves of Lactococcus lactis subsp. lactis bacteriophage pll98 inactivated by heat were obtained at seven temperature values (50-80 degrees C) in M17 broth and skim milk. Deviations from first-order kinetics in both media were observed as sigmoidal shapes in the survival curves of pll98. An empirical model with four parameters was used to define the thermal inactivation. Number of parameters of the model was reduced from four to two in order to increase the robustness of the model. The reduced model produced comparable fits to the full model. Both the survival data and the calculations done using the reduced model (time necessary to reduce the number of phage pll98 six- or seven- log10) indicated that skim milk is a more protective medium than M17 broth within the assayed temperature range.

In Vitro Assessment of the Probiotic Potential of Lactococcus lactis LMG 7930 against Ruminant Mastitis-Causing Pathogens.

PubMed

Armas, Federica; Camperio, Cristina; Marianelli, Cinzia

2017-01-01

Mastitis in dairy ruminants is considered to be the most expensive disease to farmers worldwide. Recently, the intramammary infusion of lactic acid bacteria has emerged as a potential new alternative to antibiotics for preventing and treating bovine mastitis. In this study we have investigated in vitro the probiotic potential of Lactococcus lactis LMG 7930, a food-grade and nisin-producing strain, against mastitis-causing pathogens. We have characterized its carbohydrate fermentation and antibiotic susceptibility profiles, cell surface properties and antimicrobial activity, as well as its capabilities to adhere to and inhibit the invasion of pathogens into the bovine mammary epithelial cell line BME-UV1d. We found that L. lactis LMG 7930 was sensitive to tested drugs, according to the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), and showed an improved carbohydrate fermentation capacity compared to starter strains. Moreover, the strain exhibited antagonistic properties towards many of the pathogens tested. It presented medium surface hydrophobicity, a low basic property and no electron acceptor capability. It showed low auto-aggregation and no co-aggregation abilities towards any of the tested pathogens. The strain was one of the most adhesive to bovine mammary epithelial cells among tested bacteria, but its internalisation was low. The strain did not affect significantly pathogen invasion; however, a trend to decrease internalization of some pathogens tested was observed. In conclusion, our results suggest that this strain might be a promising candidate for the development of new strategies of mastitis control in ruminants. Future investigations are needed to evaluate its safety and efficacy under field conditions.

In Vitro Assessment of the Probiotic Potential of Lactococcus lactis LMG 7930 against Ruminant Mastitis-Causing Pathogens

PubMed Central

Armas, Federica; Camperio, Cristina

2017-01-01

Mastitis in dairy ruminants is considered to be the most expensive disease to farmers worldwide. Recently, the intramammary infusion of lactic acid bacteria has emerged as a potential new alternative to antibiotics for preventing and treating bovine mastitis. In this study we have investigated in vitro the probiotic potential of Lactococcus lactis LMG 7930, a food-grade and nisin-producing strain, against mastitis-causing pathogens. We have characterized its carbohydrate fermentation and antibiotic susceptibility profiles, cell surface properties and antimicrobial activity, as well as its capabilities to adhere to and inhibit the invasion of pathogens into the bovine mammary epithelial cell line BME-UV1d. We found that L. lactis LMG 7930 was sensitive to tested drugs, according to the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), and showed an improved carbohydrate fermentation capacity compared to starter strains. Moreover, the strain exhibited antagonistic properties towards many of the pathogens tested. It presented medium surface hydrophobicity, a low basic property and no electron acceptor capability. It showed low auto-aggregation and no co-aggregation abilities towards any of the tested pathogens. The strain was one of the most adhesive to bovine mammary epithelial cells among tested bacteria, but its internalisation was low. The strain did not affect significantly pathogen invasion; however, a trend to decrease internalization of some pathogens tested was observed. In conclusion, our results suggest that this strain might be a promising candidate for the development of new strategies of mastitis control in ruminants. Future investigations are needed to evaluate its safety and efficacy under field conditions. PMID:28068371

Transformation of Streptococcus lactis Protoplasts by Plasmid DNA †

PubMed Central

Kondo, Jeffery K.; McKay, Larry L.

1982-01-01

Polyethylene glycol-treated protoplasts prepared from Streptococcus lactis LM3302, a lactose-negative (Lac−) derivative of S. lactis ML3, were transformed to lactose-fermenting ability by a transductionally shortened plasmid (pLM2103) coding for lactose utilization. Images PMID:16346019

Isolation and characterization of new strains of methanogens from cold terrestrial habitats.

PubMed

Simankova, Maria V; Kotsyurbenko, Oleg R; Lueders, Tillmann; Nozhevnikova, Alla N; Wagner, Bianca; Conrad, Ralf; Friedrich, Michael W

2003-06-01

Five strains of methanogenic archaea (MT, MS, MM, MSP, ZB) were isolated from permanently and periodically cold terrestrial habitats. Physiological and morphological studies, as well as phylogenetic analyses of the new isolates were performed. Based on sequences of the 16S rRNA and methyl-coenzyme M reductase a-subunit (mcrA) genes all new isolates are closely related to known mesophilic and psychrotolerant methanogens. Both, phylogenetic analyses and phenotypic properties allow to classify strains MT, MS, and MM as members of the genus Methanosarcina. Strain MT is a new ecotype of Methanosarcina mazei, whereas strains MM and MS are very similar to each other and can be assigned to the recently described psychrotolerant species Methanosarcina lacustris. The hydrogenotrophic strain MSP is a new ecotype of the genus Methanocorpusculum. The obligately methylotrophic strain ZB is closely related to Methanomethylovorans hollandica and can be classified as new ecotype of this species. All new isolates, including the strains from permanently cold environments, are not true psychrophiles according to their growth temperature characteristics. In spite of the ability of all isolates to grow at temperatures as low as 1-5 degrees C, all of them have their growth optima in the range of moderate temperatures (25-35 degrees C). Thus, they can be regarded as psychrotolerant organisms. Psychrotolerant methanogens are thought to play an important role in methane production in both, habitats under seasonal temperature variations or from permanently cold areas.

High Incidence of Pathogenic Streptococcus agalactiae ST485 Strain in Pregnant/Puerperal Women and Isolation of Hyper-Virulent Human CC67 Strain

PubMed Central

Li, Liping; Wang, Rui; Huang, Yan; Huang, Ting; Luo, Fuguang; Huang, Weiyi; Yang, Xiuying; Lei, Aiying; Chen, Ming; Gan, Xi

2018-01-01

Group B streptococcus (GBS) is the major pathogen causing diseases in neonates, pregnant/puerperal women, cows and fish. Recent studies have shown that GBS may be infectious across hosts and some fish GBS strain might originate from human. The purpose of this study is to investigate the genetic relationship of CC103 strains that recently emerged in cows and humans, and explore the pathogenicity of clinical GBS isolates from human to tilapia. Ninety-two pathogenic GBS isolates were identified from 19 patients with different diseases and their evolution and pathogenicity to tilapia were analyzed. The multilocus sequence typing revealed that clonal complex (CC) 103 strain was isolated from 21.74% (20/92) of patients and ST485 strain was from 14.13% (13/92) patients with multiple diseases including neonates. Genomic evolution analysis showed that both bovine and human CC103 strains alternately form independent evolutionary branches. Three CC67 isolates carried gbs2018-C gene and formed one evolutionary branch with ST61 and ST67 strains that specifically infect dairy cows. Studies of interspecies transmission to tilapia found that 21/92 (22.83%) isolates including all ST23 isolates were highly pathogenic to tilapia and demonstrated that streptococci could break through the blood-brain barrier into brain tissue. In conclusions, CC103 strains are highly prevalent among pathogenic GBS from humans and have evolved into the highly pathogenic ST485 strains specifically infecting humans. The CC67 strains isolated from cows are able to infect humans through evolutionary events of acquiring CC17-specific type C gbs2018 gene and others. Human-derived ST23 pathogenic GBS strains are highly pathogenic to tilapia. PMID:29467722

Different distribution patterns of ten virulence genes in Legionella reference strains and strains isolated from environmental water and patients.

PubMed

Zhan, Xiao-Yong; Hu, Chao-Hui; Zhu, Qing-Yi

2016-04-01

Virulence genes are distinct regions of DNA which are present in the genome of pathogenic bacteria and absent in nonpathogenic strains of the same or related species. Virulence genes are frequently associated with bacterial pathogenicity in genus Legionella. In the present study, an assay was performed to detect ten virulence genes, including iraA, iraB, lvrA, lvrB, lvhD, cpxR, cpxA, dotA, icmC and icmD in different pathogenicity islands of 47 Legionella reference strains, 235 environmental strains isolated from water, and 4 clinical strains isolated from the lung tissue of pneumonia patients. The distribution frequencies of these genes in reference or/and environmental L. pneumophila strains were much higher than those in reference non-L. pneumophila or/and environmental non-L. pneumophila strains, respectively. L. pneumophila clinical strains also maintained higher frequencies of these genes compared to four other types of Legionella strains. Distribution frequencies of these genes in reference L. pneumophila strains were similar to those in environmental L. pneumophila strains. In contrast, environmental non-L. pneumophila maintained higher frequencies of these genes compared to those found in reference non-L. pneumophila strains. This study illustrates the association of virulence genes with Legionella pathogenicity and reveals the possible virulence evolution of non-L. pneumophia strains isolated from environmental water.

Trimming of two major type 1 diabetes driving antigens, GAD65 and IA-2, allows for successful expression in Lactococcus lactis.

PubMed

Robert, S; Van Huynegem, K; Gysemans, C; Mathieu, C; Rottiers, P; Steidler, L

2015-01-01

Type 1 diabetes (T1D) is a chronic autoimmune disease characterised by excessive immune reactions against auto-antigens of pancreatic β-cells. Restoring auto-antigen tolerance remains the superior therapeutic strategy. Oral auto-antigen administration uses the tolerogenic nature of the gut-associated immune system to induce antigen-specific tolerance. However, due to gastric degradation, proper mucosal product delivery often imposes a challenge. Recombinant Lactococcus lactis have proven to be effective and safe carriers for gastrointestinal delivery of therapeutic products: L. lactis secreting diabetes-associated auto-antigens in combination with interleukin (IL)-10 have demonstrated therapeutic efficacy in a well-defined mouse model for T1D. Here, we describe the construction of recombinant L. lactis secreting the 65 kDa isoform of glutamic acid decarboxylase (GAD65) and tyrosine phosphatase-like protein ICA512 (IA-2), two major T1D-related auto-antigens. Attempts to secrete full size human GAD65 and IA-2 protein by L. lactis were unsuccessful. Trimming of GAD65 and IA-2 was investigated to optimise antigen secretion while maintaining sufficient bacterial growth. GAD65370-575 and IA-2635-979 showed to be efficiently secreted by recombinant L. lactis. Antigen secretion was verified by immunoblotting. Plasmid-derived GAD65 and IA-2 expression was combined in single strains with human IL-10 expression, a desired combination to allow tolerance induction. This study reports the generation of recombinant L. lactis secreting two major diabetes-related auto-antigens: human GAD65 and IA-2, by themselves or combined with the anti-inflammatory cytokine human IL-10. Prohibitive sequence obstacles hampering antigen secretion were resolved by trimming the full size proteins.

Isolation of digested sludge-assimilating fungal strains and their potential applications.

PubMed

Fujii, K; Kai, Y; Matsunobu, S; Sato, H; Mikami, A

2013-09-01

Digested sludge (DS) is a major waste product of anaerobic digestion of sewage sludge and is resistant to biodegradation. In this study, we isolated and characterized DS-assimilating fungi from soil. We tried to isolate DS-assimilating strains by enrichment culture using DS as the nutrient source, but microbial growth was not observed in any culture. To eliminate the inhibitory effect of metals in DS on microbial growth, acid-treated DS was subsequently used for enrichment, and eight fungal strains were isolated from the subcultures. At least 10-30% reduction in sludge was observed after 1-week cultivation, and prolonged cultivation led to further sludge reduction. All isolates produced xylanase, chitinase and keratinase. Phylogenetic analysis revealed that the isolates were Penicillium, Fusarium, Chaetomium, Cunninghamella, Neosartorya and Umbelopsis. Some isolates were suggested novel species. To the best of our knowledge, our study is the first to report the isolation of DS-assimilating strains. These isolates may be useful for commercial production of microbial enzymes using DS as the substrate. Because xylan, chitin and keratin in sludge-hyphae complexes are considered to be partially depolymerized, this material could also be utilized as a readily available fertilizer. © 2013 The Society for Applied Microbiology.

Plasmids in Vibrio parahemolyticus strains isolated in Japan and Bangladesh with special reference to different distributions.

PubMed

Arai, T; Ando, T; Kusakabe, A; Ullah, M A

1983-01-01

We surveyed plasmids in naturally occurring Vibrio parahemolyticus strains isolated in Japan and Bangladesh. Among the strains isolated in Japan, about half of the strains isolated from stools of patients of domestic diarrhea outbreaks as well as of travelers returning from East Asia were found to have plasmids, but no strains from foods had plasmids. In contrast, among the strains isolated in Bangladesh, none of the four strains isolated from patients had plasmids, but two out of eight strains isolated from water had plasmids, suggesting that plasmids are common in strains from the water in Bangladesh. All plasmids so far reported in V. parahemolyticus were detected in strains isolated from stools of patients. Incidences of plasmids in this organism were not so high in either area. In Japan, all plasmids were detected in strains from human intestines at 37 C, but in Bangladesh, where the temperature is around 30-40 C, the plasmids were detected in strains from the natural environment. These results suggested the possibility that these plasmids can come from different bacteria under rather high temperatures and that incidences of plasmids are influenced by the incidences of plasmids in bacteria present in the vicinity of V. parahemolyticus strains. None of these plasmids were found to have any relation to the biological characters tested.

21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

Code of Federal Regulations, 2012 CFR

2012-04-01

..., nontoxicogenic yeast Kluyveromyces lactis (previously named Saccharomyces lactis). It contains the enzyme β... prepared from yeast that has been grown in a pure culture fermentation and by using materials that are...

21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

Code of Federal Regulations, 2013 CFR

2013-04-01

..., nontoxicogenic yeast Kluyveromyces lactis (previously named Saccharomyces lactis). It contains the enzyme β... prepared from yeast that has been grown in a pure culture fermentation and by using materials that are...

Detection of diarrheagenic Escherichia coli strains isolated from dogs and cats in Brazil.

PubMed

Puño-Sarmiento, Juan; Medeiros, Leonardo; Chiconi, Carolina; Martins, Fernando; Pelayo, Jacinta; Rocha, Sérgio; Blanco, Jorge; Blanco, Miguel; Zanutto, Marcelo; Kobayashi, Renata; Nakazato, Gerson

2013-10-25

Escherichia coli are gut microbiota bacteria that can cause disease in some humans and other animals, including dogs and cats that humans often keep as pets. Diarrheagenic E. coli (DEC) strains are classified into six categories: enteropathogenic (EPEC), enterotoxigenic (ETEC), Shiga toxin-producing (STEC), enteroinvasive (EIEC), enteroaggregative (EAEC), and diffuse-adhering E. coli (DAEC). In this study 144 and 163 E. coli colonies were isolated from the fecal samples of 50 dogs and 50 cats, respectively, with and without diarrhea from a Veterinary Hospital (clinical isolates). The virulence factors were determined using multiplex Polymerase Chain Reaction. Adherence assays, antibacterial susceptibility and serotyping (somatic or flagellar antigens) were performed on DEC isolates. We found 25 (17.4%) and 4 (2.5%) DEC strains isolated from dogs and cats, respectively. Only the EPEC and EAEC pathotypes were found in both animals. Meanwhile, genes from other pathotypes (STEC, EIEC, and ETEC) were not found in these clinical isolates. All of the DEC strains showed mannose-resistant adherence to HEp-2 and HeLa cells, and aggregative adherence was predominant in these isolates. Multiresistant strains to antimicrobials were found in most DEC strains including usual and unusual antimicrobials in veterinary practices. The serotypes of these DEC isolates were variable. The ONT serotype was predominant in these isolates. Some serotypes found in our study were described to human DEC. Here, we demonstrate that pets carry virulent DEC genes, which are mainly strains of EPECs and EAECs. The presence of these virulence factors in isolates from animals without diarrhea suggests that pets can act as a reservoir for human infection. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular characterization of Streptococcus agalactiae strains isolated from fishes in Malaysia.

PubMed

Amal, M N A; Zamri-Saad, M; Siti-Zahrah, A; Zulkafli, A R; Nur-Nazifah, M

2013-07-01

The aim of this study was to characterize Streptococcus agalactiae strains that were isolated from fishes in Malaysia using random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (REP-PCR) techniques. A total of 181 strains of Strep. agalactiae isolated from red hybrid tilapia (Oreochromis sp.) and golden pompano (Trachinotus blochii) were characterized using RAPD and REP-PCR techniques. Both the fingerprinting techniques generated reproducible band patterns, differing in the number and molecular mass amplicons. The RAPD technique displayed greater discriminatory power by its production of more complex binding pattern and divided all the strains into 13 groups, compared to 9 by REP-PCR technique. Both techniques showed the availability to differentiate the genetic profiles of the strains according to their geographical location of origin. Three strains of Strep. agalactiae that were recovered from golden pompano showed a genetic dissimilarity from the strains isolated from red hybrid tilapia, while the strain of ATCC 27956 that recovered from bovine displayed a unique profile for both methods. Both techniques possess excellent discriminative capabilities and can be used as a rapid means of comparing Strep. agalactiae strains for future epidemiological investigation. Framework as the guideline in traceability of this disease and in the search for potential local vaccine candidates for streptococcosis in this country. Journal of Applied Microbiology © 2013 The Society for Applied Microbiology.

Lactococcus lactis-based vaccines: current status and future perspectives.

PubMed

Bahey-El-Din, Mohammed; Gahan, Cormac G M

2011-01-01

Lactococcus lactis offers significant potential as a platform for the delivery of vaccines especially via mucosal routes of administration. The organism has an established history of safe use in the food industry and is highly amenable to genetic manipulation, with many systems available for efficient production of secreted and surface-expressed proteins. Here we describe the benefits of using this organism as a vaccine delivery platform and outline how L. lactis based antigen delivery may be improved. Finally we discuss the safe use of L. lactis vectors and outline the potential for use of biological containment systems and killed lactococcal preparations.

Relationship between Glycolysis and Exopolysaccharide Biosynthesis in Lactococcus lactis

PubMed Central

Ramos, Ana; Boels, Ingeborg C.; de Vos, Willem M.; Santos, Helena

2001-01-01

The relationships between glucose metabolism and exopolysaccharide (EPS) production in a Lactococcus lactis strain containing the EPS gene cluster (Eps+) and in nonproducer strain MG5267 (Eps−) were characterized. The concentrations of relevant phosphorylated intermediates in EPS and cell wall biosynthetic pathways or glycolysis were determined by 31P nuclear magnetic resonance. The concentrations of two EPS precursors, UDP-glucose and UDP-galactose, were significantly lower in the Eps+ strain than in the Eps− strain. The precursors of the peptidoglycan pathway, UDP-N-acetylglucosamine and UDP-N-acetylmuramoyl-pentapeptide, were the major UDP-sugar derivatives detected in the two strains examined, but the concentration of the latter was greater in the Eps+ strain, indicating that there is competition between EPS synthesis and cell growth. An intermediate in biosynthesis of histidine and nucleotides, 5-phosphorylribose 1-pyrophosphate, accumulated at concentrations in the millimolar range, showing that the pentose phosphate pathway was operating. Fructose 1,6-bisphosphate and glucose 6-phosphate were the prominent glycolytic intermediates during exponential growth of both strains, whereas in the stationary phase the main metabolites were 3-phosphoglyceric acid, 2-phosphoglyceric acid, and phosphoenolpyruvate. The activities of relevant enzymes, such as phosphoglucose isomerase, α-phosphoglucomutase, and UDP-glucose pyrophosphorylase, were identical in the two strains. 13C enrichment on the sugar moieties of pure EPS showed that glucose 6-phosphate is the key metabolite at the branch point between glycolysis and EPS biosynthesis and ruled out involvement of the triose phosphate pool. This study provided clues for ways to enhance EPS production by genetic manipulation. PMID:11133425

Hsp65-producing Lactococcus lactis prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells

PubMed Central

Rezende, Rafael M.; Oliveira, Rafael P.; Medeiros, Samara R.; Gomes-Santos, Ana C.; Alves, Andrea C.; Loli, Flávia G.; Guimarães, Mauro A.F.; Amaral, Sylvia S.; da Cunha, André P.; Weiner, Howard L.; Azevedo, Vasco; Miyoshi, Anderson; Faria, Ana M.C.

2013-01-01

Heat shock proteins (Hsps) participate in the cellular response to stress and they are hiperexpressed in inflammatory conditions. They are also known to play a major role in immune modulation, controlling, for instance, autoimmune responses. In this study, we showed that oral administration of a recombinant Lactococcus lactis strain that produces and releases LPS-free Hsp65 prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. This was confirmed by the reduced inflammatory cell infiltrate and absence of injury signs in the spinal cord. The effect was associated with reduced IL-17 and increased IL-10 production in mesenteric lymph node and spleen cell cultures. Hsp65-producing-L. lactis-fed mice had a remarkable increase in the number of natural and inducible CD4+Foxp3+ regulatory T (Treg) cells and CD4+LAP+ (Latency-associated peptide) Tregs - which express the membrane-bound TGF-β - in spleen, inguinal and mesenteric lymph nodes as well as in spinal cord. Moreover, many Tregs co-expressed Foxp3 and LAP. In vivo depletion of LAP+ cells abrogated the effect of Hsp65-producing L. lactis in EAE prevention and worsened disease in medium-fed mice. Thus, Hsp65-L.lactis seems to boost this critical regulatory circuit involved in controlling EAE development in mice. PMID:22939403

Hsp65-producing Lactococcus lactis prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells.

PubMed

Rezende, Rafael M; Oliveira, Rafael P; Medeiros, Samara R; Gomes-Santos, Ana C; Alves, Andrea C; Loli, Flávia G; Guimarães, Mauro A F; Amaral, Sylvia S; da Cunha, André P; Weiner, Howard L; Azevedo, Vasco; Miyoshi, Anderson; Faria, Ana M C

2013-02-01

Heat shock proteins (Hsps) participate in the cellular response to stress and they are hiperexpressed in inflammatory conditions. They are also known to play a major role in immune modulation, controlling, for instance, autoimmune responses. In this study, we showed that oral administration of a recombinant Lactococcus lactis strain that produces and releases LPS-free Hsp65 prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. This was confirmed by the reduced inflammatory cell infiltrate and absence of injury signs in the spinal cord. The effect was associated with reduced IL-17 and increased IL-10 production in mesenteric lymph node and spleen cell cultures. Hsp65-producing-L. lactis-fed mice had a remarkable increase in the number of natural and inducible CD4+Foxp3+ regulatory T (Treg) cells and CD4+LAP+ (Latency-associated peptide) Tregs - which express the membrane-bound TGF-β - in spleen, inguinal and mesenteric lymph nodes as well as in spinal cord. Moreover, many Tregs co-expressed Foxp3 and LAP. In vivo depletion of LAP+ cells abrogated the effect of Hsp65-producing L. lactis in EAE prevention and worsened disease in medium-fed mice. Thus, Hsp65-L.lactis seems to boost this critical regulatory circuit involved in controlling EAE development in mice. Copyright © 2012 Elsevier Ltd. All rights reserved.

Isolation and speciation of Prevotella strains from periodontal abscesses.

PubMed

Dumitriu, S; Băncescu, G; Murea, A; Skaug, N

1998-01-01

The aims of the study were to isolate and to identify at species level the Prevotella strains in pus samples collected by needle aspiration from 25 Romanian patients with periodontal abscesses. Gram-stained smears and cultures on selective and nonselective media were performed from each of the 25 pus samples. The isolates were identified on the basis of Gram staining, cultural characteristics and standard biochemical reactions. The Gram-negative anaerobic bacilli isolates were biochemically characterized and identified at species level using the Rapid ID 32 A system (Bio Mérieux, France). Fifteen Prevotella isolates belonging to one of the following species: P. melaninogenica, P. denticola, P. oralis, P. loescheii and P. bivia were recovered. All Prevotella isolates reacted similarly in 20 tests in the Rapid ID 32 A system. The P. melaninogenica strain showed approximately the same biochemical profile and only two sugar fermentation tests were not constantly positive. The study confirmed that Prevotella is often involved in periodontal abscesses (> 50% of the cases) in association with other anaerobic or/and aerobic bacteria. P. melaninogenica was the most frequently isolated Prevotella species from the investigated cases.

Phenotypic and genotypic discrepancy of Streptococcus pneumoniae strains isolated from Asian countries.

PubMed

Ko, Kwan Soo; Oh, Won Sup; Peck, Kyong Ran; Lee, Jang Ho; Lee, Nam Yong; Song, Jae-Hoon

2005-07-01

Non-typeable isolates of Streptococcus pneumoniae collected from Asian countries were characterized by optochin susceptibility test, bile solubility test, multilocus sequence typing of housekeeping genes, amplification of virulence-related genes, 16S rDNA-RsaI digestion, and 16S rDNA sequencing. Six of 54 non-typeable pneumococcal isolates showed divergence of gene sequences of recP and xpt from typical pneumococcal strains. Of these six atypical pneumococcal strains, two showed different results in optochin susceptibility or bile solubility test from typical pneumococcal strains. All six isolates showed high sequence dissimilarities of multilocus sequence typing, 16S rDNA sequences, and lytA sequences from typical S. pneumoniae strains. Data from this study suggest that classic tests such as optochin susceptibility and bile solubility tests may lead to incorrect identification of S. pneumoniae. These atypical strains may belong to different bacterial species from S. pneumoniae.

Isolation of Dickeya dadantii strains from potato disease and biocontrol by their bacteriophages.

PubMed

Soleimani-Delfan, Abbas; Etemadifar, Zahra; Emtiazi, Giti; Bouzari, Majid

2015-01-01

One of the most economically important bacterial pathogens of plants and plant products is Dickeya dadantii. This bacterium causes soft rot disease in tubers and other parts of the potato and other plants of the Solanaceae family. The application of restricted host range bacteriophages as biocontrol agents has recently gained widespread interest. This study purposed to isolate the infectious agent of the potato and evaluate its biocontrol by bacteriophages. Two phytopathogenic strains were isolated from infected potatoes, identified based on biochemical and 16S rRNA gene sequencing, and submitted to GenBank as D. dadantii strain pis3 (accession no. HQ423668) and D. dadantii strain sip4 (accession no. HQ423669). Their bacteriophages were isolated from Caspian Sea water by enriching the water filtrate with D. dadantii strains as hosts using spot or overlay methods. On the basis of morphotypes, the isolated bacteriophages were identified as members of the Myoviridae and Siphoviridae families and could inhibit the growth of antibiotic resistant D. dadantii strains in culture medium. Moreover, in Dickeya infected plants treated with bacteriophage, no disease progression was detected. No significant difference was seen between phage-treated and control plants. Thus, isolated bacteriophages can be suggested for the biocontrol of plant disease caused by Dickeya strains.

Isolation and Preliminary Screening of a Weissella confusa Strain from Giant Panda (Ailuropoda melanoleuca).

PubMed

Xiong, Lvchen; Ni, Xueqin; Niu, Lili; Zhou, Yi; Wang, Qiang; Khalique, Abdul; Liu, Qian; Zeng, Yan; Shu, Gang; Pan, Kangcheng; Jing, Bo; Zeng, Dong

2018-04-13

Weissella confusa has recently received attention for its probiotic potential. Some W. confusa and Weissella cibaria strains isolated from fermented foods show favorable probiotic effects. However, the probiotic properties of W. confusa isolated from giant panda remain unreported to date. Thus, this study isolated a W. confusa strain from giant panda feces and then investigated its characteristics and probiotic properties. A lactic acid bacteria strain was isolated from giant panda fecal samples. The isolated strain was screened by in vitro probiotic property tests, including in vitro antimicrobial test, antioxidant test, surface hydrophobicity, and stress resistance. On the basis of biochemical identification and 16S rDNA sequencing, the W. confusa strain was identified as BSP201703. This Weissella confusa strain can survive at pH 2 and 0.3% (w/v) concentration of bile salt environment and inhibit common intestinal pathogens. It also possesses an in vitro antioxidant capacity, a high auto-aggregation ability, and a high surface hydrophobicity. BSP201703 might serve as a probiotic to giant pandas.

Molecular Characterization of Shiga Toxin-Producing Escherichia coli Strains Isolated in Poland.

PubMed

Januszkiewicz, Aleksandra; Rastawicki, Waldemar

2016-08-26

Shiga toxin-producing Escherichia coli (STEC) strains also called verotoxin-producing E. coli (VTEC) represent one of the most important groups of food-borne pathogens that can cause several human diseases such as hemorrhagic colitis (HC) and hemolytic - uremic syndrome (HUS) worldwide. The ability of STEC strains to cause disease is associated with the presence of wide range of identified and putative virulence factors including those encoding Shiga toxin. In this study, we examined the distribution of various virulence determinants among STEC strains isolated in Poland from different sources. A total of 71 Shiga toxin-producing E. coli strains isolated from human, cattle and food over the years 1996-2010 were characterized by microarray and PCR detection of virulence genes. As stx1a subtype was present in all of the tested Shiga toxin 1 producing E. coli strains, a greater diversity of subtypes was found in the gene stx2, which occurred in five subtypes: stx2a, stx2b, stx2c, stx2d, stx2g. Among STEC O157 strains we observed conserved core set of 14 virulence factors, stable in bacteria genome at long intervals of time. There was one cattle STEC isolate which possessed verotoxin gene as well as sta1 gene encoded heat-stable enterotoxin STIa characteristic for enterotoxigenic E. coli. To the best of our knowledge, this is the first comprehensive analysis of virulence gene profiles identified in STEC strains isolated from human, cattle and food in Poland. The results obtained using microarrays technology confirmed high effectiveness of this method in determining STEC virulotypes which provides data suitable for molecular risk assessment of the potential virulence of this bacteria. virulence factors including those encoding Shiga toxin. In this study, we examined the distribution of various virulence determinants among STEC strains isolated in Poland from different sources. A total of 71 Shiga toxin-producing E. coli strains isolated from human, cattle and food over

Acetobacter strains isolated during the acetification of blueberry (Vaccinium corymbosum L.) wine.

PubMed

Hidalgo, C; García, D; Romero, J; Mas, A; Torija, M J; Mateo, E

2013-09-01

Highbush blueberries (Vaccinium corymbosum L.) are known to have positive health benefits. The production of blueberry vinegar is one method to preserve this seasonal fruit and allow extended consumption. In this study, blueberry wine acetification was performed with naturally occurring micro-organisms and with an inoculated Acetobacter cerevisiae strain. Acetifications were carried out in triplicate using the Schützenbach method. The successful spontaneous processes took up to 66% more time than the processes involving inoculation. The isolation of acetic acid bacteria (AAB) and the analysis of these AAB using molecular methods allowed the identification of the main genotypes responsible of the blueberry acetification. Although the Acet. cerevisiae strain was the predominant strain isolated from the inoculated process samples, Acetobacter pasteurianus was isolated from samples for both processes and was the only species present in the spontaneous acetification samples. To the best of our knowledge, this is the first report describing the identification and variability of AAB isolated during blueberry acetification. The isolated Acet. pasteurianus strains could be used for large-scale blueberry vinegar production or as a starter culture in studies of other vinegar production methods. © 2013 The Society for Applied Microbiology.

Development of a systematic feedback isolation approach for targeted strains from mixed culture systems.

PubMed

Poudel, Pramod; Tashiro, Yukihiro; Miyamoto, Hirokuni; Miyamoto, Hisashi; Okugawa, Yuki; Sakai, Kenji

2017-01-01

Elucidation of functions of bacteria in a mixed culture system (MCS) such as composting, activated sludge system is difficult, since the system is complicating with many unisolated bacteria. Here, we developed a systematic feedback isolation strategy for the isolation and rapid screening of multiple targeted strains from MCS. Six major strains (Corynebacterium sphenisci, Bacillus thermocloacae, Bacillus thermoamylovorans, Bacillus smithii, Bacillus humi, and Bacillus coagulans), which are detected by denaturing gradient gel electrophoresis (DGGE) analysis in our previous study on MCS for l-lactic acid production, were targeted for isolation. Based on information of suitable cultivation conditions (e.g., media, pH, temperature) from the literature, feedback isolation was performed to form 136 colonies. The following direct colony matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was optimised as the second screening to narrow down 20 candidate colonies from similar spectra patterns with six closest type strains. This step could distinguish bacteria at the species level with distance similarity scores ≥0.55 corresponding to 16S rRNA gene sequence similarity ≥98.2%, suggesting that this is an effective technique to minimize isolates close to targeted type strains. Analysis of 16S rRNA gene sequences indicated that two targeted strains and one strain related to the target had successfully been isolated, showing high similarities (99.5-100%) with the sequences from the DGGE bands, and that the other candidates were affiliated with three strains that were closely related to the target species. This study proposes a new method for systematic feedback isolation that may be useful for isolating targeted strains from MCS for further investigation. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Antibiotic Resistance Determinants in a Pseudomonas putida Strain Isolated from a Hospital

PubMed Central

Duque, Estrella; Fernández, Matilde; Molina-Santiago, Carlos; Roca, Amalia; Porcel, Mario; de la Torre, Jesús; Segura, Ana; Plesiat, Patrick; Jeannot, Katy; Ramos, Juan-Luis

2014-01-01

Environmental microbes harbor an enormous pool of antibiotic and biocide resistance genes that can impact the resistance profiles of animal and human pathogens via horizontal gene transfer. Pseudomonas putida strains are ubiquitous in soil and water but have been seldom isolated from humans. We have established a collection of P. putida strains isolated from in-patients in different hospitals in France. One of the isolated strains (HB3267) kills insects and is resistant to the majority of the antibiotics used in laboratories and hospitals, including aminoglycosides, ß-lactams, cationic peptides, chromoprotein enediyne antibiotics, dihydrofolate reductase inhibitors, fluoroquinolones and quinolones, glycopeptide antibiotics, macrolides, polyketides and sulfonamides. Similar to other P. putida clinical isolates the strain was sensitive to amikacin. To shed light on the broad pattern of antibiotic resistance, which is rarely found in clinical isolates of this species, the genome of this strain was sequenced and analysed. The study revealed that the determinants of multiple resistance are both chromosomally-borne as well as located on the pPC9 plasmid. Further analysis indicated that pPC9 has recruited antibiotic and biocide resistance genes from environmental microorganisms as well as from opportunistic and true human pathogens. The pPC9 plasmid is not self-transmissible, but can be mobilized by other bacterial plasmids making it capable of spreading antibiotic resistant determinants to new hosts. PMID:24465371

Cell-Wall-Bound Proteinase of Lactobacillus delbrueckii subsp. lactis ACA-DC 178: Characterization and Specificity for β-Casein

PubMed Central

Tsakalidou, E.; Anastasiou, R.; Vandenberghe, I.; van Beeumen, J.; Kalantzopoulos, G.

1999-01-01

Lactobacillus delbrueckii subsp. lactis ACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca2+-free buffer. The crude proteinase extract shows its highest activity at pH 6.0 and 40°C. It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the enzyme is similar to the lactococcal PI-type proteinases, since it hydrolyzes β-casein mainly and α- and κ-caseins to a much lesser extent. The cell-wall-bound proteinase from L. delbrueckii subsp. lactis ACA-DC 178 liberates four main peptides from β-casein, which have been identified. PMID:10223997

Some space shuttle tile/strain-isolator-pad sinusoidal vibration tests

NASA Technical Reports Server (NTRS)

Miserentino, R.; Pinson, L. D.; Leadbetter, S. A.

1980-01-01

Vibration tests were performed on the tile/strain-isolator-pad system used as thermal protection for the space shuttle orbiter. Experimental data on normal and in-plane vibration response and damping properties are presented. Three test specimens exhibited shear type motion during failures that occurred in the tile near the tile/strain-isolator-pad bond-line. A dynamic instability is described which has large in-plane motion at a frequency one-half that of the nominal driving frequency. Analysis shows that this phenomenon is a parametric response.

Diversity of Geotrichum candidum Strains Isolated from Traditional Cheesemaking Fabrications in France

PubMed Central

Marcellino, N.; Beuvier, E.; Grappin, R.; Guéguen, M.; Benson, D. R.

2001-01-01

The diversity of French fungus-ripened cheeses is due partly to the succession of fungi that colonize the cheese during ripening. Geotrichum candidum appears in the early stages of ripening on soft cheeses such as Camembert and semihard cheeses such as St. Nectaire and Reblochon. Its lipases and proteases promote flavor development, and its aminopeptidases reduce bitterness imparted by low-molecular-weight peptides in cheese. We assessed the genetic diversity of G. candidum strains by using random amplification of polymorphic DNA (RAPD)-PCR correlated with phenotypic tests for carbon assimilation and salt tolerance. Strains were isolated from milk, curd, and cheese collected in seven major cheesemaking regions of France. Sixty-four isolates were characterized. We found high genetic diversity of G. candidum even within the same cheesemaking regions. Strains did not group according to region. All of the strains from the Haute-Savoie were able to assimilate lactate as the sole source of carbon, while lactate assimilation varied among strains from the Auvergne. Strains varied in d-mannitol assimilation, and none used citrate as the sole source of carbon. Yeast-like colony morphology predominated in Reblochon, while all of the strains isolated from St. Nectaire were filamentous. The RAPD-PCR technique readily differentiated Geotrichum fragrans isolated from milk and curd in a St. Nectaire cheesemaking facility. This study reveals an enormous diversity of G. candidum that has been empirically selected through the centuries by the cheesemakers of France. PMID:11571181

Diversity of Geotrichum candidum strains isolated from traditional cheesemaking fabrications in France.

PubMed

Marcellino, N; Beuvier, E; Grappin, R; Guéguen, M; Benson, D R

2001-10-01

The diversity of French fungus-ripened cheeses is due partly to the succession of fungi that colonize the cheese during ripening. Geotrichum candidum appears in the early stages of ripening on soft cheeses such as Camembert and semihard cheeses such as St. Nectaire and Reblochon. Its lipases and proteases promote flavor development, and its aminopeptidases reduce bitterness imparted by low-molecular-weight peptides in cheese. We assessed the genetic diversity of G. candidum strains by using random amplification of polymorphic DNA (RAPD)-PCR correlated with phenotypic tests for carbon assimilation and salt tolerance. Strains were isolated from milk, curd, and cheese collected in seven major cheesemaking regions of France. Sixty-four isolates were characterized. We found high genetic diversity of G. candidum even within the same cheesemaking regions. Strains did not group according to region. All of the strains from the Haute-Savoie were able to assimilate lactate as the sole source of carbon, while lactate assimilation varied among strains from the Auvergne. Strains varied in D-mannitol assimilation, and none used citrate as the sole source of carbon. Yeast-like colony morphology predominated in Reblochon, while all of the strains isolated from St. Nectaire were filamentous. The RAPD-PCR technique readily differentiated Geotrichum fragrans isolated from milk and curd in a St. Nectaire cheesemaking facility. This study reveals an enormous diversity of G. candidum that has been empirically selected through the centuries by the cheesemakers of France.

Tannic acid degradation by Klebsiella strains isolated from goat feces

PubMed Central

Tahmourespour, Arezoo; Tabatabaee, Nooroldin; Khalkhali, Hossein; Amini, Imane

2016-01-01

Background and Objectives: Tannins are toxic polyphenols that either bind and precipitate or condense proteins. The high tannin content of some plants is the preliminary limitation of using them as a ruminant feed. So, the aim of this study was the isolation and characterization of tannic acid degrading bacterial strains from goat feces before and after feeding on Pistachio-Soft Hulls as tannin rich diet (TRD). Materials and Methods: Bacterial strains capable of utilizing tannic acid as sole carbon and energy source were isolated and characterized from goat feces before and after feeding on TRD. Tannase activity, maximum tolerable concentration and biodegradation potential were assessed. Results: Four tannase positive isolates were identified as Klebsiella pneumoniae. Isolated strains showed the maximum tolerable concentration of 64g/L of tannin. The tannic acid degradation percentage at a concentration of 15.0 g/L reached a maximum of 68% after 24 h incubation, and more than 98% after 72 h incubation. The pH of the medium also decreased along with tannic acid utilization. Conclusions: It is obvious that TRD induced adaptive responses. Thus, while the bacteria were able to degrade and detoxify the tannic acids, they had to adapt in the presence of high concentrations of tannic acid. So, these isolates have an amazing potential for application in bioremediation, waste water treatment, also reduction of tannins antinutritional effects in animal feeds. PMID:27092220

Isolation of bisphenol A-tolerant/degrading Pseudomonas monteilii strain N-502.

PubMed

Masuda, Midori; Yamasaki, Yoshiki; Ueno, Shun; Inoue, Akira

2007-03-01

Bisphenol A (BPA) is a highly biotoxic compound that kills many microorganisms at a low concentration (1,000 ppm). We isolated BPA-tolerant/degrading Pseudomonas monteilii strain N-502 from about 1,000 samples collected from a field, sewage, and pond water. The isolated strain had strong BPA tolerance and high BPA-degrading activity. This strain was able to grow in a minimum medium containing BPA as the sole carbon source. Strain N-502 is an aerobic, motile, gram-negative, nonspore-forming, rod-shaped bacterium and was identified as P. monteilii, based on 16 S rRNA gene analysis. Strain N-502 completely degraded BPA 500 ppm in a 10-day, in culture system and was able to degrade BPA 100 ppm in a 2-h resting cell system. This strain also showed potent ability to degrade BPA 500 and 1,000 ppm in the resting cell system. Moreover, the initial BPA degradation rate was accelerated with the addition of Ca(2+), Mg(2+), and folic acid.

Isolation and in vitro selection of actinomycetes strains as potential probiotics for aquaculture

PubMed Central

Bernal, Milagro García; Campa-Córdova, Ángel Isidro; Saucedo, Pedro Enrique; González, Marlen Casanova; Marrero, Ricardo Medina; Mazón-Suástegui, José Manuel

2015-01-01

Aim: This study was designed to describe a series of in vitro tests that may aid the discovery of probiotic strains from actinomycetes. Materials and Methods: Actinomycetes were isolated from marine sediments using four different isolation media, followed by antimicrobial activity and toxicity assessment by the agar diffusion method and the hemolysis of human blood cells, respectively. Extracellular enzymatic production was monitored by the hydrolysis of proteins, lipids and carbohydrates. Tolerance to different pH values and salt concentrations was also determined, followed by hydrophobicity analysis and genetic identification of the most promising strains. Results: Five out of 31 isolated strains showed antimicrobial activity against three Vibrio species. Three non-hemolytic strains (N7, RL8 and V4) among these active isolates yielded positive results in hydrophobicity tests and exhibited good growth at salt concentrations ranging from 0% to 10%, except strain RL8, which required a salt concentration >0.6%. Although these strains did not grow at pH<3, they showed different enzymatic activities. Phylogenetic analysis revealed that strains N7 and V4 have more than 99% identity with several Streptomyces species, whereas the closest matches to strain RL8 are Streptomyces panacagri and Streptomyces flocculus, with 98% and 98.2% similarity, respectively. Conclusion: Three actinomycetes strains showing probiotic-like properties were discovered using several in vitro tests that can be easily implemented in different institutions around the world. PMID:27047067

Isolation and Identification of Lactic Acid Bacteria from Traditional Dairy Products in Baotou and Bayannur of Midwestern Inner Mongolia and q-PCR Analysis of Predominant Species

PubMed Central

2016-01-01

In this study, traditional culture method and 16S rRNA gene analysis were applied to reveal the composition and diversity of lactic acid bacteria (LAB) of fermented cow milk, huruud and urum from Baotou and Bayannur of midwestern Inner Mongolia. Also, the quantitative results of dominant LAB species in three different types of dairy products from Baotou and Bayannur were gained by quantitative polymerase chain reaction (q-PCR) technology. Two hundred and two LAB strains isolated from sixty-six samples were identified and classified into four genera, namely Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, and twenty-one species and subspecies. From these isolates, Lactococcus lactis subsp. lactis (32.18%), Lactobacillus plantarum (12.38%) and Leuconosto mesenteroides (11.39%) were considered as the dominated LAB species under the condition of cultivating in MRS and M17 medium. And the q-PCR results revealed that the number of dominant species varied from samples to samples and from region to region. This study clearly shows the composition and diversity of LAB existing in fermented cow milk, huruud and urum, which could be considered as valuable resources for LAB isolation and further probiotic selection. PMID:27621691

Epidemic Clostridium difficile Strains Demonstrate Increased Competitive Fitness Compared to Nonepidemic Isolates

PubMed Central

Robinson, Catherine D.; Auchtung, Jennifer M.; Collins, James

2014-01-01

Clostridium difficile infection is the most common cause of severe cases of antibiotic-associated diarrhea (AAD) and is a significant health burden. Recent increases in the rate of C. difficile infection have paralleled the emergence of a specific phylogenetic clade of C. difficile strains (ribotype 027; North American pulsed-field electrophoresis 1 [NAP1]; restriction endonuclease analysis [REA] group BI). Initial reports indicated that ribotype 027 strains were associated with increased morbidity and mortality and might be hypervirulent. Although subsequent work has raised some doubt as to whether ribotype 027 strains are hypervirulent, the strains are considered epidemic isolates that have caused severe outbreaks across the globe. We hypothesized that one factor that could lead to the increased prevalence of ribotype 027 strains would be if these strains had increased competitive fitness compared to strains of other ribotypes. We developed a moderate-throughput in vitro model of C. difficile infection and used it to test competition between four ribotype 027 clinical isolates and clinical isolates of four other ribotypes (001, 002, 014, and 053). We found that ribotype 027 strains outcompeted the strains of other ribotypes. A similar competitive advantage was observed when two ribotype pairs were competed in a mouse model of C. difficile infection. Based upon these results, we conclude that one possible mechanism through which ribotype 027 strains have caused outbreaks worldwide is their increased ability to compete in the presence of a complex microbiota. PMID:24733099

Molecular characterisation of Mycobacterium caprae strains isolated in Poland.

PubMed

Krajewska-Wędzina, Monika; Kozińska, Monika; Orłowska, Blanka; Weiner, Marcin; Szulowski, Krzysztof; Augustynowicz-Kopeć, Ewa; Anusz, Krzysztof; Smith, Noel H

2018-03-10

Bovine tuberculosis (bovine TB, bTB) is caused by bovine bacilli: Mycobacterium bovis and M caprae The studies conducted in Poland, in the National Bovine Tuberculosis Reference Laboratory in the Department of Microbiology of the National Veterinary Research Institute in Pulawy, show that animal tuberculosis in Poland is also caused by M caprae We here describe the identification and genotypic assessment of 52 isolates of M caprae obtained from Polish cattle and wild animals over the last five years. We show that strains isolated from bison have significant genotypic diversity and are distinct compared with the genotypes of strains isolated from cattle. Similarly, isolates from cattle herds can be highly genotypically variable. Formal designation of the members of the Mycobacterium tuberculosis complex is controversial in Poland; there is a gap in veterinary legislation with regard to bTB and no explicit mention of M caprae causing tuberculosis in animal. © British Veterinary Association (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Selection of Streptococcus lactis Mutants Defective in Malolactic Fermentation

PubMed Central

Renault, Pierre P.; Heslot, Henri

1987-01-01

An enrichment medium and a new sensitive medium were developed to detect malolactic variants in different strains of lactic bacteria. Factors such as the concentration of glucose and l-malate, pH level, and the type of indicator dye used are discussed with regard to the kinetics of malic acid conversion to lactic acid. Use of these media allowed a rapid and easier screening of mutagenized streptococcal cells unable to ferment l-malate. A collection of malolactic-negative mutants of Streptococcus lactis induced by UV, nitrosoguanidine, or transposonal mutagenesis were characterized. The results showed that several mutants were apparently defective in the structural gene of malolactic enzyme, whereas others contained mutations which may either inactivate a putative permease or affect a regulatory sequence. PMID:16347282

Screening, identification and characterization of bacteriocins produced by wine-isolated LAB strains.

PubMed

Ndlovu, B; Schoeman, H; Franz, C M A P; du Toit, M

2015-04-01

To screen and identify wine-isolated LAB strains for bacteriocin production, and to identify and characterize bacteriocins. One hundred and fifty-five LAB strains isolated from South African red wines undergoing spontaneous malolactic fermentation were screened for bacteriocin production. Eight isolates were identified to be bacteriocin producers and were identified as Enterococcus faecium. All eight isolates had the same phenotypic and genotypic profiles. The peptides were preliminarily identified as enterocin P using mass spectrometry and further confirmed by PCR-amplifying enterocin P gene. The enterocin activity was inhibited by α-Chymotrypsin, papain and proteinase K treatments. It was heat stable at 37, 60, 80 and 100°C and showed activity over a broad pH range of 2-10. The production of the enterocin followed that of primary metabolite kinetics and, it showed bactericidal effect to some wine spoilage LAB strains. Our study identified the presence of the enterocin-producing Enterococcus in wine. The enterocin was heat stable; with broad pH range and bactericidal effects to sensitive strains. This is one of very few studies that isolated Enterococcus species from wine. It is, however, the first to report presence of bacteriocin-producing Enterococcus in wine fermentation. © 2015 The Society for Applied Microbiology.

Draft Genome Sequences of Two Aspergillus fumigatus Strains, Isolated from the International Space Station.

PubMed

Singh, Nitin Kumar; Blachowicz, Adriana; Checinska, Aleksandra; Wang, Clay; Venkateswaran, Kasthuri

2016-07-14

Draft genome sequences of Aspergillus fumigatus strains (ISSFT-021 and IF1SW-F4), opportunistic pathogens isolated from the International Space Station (ISS), were assembled to facilitate investigations of the nature of the virulence characteristics of the ISS strains to other clinical strains isolated on Earth. Copyright © 2016 Singh et al.

Isolation and characterization of new strains of cholesterol-reducing bacteria from baboons.

PubMed

Brinkley, A W; Gottesman, A R; Mott, G E

1982-01-01

We isolated and characterized nine new strains of cholesterol-reducing bacteria from feces and intestinal contents of baboons. Cholesterol-brain agar was used for the primary isolation, and subsequent biochemical tests were done in a lecithin-cholesterol broth containing plasmenylethanolamine and various substrates. All strains had similar colony and cell morphology, hydrolyzed the beta-glucosides esculin and amygdalin, metabolized pyruvate, and produced acetate and acetoin. Unlike previously reported strains, the nine new strains did not require cholesterol and an alkenyl ether lipid (e.g., plasmalogen) for growth; however, only two strains reduced cholesterol in the absence of the plasmalogen. These two strains also produced succinate as an end product. Carbohydrate fermentation was variable; some strains produced weak acid (pH 5.5 to 6.0) from only a few carbohydrates, whereas other strains produced strong acid reactions (pH less than or equal to 5.5) from a wide variety of carbohydrates.

Molecular characterization of Mycobacterium bovis strains isolated from cattle slaughtered at two abattoirs in Algeria

PubMed Central

Sahraoui, Naima; Müller, Borna; Guetarni, Djamel; Boulahbal, Fadéla; Yala, Djamel; Ouzrout, Rachid; Berg, Stefan; Smith, Noel H; Zinsstag, Jakob

2009-01-01

Background Bovine Tuberculosis is prevalent in Algeria despite governmental attempts to control the disease. The objective of this study was to conduct, for the first time, molecular characterization of a population sample of Mycobacterium bovis strains isolated from slaughter cattle in Algeria. Between August and November 2007, 7250 animals were consecutively screened at the abattoirs of Algiers and Blida. In 260 animals, gross visible granulomatous lesions were detected and put into culture. Bacterial isolates were subsequently analysed by molecular methods. Results Altogether, 101 bacterial strains from 100 animals were subjected to molecular characterization. M. bovis was isolated from 88 animals. Other bacteria isolated included one strain of M. caprae, four Rhodococcus equi strains, three Non-tuberculous Mycobacteria (NTM) and five strains of other bacterial species. The M. bovis strains isolated showed 22 different spoligotype patterns; four of them had not been previously reported. The majority of M. bovis strains (89%) showed spoligotype patterns that were previously observed in strains from European cattle. Variable Number of Tandem Repeat (VNTR) typing supported a link between M. bovis strains from Algeria and France. One spoligotype pattern has also been shown to be frequent in M. bovis strains from Mali although the VNTR pattern of the Algerian strains differed from the Malian strains. Conclusion M. bovis infections account for a high amount of granulomatous lesions detected in Algerian slaughter cattle during standard meat inspection at Algiers and Blida abattoir. Molecular typing results suggested a link between Algerian and European strains of M. bovis. PMID:19173726

Modeling of the Competitive Growth of Listeria monocytogenes and Lactococcus lactis in Vegetable Broth

PubMed Central

Breidt, Frederick; Fleming, Henry P.

1998-01-01

Current mathematical models used by food microbiologists do not address the issue of competitive growth in mixed cultures of bacteria. We developed a mathematical model which consists of a system of nonlinear differential equations describing the growth of competing bacterial cell cultures. In this model, bacterial cell growth is limited by the accumulation of protonated lactic acid and decreasing pH. In our experimental system, pure and mixed cultures of Lactococcus lactis and Listeria monocytogenes were grown in a vegetable broth medium. Predictions of the model indicate that pH is the primary factor that limits the growth of L. monocytogenes in competition with a strain of L. lactis which does not produce the bacteriocin nisin. The model also predicts the values of parameters that affect the growth and death of the competing populations. Further development of this model will incorporate the effects of additional inhibitors, such as bacteriocins, and may aid in the selection of lactic acid bacterium cultures for use in competitive inhibition of pathogens in minimally processed foods. PMID:9726854

Full-Genome Sequence Analysis of a Multirecombinant Echovirus 3 Strain Isolated from Sewage in Greece▿

PubMed Central

Kyriakopoulou, Zaharoula; Dedepsidis, Evaggelos; Pliaka, Vaia; Tsakogiannis, Dimitris; Pratti, Anastassia; Levidiotou-Stefanou, Stamatina; Markoulatos, Panayotis

2010-01-01

An echovirus 3 (Echo3) strain (strain LR31G7) was isolated from a sewage treatment plant in Greece in 2005. Full-genome molecular, phylogenetic, and SimPlot analyses were conducted in order to reveal the evolutionary pathways of the isolate. Nucleotide and phylogenetic analyses of part of the VP1 genomic region revealed that the isolated strain correlates with Echo3 strains isolated during the same year in France and Japan, implying that the same virus circulated in Europe and Asia. LR31G7 was found to be a recombinant that shares the 3′ part of its genome with an Echo25 strain isolated from asymptomatic infants in Norway in 2003. Nucleotide and SimPlot analyses of the VP1-2A junction, where the recombination was located, revealed the exact recombination breakpoint (nucleotides 3357 to 3364). Moreover, there is evidence that recombination events had occurred in 3B-3D region in the evolutionary history of the isolate. Our study indicates that recombination events play major roles in enterovirus evolution and that the circulation of multirecombinant strains with unknown properties could be potentially dangerous for public health. PMID:20129960

Urease-positive thermophilic strains of Campylobacter isolated from seagulls (Larus spp.).

PubMed

Kaneko, A; Matsuda, M; Miyajima, M; Moore, J E; Murphy, P G

1999-07-01

Three strains of urease-positive thermophilic Campylobacter (UPTC), designated A1, A2 and A3, were identified by biochemical characterization after isolation from faeces of seagulls in Northern Ireland in 1996. The biochemical characteristics of the strains were identical to those of strains described previously. Analysis by pulsed-field gel electrophoresis (PFGE) after separate digestion with ApaI and SmaI demonstrated that the respective PFGE profiles were indistinguishable. The PFGE analysis also suggested that the genomes were approximately 1810 kb in length. This is the first example of the isolation of UPTC from flying homoiothermal animals, i.e. from seagulls (Larus spp.).

Isolating and evaluating lactic acid bacteria strains for effectiveness of Leymus chinensis silage fermentation.

PubMed

Zhang, Q; Li, X J; Zhao, M M; Yu, Z

2014-10-01

Five LAB strains were evaluated using the acid production ability test, morphological observation, Gram staining, physiological, biochemical and acid tolerance tests. All five strains (LP1, LP2, LP3, LC1 and LC2) grew at pH 4·0, and LP1 grew at 15°C. Strains LP1, LP2 and LP3 were identified as Lactobacillus plantarum, whereas LC1 and LC2 were classified as Lactobacillus casei by sequencing 16S rDNA. The five isolated strains and two commercial inoculants (PS and CL) were added to native grass and Leymus chinensis (Trin.) Tzvel. for ensiling. All five isolated strains decreased the pH and ammonia nitrogen content, increased the lactic acid content and LP1, LP2 and LP3 increased the acetic content and lactic/acetic acid ratio of L. chinensis silage significantly. The five isolated strains and two commercial inoculants decreased the butyric acid content of the native grass silage. LP2 treatment had lower butyric acid content and ammonia nitrogen content than the other treatments. The five isolated strains improved the quality of L. chinensis silage. The five isolated strains and the two commercial inoculants were not effective in improving the fermentation quality of the native grass silage, but LP2 performed better comparatively. Significance and impact of the study: Leymus chinensis is an important grass in China and Russia, being the primary grass of the short grassland 'steppe' regions of central Asia. However, it has been difficult to make high-quality silage of this species because of low concentration of water-soluble carbohydrates (WSC). Isolating and evaluating lactic acid bacteria strains will be helpful for improving the silage quality of this extensively grown species. © 2014 The Society for Applied Microbiology.

Galactose transport in Kluyveromyces lactis: major role of the glucose permease Hgt1.

PubMed

Baruffini, Enrico; Goffrini, Paola; Donnini, Claudia; Lodi, Tiziana

2006-12-01

In Kluyveromyces lactis, galactose transport has been thought to be mediated by the lactose permease encoded by LAC12. In fact, a lac12 mutant unable to grow on lactose did not grow on galactose either and showed low and uninducible galactose uptake activity. The existence of other galactose transport systems, at low and at high affinity, had, however, been hypothesized on the basis of galactose uptake kinetics studies. Here we confirmed the existence of a second galactose transporter and we isolated its structural gene. It turned out to be HGT1, previously identified as encoding the high-affinity glucose carrier. Analysis of galactose transporter mutants, hgt1 and lac12, and the double mutant hgt1lac12, suggested that Hgt1 was the high-affinity and Lac12 was the low-affinity galactose transporter. HGT1 expression was strongly induced by galactose and insensitive to glucose repression. This could explain the rapid adaptation to galactose observed in K. lactis after a shift from glucose to galactose medium.

Variable characteristics of bacteriocin-producing Streptococcus salivarius strains isolated from Malaysian subjects.

PubMed

Barbour, Abdelahhad; Philip, Koshy

2014-01-01

Salivaricins are bacteriocins produced by Streptococcus salivarius, some strains of which can have significant probiotic effects. S. salivarius strains were isolated from Malaysian subjects showing variable antimicrobial activity, metabolic profile, antibiotic susceptibility and lantibiotic production. In this study we report new S. salivarius strains isolated from Malaysian subjects with potential as probiotics. Safety assessment of these strains included their antibiotic susceptibility and metabolic profiles. Genome sequencing using Illumina's MiSeq system was performed for both strains NU10 and YU10 and demonstrating the absence of any known streptococcal virulence determinants indicating that these strains are safe for subsequent use as probiotics. Strain NU10 was found to harbour genes encoding salivaricins A and 9 while strain YU10 was shown to harbour genes encoding salivaricins A3, G32, streptin and slnA1 lantibiotic-like protein. Strain GT2 was shown to harbour genes encoding a large non-lantibiotic bacteriocin (salivaricin-MPS). A new medium for maximum biomass production buffered with 2-(N-morpholino)ethanesulfonic acid (MES) was developed and showed better biomass accumulation compared with other commercial media. Furthermore, we extracted and purified salivaricin 9 (by strain NU10) and salivaricin G32 (by strain YU10) from S. salivarius cells grown aerobically in this medium. In addition to bacteriocin production, S. salivarius strains produced levan-sucrase which was detected by a specific ESI-LC-MS/MS method which indicates additional health benefits from the developed strains. The current study established the bacteriocin, levan-sucrase production and basic safety features of S. salivarius strains isolated from healthy Malaysian subjects demonstrating their potential for use as probiotics. A new bacteriocin-production medium was developed with potential scale up application for pharmaceuticals and probiotics from S. salivarius generating different

The structure and immunoreactivity of exopolysaccharide isolated from Lactobacillus johnsonii strain 151.

PubMed

Górska-Frączek, Sabina; Sandström, Corine; Kenne, Lennart; Paściak, Mariola; Brzozowska, Ewa; Strus, Magdalena; Heczko, Piotr; Gamian, Andrzej

2013-08-30

The exopolysaccharide (EPS) structure from Lactobacillus johnsonii strain 151 isolated from the intestinal tract of mice was investigated. Sugar and methylation analyses together with (1)H and (13)C NMR spectroscopy, including two-dimensional (1)H,(1)H COSY, TOCSY, NOESY, and (1)H,(13)C HSQC, HMBC experiments, revealed that the repeating unit of the EPS is the linear pentasaccharide: →6)-α-d-Galp-(1→6)-α-d-Glcp-(1→3)-β-d-Galf-(1→3)-α-d-Glcp-(1→2)-β-d-Galf-(1→ The immunoreactivity of two structurally different exopolysaccharides isolated from L. johnsonii, 151 and 142 (Carbohydr. Res. 2010, 345, 108-114), was compared. Both EPSs differed in their reactivity with antisera. EPS from L. johnsonii 151 reacted with anti-Lactobacillus polyclonal sera against cells of five different strains, while EPS from L. johnsonii 142 was found to react only with its own antiserum. The broader specificity and higher reactivity of EPS from 151 strain than EPS from 142 strain were also observed with human sera. The physiological antibodies recognizing polysaccharide antigens were present in both adults and umbilical cord blood sera. A highly specific EPS 142 bearing strain was isolated from experimentally induced inflammatory bowel disease (IBD) mice, while a strain with EPS 151 isolated from the intestinal tract of healthy mice is characterized by a broad immune reactivity common structure. Copyright © 2013 Elsevier Ltd. All rights reserved.

Antimicrobial properties of lactic acid bacteria isolated from traditional yogurt and milk against Shigella strains.

PubMed

Zare Mirzaei, Elnaze; Lashani, Elahe; Davoodabadi, Abolfazl

2018-01-01

Background: Lactic acid bacteria (LAB) are normal flora of the mouth, intestines and the female genital tract. They are also frequently found in meat, vegetables, and dairy products. Most of probiotic bacteria belong to the LAB group. Some probiotic LAB are useful in prevention and treatment of diarrheal diseases. The aim of this study was to investigate the antimicrobial properties of LAB isolated from traditional yogurt and milk against Shigella strains. Materials and methods: Forty LAB strains were isolated from traditional yogurt and milk. The antimicrobial activity of LAB against Shigella strains (eight S. flexneri , four S. sonnei ) was examined using the agar-well diffusion assay. LAB strains with antimicrobial effect against all Shigella strains were identified by 16S rRNA gene sequencing. Results: Six LAB strains inhibited the growth of all 12 Shigella strains. Lb. paracasei Y1-3, Lb. paracasei Y8-1 and Lb. fermentum Y2-2 were isolated from yogurt. Lb. paracasei M18-1, Lb. parelimentarius M4-3 and Lb. plantarum M19-1 were isolated from milk. Conclusion: This study showed that Lactobacillus strains with good inhibitory activity against S. flexneri and S. sonnei could be isolated from traditional yogurt and milk.

Isolation and identification of biocellulose-producing bacterial strains from Malaysian acidic fruits.

PubMed

Voon, W W Y; Rukayadi, Y; Meor Hussin, A S

2016-05-01

Biocellulose (BC) is pure extracellular cellulose produced by several species of micro-organisms that has numerous applications in the food, biomedical and paper industries. However, the existing biocellulose-producing bacterial strain with high yield was limited. The aim of this study was to isolate and identify the potential biocellulose-producing bacterial isolates from Malaysian acidic fruits. One hundred and ninety-three bacterial isolates were obtained from 19 local acidic fruits collected in Malaysia and screened for their ability to produce BC. A total of 15 potential bacterial isolates were then cultured in standard Hestrin-Schramm (HS) medium statically at 30°C for 2 weeks to determine the BC production. The most potent bacterial isolates were identified using 16S rRNA gene sequence analysis, morphological and biochemical characteristics. Three new and potent biocellulose-producing bacterial strains were isolated from soursop fruit and identified as Stenotrophomonas maltophilia WAUPM42, Pantoea vagans WAUPM45 and Beijerinckia fluminensis WAUPM53. Stenotrophomonas maltophilia WAUPM42 was the most potent biocellulose-producing bacterial strain that produced the highest amount of BC 0·58 g l(-1) in standard HS medium. Whereas, the isolates P. vagans WAUPM45 and B. fluminensis WAUPM53 showed 0·50 and 0·52 g l(-1) of BC production, respectively. Biocellulose (BC) is pure extracellular cellulose that is formed by many micro-organisms in the presence of carbon source and acidic condition. It can replace plant-based cellulose in multifarious applications due to its unique characteristics. In this study, three potential biocellulose-producing bacterial strains were obtained from Malaysian acidic fruits and identified as Stenotrophomonas maltophilia WAUPM42, Pantoea vagans WAUPM45 and Beijerinckia fluminensis WAUPM53. This study reports for the first time the new biocellulose-producing bacterial strains isolated from Malaysian acidic fruits. © 2016 The

Lactococcus lactis As a Versatile Vehicle for Tolerogenic Immunotherapy

PubMed Central

Cook, Dana P.; Gysemans, Conny; Mathieu, Chantal

2018-01-01

Genetically modified Lactococcus lactis bacteria have been engineered as a tool to deliver bioactive proteins to mucosal tissues as a means to exert both local and systemic effects. They have an excellent safety profile, the result of years of human consumption in the food industry, as well as a lack of toxicity and immunogenicity. Also, containment strategies have been developed to promote further application as clinical protein-based therapeutics. Here, we review technological advancements made to enhanced the potential of L. lactis as live biofactories and discuss some examples of tolerogenic immunotherapies mediated by mucosal drug delivery via L. lactis. Additionally, we highlight their use to induce mucosal tolerance by targeted autoantigen delivery to the intestine as an approach to reverse autoimmune type 1 diabetes. PMID:29387056

Secretory expression of a heterologous nattokinase in Lactococcus lactis.

PubMed

Liang, Xiaobo; Zhang, Lixin; Zhong, Jin; Huan, Liandong

2007-05-01

Nattokinase has been reported as an oral health product for the prevention of atherosclerosis. We developed a novel strategy to express a nattokinase from Bacillus subtilis in a live delivery vehicle, Lactococcus lactis. Promoter P( nisZ) and signal peptide SP(Usp) were used for inducible and secretory expression of nattokinase in L. lactis. Western blotting analysis demonstrated that nattokinase was successfully expressed, and about 94% of the enzyme was secreted to the culture. The recombinant nattokinase showed potent fibrinolytic activity, equivalent to 41.7 urokinase units per milliliter culture. Expression and delivery of such a fibrinolytic enzyme in the food-grade vehicle L. lactis would facilitate the widespread application of nattokinase in the control and prevention of thrombosis diseases.

Isolation of Thermus strains from hot composts (60 to 80 degrees C).

PubMed Central

Beffa, T; Blanc, M; Lyon, P F; Vogt, G; Marchiani, M; Fischer, J L; Aragno, M

1996-01-01

High numbers (10(7) to 10(10) cells per g [dry weight]) of heterotrophic, gram-negative, rod-shaped, non-sporeforming, aerobic, thermophilic bacteria related to the genus Thermus were isolated from thermogenic composts at temperatures between 65 and 82 degrees C. These bacteria were present in different types of wastes (garden and kitchen wastes and sewage sludge) and in all the industrial composting systems studied (open-air windows, boxes with automated turning and aeration, and closed bioreactors with aeration). Isolates grew fast on a rich complex medium at temperatures between 40 and 80 degrees C, with optimum growth between 65 and 75 degrees C. Nutritional characteristics, total protein profiles, DNA-DNA hybridization (except strain JT4), and restriction fragment length polymorphism profiles of the DNAs coding for the 16S rRNAs (16S rDNAs) showed that Thermus strains isolated from hot composts were closely related to Thermus thermophilus HB8. These newly isolated T. thermophilus strains have probably adapted to the conditions in the hot-compost ecosystem. Heterotrophic, ovalspore-forming, thermophilic bacilli were also isolated from hot composts, but none of the isolates was able to grow at temperatures above 70 degrees C. This is the first report of hot composts as habitats for a high number of thermophilic bacteria related to the genus Thermus. Our study suggests that Thermus strains play an important role in organic-matter degradation during the thermogenic phase (65 to 80 degrees C) of the composting process. PMID:8633870

[Molecular typing of 12 Brucella strains isolated in Guizhou province in 2010-2013].

PubMed

Wang, Yue; Chen, Hong; Liu, Ying; Zhou, Jingzhu; Li, Shijun; Hang, Yan; Tang, Guangpeng; Wang, Dingming; Chen, Guichun

2015-09-01

To identify and characterize the Brucella strains from Guizhou province in 2010-2013. A total of 12 strains of Brucella suspicious bacteria were isolated in Guizhou province from 2010 to 2013. Four strains (GZLL3, GZLL4, GZLL11 and SH2) were isolated from goat blood samples and eight strains (SH4, GZZY, GZSQ, GZZA, BR13001, BR13004, BR13005 and BR13006) were isolated from blood samples of patient 12 Brucella suspicious strains were identified and characterized using conventional methods. Brucella genus specific gene BCSP31-based PCR (BCSP31-PCR) was used to identify the genus of Brucella and IS711 insert sequence-based PCR (AMOS-PCR) was applied to identify the species of Brucella strains. Goats and patients originated Brucella strains were comparatively analysed using Pulse-field Gel Electrophoresis (PFGE). Both of conventional methods and PCR identified the 12 Brucella suspicious strains as B. melitensis biotype 3. BCSP31-PCR identification results showed that a specific DNA bands (223 bp) were detected in all the 12 strains and positive control samples with no DNA band in negative samples. AMOS-PCR amplified a 731 bp-DNA bands in all the 12 strains, with 731 bp, 498 bp and 275 bp in M5, S2 and A19 strains, respectively, and no DNA band was detected in the negative control samples. PFGE analysis showed that 12 Brucella isolates from patients and goats showed consistent PFGE patterns with the digestion of restriction enzyme Xba I. The epidemic species/type of Brucella in both human and animal in Guizhou province was B. melitensis biotype 3 and goat was the main animal source of infection of brucellosis in Guizhou province.

Cytoplasmic expression of a thermostable invertase from Thermotoga maritima in Lactococcus lactis.

PubMed

Pek, Han Bin; Lim, Pei Yu; Liu, Chengcheng; Lee, Dong-Yup; Bi, Xuezhi; Wong, Fong Tian; Ow, Dave Siak-Wei

2017-05-01

To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis. The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively. Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.

Genetic diversity of Mycobacterium tuberculosis strains isolated in Algeria: Results of spoligotyping.

PubMed

Ifticene, Malika; Kaïdi, Saïd; Khechiba, Mesbah-Mounir; Yala, Djamel; Boulahbal, Fadila

2015-12-01

Molecular typing tools, including spoligotyping, are currently widely used in the monitoring and study of the dynamics of tuberculosis epidemics. A study of the molecular profile of a sample of 129 Myobacterium tuberculosis strains isolated during 2011 was carried out in the National Reference Laboratory for Tuberculosis and Mycobacteria at the Pasteur Institute of Algeria. This sample was selected at random from a set of 350 strains isolated from tuberculosis patients from central and eastern areas of the country. Genotypic analysis helped to clarify the frequencies of the different genotypes in the current study population: H family, 29%; LAM family, 26%; T family, 25%; S family, 5%, and other genomic families, including orphan strains, 15%. The study of strains isolated between January and December 2011 has allowed insight into the frequency of different genomic families and the importance of existing clusters in the population of central and eastern Algeria. Copyright © 2015 Asian African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

Lactobacillus delbrueckii subsp. jakobsenii subsp. nov., isolated from dolo wort, an alcoholic fermented beverage in Burkina Faso.

PubMed

Adimpong, David B; Nielsen, Dennis S; Sørensen, Kim I; Vogensen, Finn K; Sawadogo-Lingani, Hagrétou; Derkx, Patrick M F; Jespersen, Lene

2013-10-01

Lactobacillus delbrueckii is divided into five subspecies based on phenotypic and genotypic differences. A novel isolate, designated ZN7a-9(T), was isolated from malted sorghum wort used for making an alcoholic beverage (dolo) in Burkina Faso. The results of 16S rRNA gene sequencing, DNA-DNA hybridization and peptidoglycan cell-wall structure type analyses indicated that it belongs to the species L. delbrueckii. The genome sequence of isolate ZN7a-9(T) was determined by Illumina-based sequencing. Multilocus sequence typing (MLST) and split-decomposition analyses were performed on seven concatenated housekeeping genes obtained from the genome sequence of strain ZN7a-9(T) together with 41 additional L. delbrueckii strains. The results of the MLST and split-decomposition analyses could not establish the exact subspecies of L. delbrueckii represented by strain ZN7a-9(T) as it clustered with L. delbrueckii strains unassigned to any of the recognized subspecies of L. delbrueckii. Strain ZN7a-9(T) additionally differed from the recognized type strains of the subspecies of L. delbrueckii with respect to its carbohydrate fermentation profile. In conclusion, the cumulative results indicate that strain ZN7a-9(T) represents a novel subspecies of L. delbrueckii closely related to Lactobacillus delbrueckii subsp. lactis and Lactobacillus delbrueckii subsp. delbrueckii for which the name Lactobacillus delbrueckii subsp. jakobsenii subsp. nov. is proposed. The type strain is ZN7a-9(T) = DSM 26046(T) = LMG 27067(T).

Quantification of Siderophore and Hemolysin from Stachybotrys chartarum Strains, Including a Strain Isolated from the Lung of a Child with Pulmonary Hemorrhage and Hemosiderosis

PubMed Central

Vesper, Stephen J.; Dearborn, Dorr G.; Elidemir, Okan; Haugland, Richard A.

2000-01-01

A strain of Stachybotrys chartarum was recently isolated from the lung of a pulmonary hemorrhage and hemosiderosis (PH) patient in Texas (designated the Houston strain). This is the first time that S. chartarum has been isolated from the lung of a PH patient. In this study, the Houston strain and 10 strains of S. chartarum isolated from case (n = 5) or control (n = 5) homes in Cleveland were analyzed for hemolytic activity, siderophore production, and relatedness as measured by random amplified polymorphic DNA analysis. PMID:10831457

Isolation and Evaluation of Bacillus Strains for Industrial Production of 2,3-Butanediol.

PubMed

Song, Chan Woo; Rathnasingh, Chelladurai; Park, Jong Myoung; Lee, Julia; Song, Hyohak

2018-03-28

Biologically produced 2,3-butanediol (2,3-BDO) has diverse industrial applications. In this study, schematic isolation and screening procedures were designed to obtain generally regarded as safe (GRAS) and efficient 2,3-BDO producers. Over 4,000 candidate strains were isolated by pretreatment and enrichment, and the isolated Bacillus strains were further screened by morphological, biochemical, and genomic analyses. The screened strains were then used to test the utilization of the most common carbon (glucose, xylose, fructose, sucrose) and nitrogen (yeast extract, corn steep liquor) sources for the economical production of 2,3-BDO. Two-stage fed-batch fermentation was finally carried out to enhance 2,3-BDO production. In consequence, a newly isolated Bacillus licheniformis GSC3102 strain produced 92.0 g/l of total 2,3-BDO with an overall productivity and yield of 1.40 g/l/h and 0.423 g/g glucose, respectively, using a cheap and abundant nitrogen source. These results strongly suggest that B. licheniformis , which is found widely in nature, can be used as a host strain for the industrial fermentative production of 2,3-BDO.

Genetic analysis of Saccharomyces cerevisiae strains isolated from palm wine in eastern Nigeria. Comparison with other African strains.

PubMed

Ezeronye, O U; Legras, J-L

2009-05-01

To study the yeast diversity of Nigerian palm wines by comparison with other African strains. Twenty-three Saccharomyces cerevisiae strains were obtained from palm wine samples collected at four locations in eastern Nigeria, and characterized using different molecular techniques: internal transcribed spacer restriction fragment length polymorphism and sequence analysis, pulsed field gel electrophoresis, inter delta typing and microsatellite multilocus analysis. These techniques revealed that palm wine yeasts represent a group of closely related strains that includes other West African isolates (CBS400, NCYC110, DVPG6044). Population analysis revealed an excess of homozygote strains and an allelic richness similar to wine suggestive of local domestication. Several other African yeast strains were not connected to this group. Ghana sorghum beer strains and other African strains (DBVPG1853 and MUCL28071) displayed strikingly high relatedness with European bread, beer or wine strains, and the genome of strain MUCL30909 contained African and wine-type alleles, indicating its hybrid origin. Nigerian palm wine yeast represents a local specific yeast flora, whereas a European origin or hybrid was suspected for several other Africa isolates. This study presents the first genetic characterization of an autochthonous African palm wine yeast population and confirms the idea that human intervention has favoured yeast migration.

[Molecular-genetic characteristics of Mycobacterium tuberculosis strains isolated from patients with tuberculous spondylitis].

PubMed

Viazovaia, A A; Solov'eva, N S; Zhuravlev, V Iu; Mokrousov, I V; Manicheva, O A; Vishnevskiĭ, B I; Narvskaia, O V

2013-01-01

Molecular-genetic characteristic of M. tuberculosis strains isolated from operation material of patients with tuberculous spondylitis. 107 strains of M. tuberculosis isolated in 2007 - 2011 from patients with spine tuberculosis were studied by methods of spoligotyping and MIRU-VNTR by 12 and 24 loci. Strains of genetic family Beijing dominated (n = 80), 78% of those had multiple drug resistance (MDR). Strains of genetic families T, H3 (Ural), LAM, Manu, H4 and S were also detected. Differentiating of 80 strains of Beijing genotype by MIRU-VNTR method by 24 loci revealed 24 variants (HGI = 0.83) including 7 clusters, the largest of those (100-32) included 23 strains (87% MDR). The leading role of Beijing genotype M. tuberculosis strains in development of tuberculous spondylitis with multiple drug resistance of the causative agent is shown.

Phenotypic and genetic diversity of chlorine-resistant Methylobacterium strains isolated from various environments.

PubMed Central

Hiraishi, A; Furuhata, K; Matsumoto, A; Koike, K A; Fukuyama, M; Tabuchi, K

1995-01-01

Strains of pink-pigmented facultative methylotrophs which were isolated previously from various environments and assigned tentatively to the genus Methylobacterium were characterized in comparison with authentic strains of previously known species of this genus. Most of the isolates derived from chlorinated water supplies exhibited resistance to chlorine, whereas 29 to 40% of the isolates from air, natural aquatic environments, and clinical materials were chlorine resistant. None of the tested authentic strains of Methylobacterium species obtained from culture collections exhibited chlorine resistance. Numerical analysis of phenotypic profiles showed that the test organisms tested were separated from each other except M. organophilum and M. rhodesianum. The chlorine-resistant isolates were randomly distributed among all clusters. The 16S ribosomal DNA (rDNA) sequence-based phylogenetic analyses showed that representatives of the isolates together with known Methylobacterium species formed a line of descent distinct from that of members of related genera in the alpha-2 subclass of the Proteobacteria and were divided into three subclusters within the Methylobacterium group. These results demonstrate that there is phenotypic and genetic diversity among chlorine-resistant Methylobacterium strains within the genus. PMID:7793931

Phenotypic and genetic diversity of chlorine-resistant Methylobacterium strains isolated from various environments.

PubMed

Hiraishi, A; Furuhata, K; Matsumoto, A; Koike, K A; Fukuyama, M; Tabuchi, K

1995-06-01

Strains of pink-pigmented facultative methylotrophs which were isolated previously from various environments and assigned tentatively to the genus Methylobacterium were characterized in comparison with authentic strains of previously known species of this genus. Most of the isolates derived from chlorinated water supplies exhibited resistance to chlorine, whereas 29 to 40% of the isolates from air, natural aquatic environments, and clinical materials were chlorine resistant. None of the tested authentic strains of Methylobacterium species obtained from culture collections exhibited chlorine resistance. Numerical analysis of phenotypic profiles showed that the test organisms tested were separated from each other except M. organophilum and M. rhodesianum. The chlorine-resistant isolates were randomly distributed among all clusters. The 16S ribosomal DNA (rDNA) sequence-based phylogenetic analyses showed that representatives of the isolates together with known Methylobacterium species formed a line of descent distinct from that of members of related genera in the alpha-2 subclass of the Proteobacteria and were divided into three subclusters within the Methylobacterium group. These results demonstrate that there is phenotypic and genetic diversity among chlorine-resistant Methylobacterium strains within the genus.

Characterization of trh2 Harbouring Vibrio parahaemolyticus Strains Isolated in Germany

PubMed Central

Bechlars, Silke; Jäckel, Claudia; Diescher, Susanne; Wüstenhagen, Doreen A.; Kubick, Stefan; Dieckmann, Ralf; Strauch, Eckhard

2015-01-01

Background Vibrio parahaemolyticus is a recognized human enteropathogen. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) as well as the type III secretion system 2 (T3SS2) are considered as major virulence factors. As tdh positive strains are not detected in coastal waters of Germany, we focused on the characterization of trh positive strains, which were isolated from mussels, seawater and patients in Germany. Results Ten trh harbouring V. parahaemolyticus strains from Germany were compared to twenty-one trh positive strains from other countries. The complete trh sequences revealed clustering into three different types: trh1 and trh2 genes and a pseudogene Ψtrh. All German isolates possessed alleles of the trh2 gene. MLST analysis indicated a close relationship to Norwegian isolates suggesting that these strains belong to the autochthonous microflora of Northern Europe seawaters. Strains carrying the pseudogene Ψtrh were negative for T3SS2β effector vopC. Transcription of trh and vopC genes was analyzed under different growth conditions. Trh2 gene expression was not altered by bile while trh1 genes were inducible. VopC could be induced by urea in trh2 bearing strains. Most trh1 carrying strains were hemolytic against sheep erythrocytes while all trh2 positive strains did not show any hemolytic activity. TRH variants were synthesized in a prokaryotic cell-free system and their hemolytic activity was analyzed. TRH1 was active against sheep erythrocytes while TRH2 variants were not active at all. Conclusion Our study reveals a high diversity among trh positive V. parahaemolyticus strains. The function of TRH2 hemolysins and the role of the pseudogene Ψtrh as pathogenicity factors are questionable. To assess the pathogenic potential of V. parahaemolyticus strains a differentiation of trh variants and the detection of T3SS2β components like vopC would improve the V. parahaemolyticus diagnostics and could lead to a refinement of the risk

Characterization of trh2 harbouring Vibrio parahaemolyticus strains isolated in Germany.

PubMed

Bechlars, Silke; Jäckel, Claudia; Diescher, Susanne; Wüstenhagen, Doreen A; Kubick, Stefan; Dieckmann, Ralf; Strauch, Eckhard

2015-01-01

Vibrio parahaemolyticus is a recognized human enteropathogen. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) as well as the type III secretion system 2 (T3SS2) are considered as major virulence factors. As tdh positive strains are not detected in coastal waters of Germany, we focused on the characterization of trh positive strains, which were isolated from mussels, seawater and patients in Germany. Ten trh harbouring V. parahaemolyticus strains from Germany were compared to twenty-one trh positive strains from other countries. The complete trh sequences revealed clustering into three different types: trh1 and trh2 genes and a pseudogene Ψtrh. All German isolates possessed alleles of the trh2 gene. MLST analysis indicated a close relationship to Norwegian isolates suggesting that these strains belong to the autochthonous microflora of Northern Europe seawaters. Strains carrying the pseudogene Ψtrh were negative for T3SS2β effector vopC. Transcription of trh and vopC genes was analyzed under different growth conditions. Trh2 gene expression was not altered by bile while trh1 genes were inducible. VopC could be induced by urea in trh2 bearing strains. Most trh1 carrying strains were hemolytic against sheep erythrocytes while all trh2 positive strains did not show any hemolytic activity. TRH variants were synthesized in a prokaryotic cell-free system and their hemolytic activity was analyzed. TRH1 was active against sheep erythrocytes while TRH2 variants were not active at all. Our study reveals a high diversity among trh positive V. parahaemolyticus strains. The function of TRH2 hemolysins and the role of the pseudogene Ψtrh as pathogenicity factors are questionable. To assess the pathogenic potential of V. parahaemolyticus strains a differentiation of trh variants and the detection of T3SS2β components like vopC would improve the V. parahaemolyticus diagnostics and could lead to a refinement of the risk assessment in food analyses and

Antibiotic resistance assessment in S. aureus strains isolated from raw sheep's milk cheese.

PubMed

Spanu, V; Virdis, S; Scarano, C; Cossu, F; De Santis, E P L; Cosseddu, A M

2010-06-01

In vitro activities of 16 antibiotics were tested against 36 Staphylococcus aureus (SA) strains isolated from raw sheep's milk cheese from six dairies. The minimum inhibitory concentration (MIC) was determined using a broth microdilution method (CLSI). All 36 isolates were analyzed for the presence of the accessory gene regulator gene, agr (I-IV), and genes encoding resistance to methicillin (mecA), erythromycin (ermA), penicillin (blaZ), and vancomycin (vanA-B). The isolates were also analyzed for similarities in pulsed-field gel electrophoresis (PFGE) patterns. SA strains showed resistance to ampicillin (36.1%), penicillin (33.3%), tetracycline (11.1%), and cloxacillin (2.8%) but were susceptible (>or=94.4%) to 12 out of 16 tested antimicrobials. The overall susceptibility of the strains to oxacillin, vancomycin, and erythromycin was confirmed by the absence of the mecA, vanA-B, and ermA genes. The PFGE results showed that 32 strains belonged to 10 different clusters (P1-P10) while four strains were untypeable.

Different Strategies for Molecular Differentiation of Mycobacterium bovis Strains Isolated in Sardinia, Italy

PubMed Central

Sechi, Leonardo A.; Leori, Guido; Lollai, Stefano A.; Duprè, Ilaria; Molicotti, Paola; Fadda, Giovanni; Zanetti, Stefania

1999-01-01

Different genetic markers were used to analyze 22 Mycobacterium bovis strains isolated from cattle in Sardinia and one human isolate. IS6110 DNA fingerprinting differentiated the strains into six patterns, whereas with enterobacterial repetitive consensus sequence primers produced seven clusters. PCR ribotyping followed by digestion with HaeIII and PvuII produced five and seven patterns, respectively. PCR with the (GTG)5 oligonucleotide primer showed the best discriminatory power, generating eight clusters among the strains analyzed. PMID:10103282

[R factors in strains of pathogenic enterobacteria isolated from domestic animals and particularly from dogs].

PubMed

Roy, R S

1972-01-01

Strains of enterobacteria (nine Escherichia coli and two Salmonella) isolated from primary or secondary infections in the dog, cat, pig, calf and kangaroo were studied for the presence of extrachromosomal drug resistance factors (R factors). Seven strains of E. coli and two strains of Salmonella transferred resistance involving the following antibiotics: streptomycin, ampicillin, chloramphenicol, neomycin and tetracycline. All strains harboring R factors transferred streptomycin resistance and the identified resistance patterns were as follows: Sm Am, Sm Te, Sm Neo, Sm Am Te, Sm CI Neo and Sm Am CI Te. The levels of resistance observed were comparable for all donor strains and their converted recipients. Strains of E. coli harboring R factors were isolated from three dogs that had died of either otitis (followed by a generalized infection), enteritis or bronchopneumonia - secondary to distemper. The bacteria isolated from cats were recovered at the necropsy of animals that had died of purulent pleuresy and feline panleukopenia. The other strains (two Salmonella and one E. coli were isolated from fatal enteric diseases in the pig, calf and kangaroo.

Isolation and Characterization of Hydrocarbon-Degrading Yeast Strains from Petroleum Contaminated Industrial Wastewater.

PubMed

Gargouri, Boutheina; Mhiri, Najla; Karray, Fatma; Aloui, Fathi; Sayadi, Sami

2015-01-01

Two yeast strains are enriched and isolated from industrial refinery wastewater. These strains were observed for their ability to utilize several classes of petroleum hydrocarbons substrates, such as n-alkanes and aromatic hydrocarbons as a sole carbon source. Phylogenetic analysis based on the D1/D2 variable domain and the ITS-region sequences indicated that strains HC1 and HC4 were members of the genera Candida and Trichosporon, respectively. The mechanism of hydrocarbon uptaking by yeast, Candida, and Trichosporon has been studied by means of the kinetic analysis of hydrocarbons-degrading yeasts growth and substrate assimilation. Biodegradation capacity and biomass quantity were daily measured during twelve days by gravimetric analysis and gas chromatography coupled with mass spectrometry techniques. Removal of n-alkanes indicated a strong ability of hydrocarbon biodegradation by the isolated yeast strains. These two strains grew on long-chain n-alkane, diesel oil, and crude oil but failed to grow on short-chain n-alkane and aromatic hydrocarbons. Growth measurement attributes of the isolates, using n-hexadecane, diesel oil, and crude oil as substrates, showed that strain HC1 had better degradation for hydrocarbon substrates than strain HC4. In conclusion, these yeast strains can be useful for the bioremediation process and decreasing petroleum pollution in wastewater contaminated with petroleum hydrocarbons.

Quantitative analyses of the bacterial microbiota of rearing environment, tilapia and common carp cultured in earthen ponds and inhibitory activity of its lactic acid bacteria on fish spoilage and pathogenic bacteria.

PubMed

Kaktcham, Pierre Marie; Temgoua, Jules-Bocamdé; Ngoufack Zambou, François; Diaz-Ruiz, Gloria; Wacher, Carmen; Pérez-Chabela, María de Lourdes

2017-02-01

The present study aimed to evaluate the bacterial load of water, Nile Tilapia and common Carp intestines from earthen ponds, isolate lactic acid bacteria (LAB) and assess their antimicrobial activity against fish spoilage and pathogenic bacteria. Following enumeration and isolation of microorganisms the antimicrobial activity of the LAB isolates was evaluated. Taxonomic identification of selected antagonistic LAB strains was assessed, followed by partial characterisation of their antimicrobial metabolites. Results showed that high counts (>4 log c.f.u ml -1 or 8 log c.f.u g -1 ) of total aerobic bacteria were recorded in pond waters and fish intestines. The microbiota were also found to be dominated by Salmonella spp., Vibrio spp., Staphylococcus spp. and Escherichia coli. LAB isolates (5.60%) exhibited potent direct and extracellular antimicrobial activity against the host-derived and non host-derived spoilage and pathogenic bacteria. These antagonistic isolates were identified and Lactococcus lactis subsp. lactis was found as the predominant (42.85%) specie. The strains displayed the ability to produce lactic, acetic, butyric, propionic and valeric acids. Bacteriocin-like inhibitory substances with activity against Gram-positive and Gram-negative (Vibrio spp. and Pseudomonas aeruginosa) bacteria were produced by three L. lactis subsp. lactis strains. In this study, the LAB from the microbiota of fish and pond water showed potent antimicrobial activity against fish spoilage or pathogenic bacteria from the same host or ecological niche. The studied Cameroonian aquatic niche is an ideal source of antagonistic LAB that could be appropriate as new fish biopreservatives or disease control agents in aquaculture under tropical conditions in particular or worldwide in general.

Complete Genome Sequences of Three Moraxella osloensis Strains Isolated from Human Skin

PubMed Central

Lim, Jae Yun; Hwang, Ingyu; Ganzorig, Munkhtsatsral; Huang, Shir-Ly; Cho, Gyu-Sung; Franz, Charles M. A. P.

2018-01-01

ABSTRACT Here, we present the complete whole-genome sequences of three Moraxella osloensis strains with octylphenol polyethoxylate-degrading abilities. These strains were isolated from human skin. PMID:29348360

Cyclopropanation of membrane unsaturated fatty acids is not essential to the acid stress response of Lactococcus lactis subsp. cremoris.

PubMed

To, Thi Mai Huong; Grandvalet, Cosette; Tourdot-Maréchal, Raphaëlle

2011-05-01

Cyclopropane fatty acids (CFAs) are synthetized in situ by the transfer of a methylene group from S-adenosyl-L-methionine to a double bond of unsaturated fatty acid chains of membrane phospholipids. This conversion, catalyzed by the Cfa synthase enzyme, occurs in many bacteria and is recognized to play a key role in the adaptation of bacteria in response to a drastic perturbation of the environment. The role of CFAs in the acid tolerance response was investigated in the lactic acid bacterium Lactococcus lactis MG1363. A mutant of the cfa gene was constructed by allelic exchange. The cfa gene encoding the Cfa synthase was cloned and introduced into the mutant to obtain the complemented strain for homologous system studies. Data obtained by gas chromatography (GC) and GC-mass spectrometry (GC-MS) validated that the mutant could not produce CFA. The CFA levels in both the wild-type and complemented strains increased upon their entry to stationary phase, especially with acid-adapted cells or, more surprisingly, with ethanol-adapted cells. The results obtained by performing quantitative reverse transcription-PCR (qRT-PCR) experiments showed that transcription of the cfa gene was highly induced by acidity (by 10-fold with cells grown at pH 5.0) and by ethanol (by 9-fold with cells grown with 6% ethanol) in comparison with that in stationary phase. Cell viability experiments were performed after an acidic shock on the mutant strain, the wild-type strain, and the complemented strain, as a control. The higher viability level of the acid-adapted cells of the three strains after 3 h of shock proved that the cyclopropanation of unsaturated fatty acids is not essential for L. lactis subsp. cremoris survival under acidic conditions. Moreover, fluorescence anisotropy data showed that CFA itself could not maintain the membrane fluidity level, particularly with ethanol-grown cells.

Short communication: effect of exopolysaccharide isolated from "viili" on the adhesion of probiotics and pathogens to intestinal mucus.

PubMed

Ruas-Madiedo, P; Gueimonde, M; de los Reyes-Gavilán, C G; Salminen, S

2006-07-01

The strong ropy character of the Scandinavian fermented milk viili is conferred by the exopolysaccharides (EPS) produced by lactococcal strains. These biopolymers can be responsible for some health benefits. We have assessed the influence of the EPS fraction isolated from commercial viili on the adhesion of some probiotics and pathogens to human intestinal mucus. Concentrations of viili EPS greater than 0.1 mg/mL promoted a decrease in adherence of Bifidobacterium lactis Bb12 and Lactobacillus rhamnosus GG and this effect was dose-dependent. However, no modifications were detected on the adhesion levels of the pathogenic strains tested at a concentration of 1 mg/mL of EPS. Results obtained in the present work should be considered in the design of new probiotic products.

[Molecular epidemiological study on rubella virus strains isolated in Zhejiang province, China, 2005-2010].

PubMed

Feng, Yan; Zhong, Shu-ling; Xu, Chang-ping; Shi, Wen; Lu, Yi-yu

2011-09-01

To analyze the molecular epidemiological characteristic of rubella virus strains isolated in Zhejiang province from 2005 to 2010, to provide basic data for rubella prevention and control. Rubella virus strains were isolated on Vero cells from the suspected patients' specimens collected in Zhejiang province during 2005 to 2010. Partial fragments of the structural gene of Zhejiang rubella strains were amplified, using nested reverse transcription-polymerase chain reaction (RT-PCR). The amplified products were sequences and analyzed. In total, 7 rubella strains were isolated from 52 clinical specimens, of which six were classified as genotype 1E and only one was characterized as genotype 2B. In the phylogenetic tree, the Zhejiang 1E genotype rubella strains were located in the same branches with Hongkong or Hainan isolates respectively, but the Zhejiang 2B genotype strain were located in the same branch with oversea strain BuenosAires. ARG/46.08. Through p-distance analysis, results also showed that the Zhejiang 2B genotype strain was closer to the 2B strains isolated from overseas (0.011) than those strains from other provinces of China (0.023). Compared with Chinese vaccine strain BRD II, the homology on three structural genes was C > E2 > E1, but the homology of deduced amino acid sequence was E1 > C > E2, with corresponding 3, 11 and 23 amino acid mutations. There was only one amino acid on E1 gene with entropy value higher than 0.600, but seven sites on E2 gene with entropy value appeared higher than 0.600 and one with entropy value higher than 1.000. Two genotypes of rubella virus had circulated in Zhejiang province during 2005 to 2010. Genotype 1E appeared to be the predominant genotype and 2B being an imported one. Amino acid sequence of E1 gene from Zhejiang rubella strains was comparatively conserved, but E2 gene was hypervariable. Study on rubella virus E2 and C gene should be conducted in the epidemiological surveillance program of rubella.

Characterization and antimicrobial spectrum of bacteriocins produced by lactic acid bacteria isolated from traditional Bulgarian dairy products.

PubMed

Simova, E D; Beshkova, D B; Dimitrov, Zh P

2009-02-01

To isolate bacteriocin-producing lactic acid bacteria (LAB) with high wide spectrum antibacterial activity and to characterize their inhibitory peptides. Seven LAB strains [Lactobacillus casei ssp. rhamnosus (PC5), Lactobacillus delbrueckii ssp. bulgaricus (BB18), Lactococcus lactis ssp. lactis (BCM5, BK15), Enterococcus faecium (MH3), Lactobacillus plantarum (BR12), Lactobacillus casei ssp. casei (BCZ2)], isolated from authentic Bulgarian dairy products were capable of producing bacteriocins, inhibiting the widest range of pathogenic bacteria. The bacteriocins were resistant to heating at 121 degrees C for 15 min, stable at pH 2-10, sensitive to protease, insensitive to alpha-amylase and lipase. Two of bacteriocins produced by Lact. bulgaricus BB18 (bulgaricin BB18) and E. faecium MH3 (enterocin MH3) were purified and the molecular masses were determined. The N-terminal amino acid sequence of bulgaricin BB18 did not show strong homology to other known bacteriocins. Lactobacillus bulgaricus BB18 and E. faecium MH3 produce two novel bacteriocins highly similar to the pediocin-like nonlantibiotics. The two bacteriocins are potential antimicrobial agents and, in conjunction with their producers, may have use in applications to contribute a positive effect on the balance of intestinal microflora. Furthermore, bulgaricin BB18 strongly inhibits Helicobacter pylori.

Fruiting Body Formation of Cordyceps militaris from Multi-Ascospore Isolates and Their Single Ascospore Progeny Strains

PubMed Central

Shrestha, Bhushan; Han, Sang-Kuk; Sung, Jae-Mo

2012-01-01

Interest in commercial cultivation and product development of Cordyceps species has shown a recent increase. Due to its biochemical and pharmacological effects, Cordyceps militaris, commonly known as orange caterpillar fungus, is being investigated with great interest. Cultivation of C. militaris has been practiced on a large scale in order to fulfill a demand for scientific investigation and product development. Isolates of C. militaris can be easily established from both spores and tissue. For isolation of spores, ascospores released from mature stromata are trapped in sterile medium. Multi-ascospore isolates, as well as combinations of single ascospore strains, are used for production of fruiting bodies. Progeny ascospore strains can be isolated from artificial fruiting bodies, thus, the cycle of fruiting body production can be continued for a long period of time. In this study, we examined fruiting body production from multi-ascospore isolates and their progeny strains for three generations. F1 progeny strains generally produced a larger number of fruiting bodies, compared with their mother multi-ascospore isolates; however, F2 and F3 progeny strains produced fewer fruiting bodies. Optimum preservation conditions could help to increase the vitality of the progeny strains. In order to retain the fruiting ability of the strains, further testing of various methods of preservation and different methods for isolation should be performed. PMID:22870051

Production of enterocins L50A, L50B, and IT, a new enterocin, by Enterococcus faecium IT62, a strain isolated from Italian ryegrass in Japan.

PubMed

Izquierdo, Esther; Bednarczyk, Audrey; Schaeffer, Christine; Cai, Yimin; Marchioni, Eric; Van Dorsselaer, Alain; Ennahar, Saïd

2008-06-01

Enterococcus faecium IT62, isolated from ryegrass in Japan, was shown to produce three different bacteriocins, two of which had molecular masses and amino acid sequences that corresponded to those of enterocin L50A and enterocin L50B. These peptides existed, however, as chemically modified forms that were either N formylated or N formylated and oxidized at Met(24). The third bacteriocin, named enterocin IT, had a molecular mass of 6,390 Da, was made up of 54 amino acids, and did not correspond to any known bacteriocin. However, enterocin IT was identical to the C-terminal part of the 16-amino-acid-longer bacteriocin 32 (T. Inoue, H. Tomita, and Y. Ike, Antimicrob. Agents Chemother., 50:1202-1212, 2006). For the first time, the antimicrobial activity spectra for enterocins L50A and L50B were determined separately and included a wide range of gram-positive bacteria but also a few gram-negative strains that were weakly sensitive. Slight differences in the activities of enterocins L50A and L50B were observed, as gram-positive bacteria showed an overall higher level of sensitivity to L50A than to L50B, as opposed to gram-negative ones. Conversely, enterocin IT showed a very narrow antimicrobial spectrum that was limited to E. faecium strains, one strain of Bacillus subtilis, and one strain of Lactococcus lactis. This study showed that E. faecium IT62, a grass-borne strain, produces bacteriocins with very different activity features and structures that may be found in strains associated with food or those of clinical origin, which demonstrates that a particular enterocin structure may be widespread and not related to the producer's origin.

Production of Enterocins L50A, L50B, and IT, a New Enterocin, by Enterococcus faecium IT62, a Strain Isolated from Italian Ryegrass in Japan▿

PubMed Central

Izquierdo, Esther; Bednarczyk, Audrey; Schaeffer, Christine; Cai, Yimin; Marchioni, Eric; Van Dorsselaer, Alain; Ennahar, Saïd

2008-01-01

Enterococcus faecium IT62, isolated from ryegrass in Japan, was shown to produce three different bacteriocins, two of which had molecular masses and amino acid sequences that corresponded to those of enterocin L50A and enterocin L50B. These peptides existed, however, as chemically modified forms that were either N formylated or N formylated and oxidized at Met24. The third bacteriocin, named enterocin IT, had a molecular mass of 6,390 Da, was made up of 54 amino acids, and did not correspond to any known bacteriocin. However, enterocin IT was identical to the C-terminal part of the 16-amino-acid-longer bacteriocin 32 (T. Inoue, H. Tomita, and Y. Ike, Antimicrob. Agents Chemother., 50:1202-1212, 2006). For the first time, the antimicrobial activity spectra for enterocins L50A and L50B were determined separately and included a wide range of gram-positive bacteria but also a few gram-negative strains that were weakly sensitive. Slight differences in the activities of enterocins L50A and L50B were observed, as gram-positive bacteria showed an overall higher level of sensitivity to L50A than to L50B, as opposed to gram-negative ones. Conversely, enterocin IT showed a very narrow antimicrobial spectrum that was limited to E. faecium strains, one strain of Bacillus subtilis, and one strain of Lactococcus lactis. This study showed that E. faecium IT62, a grass-borne strain, produces bacteriocins with very different activity features and structures that may be found in strains associated with food or those of clinical origin, which demonstrates that a particular enterocin structure may be widespread and not related to the producer's origin. PMID:18391036

Genetic characterization of Vibrio vulnificus strains isolated from oyster samples in Mexico.

PubMed

Guerrero, Abraham; Gómez Gil Rodríguez, Bruno; Wong-Chang, Irma; Lizárraga-Partida, Marcial Leonardo

2015-01-01

Vibrio vulnificus strains were isolated from oysters that were collected at the main seafood market in Mexico City. Strains were characterized with regard to vvhA, vcg genotype, PFGE, multilocus sequence typing (MLST), and rtxA1. Analyses included a comparison with rtxA1 reference sequences. Environmental (vcgE) and clinical (vcgC) genotypes were isolated at nearly equal percentages. PFGE had high heterogeneity, but the strains clustered by vcgE or vcgC genotype. Select housekeeping genes for MLST and primers that were designed for rtxA1 domains divided the strains into two clusters according to the E or C genotype. Reference rtxA1 sequences and those from this study were also clustered according to genotype. These results confirm that this genetic dimorphism is not limited to vcg genotyping, as other studies have reported. Some environmental C genotype strains had high similarity to reference strains, which have been reported to be virulent, indicating a potential risk for oyster consumers in Mexico City.

Diversity of lactic acid bacteria associated with traditional fermented dairy products in Mongolia.

PubMed

Yu, J; Wang, W H; Menghe, B L G; Jiri, M T; Wang, H M; Liu, W J; Bao, Q H; Lu, Q; Zhang, J C; Wang, F; Xu, H Y; Sun, T S; Zhang, H P

2011-07-01

Spontaneous milk fermentation has a long history in Mongolia, and beneficial microorganisms have been handed down from one generation to the next for use in fermented dairy products. The objective of this study was to investigate the diversity of lactic acid bacteria (LAB) communities in fermented yak, mare, goat, and cow milk products by analyzing 189 samples collected from 13 different regions in Mongolia. The LAB counts in these samples varied from 3.41 to 9.03 log cfu/mL. Fermented yak and mare milks had almost identical mean numbers of LAB, which were significantly higher than those in fermented goat milk but slightly lower than those in fermented cow milk. In total, 668 isolates were obtained from these samples using de Man, Rogosa, and Sharpe agar and M17 agar. Each isolate was considered to be presumptive LAB based on gram-positive and catalase-negative properties, and was identified at the species level by 16S rRNA gene sequencing, multiplex PCR assay, and restriction fragment length polymorphism analysis. All isolates from Mongolian dairy products were accurately identified as Enterococcus faecalis (1 strain), Enterococcus durans (3 strains), Lactobacillus brevis (3 strains), Lactobacillus buchneri (2 strains), Lactobacillus casei (16 strains), Lactobacillus delbrueckii ssp. bulgaricus (142 strains), Lactobacillus diolivorans (17 strains), Lactobacillus fermentum (42 strains), Lactobacillus helveticus (183 strains), Lactobacillus kefiri (6 strains), Lactobacillus plantarum ssp. plantarum (7 strains), Lactococcus lactis ssp. lactis (7 strains), Leuconostoc lactis (22 strains), Leuconostoc mesenteroides (21 strains), Streptococcus thermophilus (195 strains), and Weissella cibaria (1 strain). The predominant LAB were Strep. thermophilus and Lb. helveticus, which were isolated from all sampling sites. The results demonstrate that traditional fermented dairy products from different regions of Mongolia have complex compositions of LAB species. Such diversity of

Complete Genome Sequences of Three Moraxella osloensis Strains Isolated from Human Skin.

PubMed

Lim, Jae Yun; Hwang, Ingyu; Ganzorig, Munkhtsatsral; Huang, Shir-Ly; Cho, Gyu-Sung; Franz, Charles M A P; Lee, Kyoung

2018-01-18

Here, we present the complete whole-genome sequences of three Moraxella osloensis strains with octylphenol polyethoxylate-degrading abilities. These strains were isolated from human skin. Copyright © 2018 Lim et al.

Biofortification of riboflavin and folate in idli batter, based on fermented cereal and pulse, by Lactococcus lactis N8 and Saccharomyces boulardii SAA655.

PubMed

Chandrasekar Rajendran, S C; Chamlagain, B; Kariluoto, S; Piironen, V; Saris, P E J

2017-06-01

Lactococcus lactis N8 and Saccharomyces boulardii SAA655 were investigated for their ability to synthesize B-vitamins (riboflavin and folate) and their functional role as microbial starters in idli fermentation. In this study, ultra-high performance liquid chromatography and microbiological assay were used to determine the total riboflavin and folate content respectively. Increased levels of folate were evident in both L. lactis N8 and S. boulardii SAA655 cultivated medium. Enhanced riboflavin levels were found only in S. boulardii SAA655 grown medium, whereas decreased riboflavin level was found in L. lactis N8 cultivated medium. To evaluate the functional role of microbial starter strains, L. lactis N8 and S. boulardii SAA655 were incorporated individually and in combination into idli batter, composed of wet grounded rice and black gram. For the experiments, naturally fermented idli batter was considered as control. The results indicated that natural idli fermentation did not enhance the riboflavin level and depleted folate levels by half. In comparison with control, L. lactis N8 and S. boulardii SAA655 incorporated idli batter (individually and in combination) increased riboflavin and folate levels by 40-90%. Apart from compensating the folate loss caused by natural fermentation, S. boulardii SAA655 fermented idli batter individually and in combination with L. lactis N8 also showed the highest leavening character. Moreover, the microbial starter incorporation did not significantly influence the pH of idli batter. Incorporation of L. lactis N8 and S. boulardii SAA655 can evidently enhance the functional and technological characteristics of idli batter. UN General Assembly declared 2016 the International Year of pulses emphasizing the importance of legumes as staple food. Furthermore, this is the first experimental report of in situ biofortifcation of riboflavin and folate using microbes in pulse based fermented staple food. The current study suggests possible

Heterologous expression of an α-amylase inhibitor from common bean (Phaseolus vulgaris) in Kluyveromyces lactis and Saccharomyces cerevisiae.

PubMed

Brain-Isasi, Stephanie; Álvarez-Lueje, Alejandro; Higgins, Thomas Joseph V

2017-06-15

Phaseolamin or α-amylase inhibitor 1 (αAI) is a glycoprotein from common beans (Phaseolus vulgaris L.) that inhibits some insect and mammalian α-amylases. Several clinical studies support the beneficial use of bean αAI for control of diabetes and obesity. Commercial extracts of P. vulgaris are available but their efficacy is still under question, mainly because some of these extracts contain antinutritional impurities naturally present in bean seeds and also exhibit a lower specific activity αAI. The production of recombinant αAI allows to overcome these disadvantages and provides a platform for the large-scale production of pure and functional αAI protein for biotechnological and pharmaceutical applications. A synthetic gene encoding αAI from the common bean (Phaseolus vulgaris cv. Pinto) was codon-optimised for expression in yeasts (αAI-OPT) and cloned into the protein expression vectors pKLAC2 and pYES2. The yeasts Kluyveromyces lactis GG799 (and protease deficient derivatives such as YCT390) and Saccharomyces cerevisiae YPH499 were transformed with the optimised genes and transformants were screened for expression by antibody dot blot. Recombinant colonies of K. lactis YCT390 that expressed and secreted functional αAI into the culture supernatants were selected for further analyses. Recombinant αAI from K. lactis YCT390 was purified using anion-exchange and affinity resins leading to the recovery of a functional inhibitor. The identity of the purified αAI was confirmed by mass spectrometry. Recombinant clones of S. cerevisiae YPH499 expressed functional αAI intracellularly, but did not secrete the protein. This is the first report describing the heterologous expression of the α-amylase inhibitor 1 (αAI) from P. vulgaris in yeasts. We demonstrated that recombinant strains of K. lactis and S. cerevisiae expressed and processed the αAI precursor into mature and active protein and also showed that K. lactis secretes functional αAI.

The effect of nisin from Lactococcus lactis subsp. lactis on refrigerated patin fillet quality

NASA Astrophysics Data System (ADS)

Adilla, S. N.; Utami, R.; Nursiwi, A.; Nurhartadi, E.

2017-04-01

The effect of nisin from Lactococcus lactis subsp. lactis with spraying method application on quality of patin fillet during refrigerated storage (4±1°C) was investigated. The quality of patin fillet based on total plate count (TPC), pH, TVB-N, and TBA values during 16 days at 4±1°C. Completely Randomized Design (CDR) was used in one factor (nisin activity) at 0 IU/ml, 500 IU/ml, 1000 IU/ml, and 2000 IU/ml. The observation was done at 0, 4th, 8th, 12th, and 16th days of storage. The result showed that variation of nisin activity significantly affected the quality of fillet according to TPC, pH, and TVB-N values, however no significant difference on the obtained of TBA value. Nisin in 500 IU/ml, 1000 IU/ml, and 2000 IU/ml could extend the shelf-life of fillet until 4th, 8th, and 12th days respectively based on standard in all parameters.

Complete Genome Sequence of Porcine Parvovirus N Strain Isolated from Guangxi, China

PubMed Central

Su, Qian-Lian; Li, Bin; Liang, Jia-Xing; He, Ying; Qin, Yi-Bin; Lu, Bing-Xia

2015-01-01

We report here the complete genomic sequence of the porcine parvovirus (PPV) N strain, isolated in 1989 from the viscera of a stillborn fetus farrowed by a gilt in Guangxi, southern China. Phylogenetic analyses suggest that the PPV-N strain is closely related to attenuated PPV NADL-2 strains. The PPV-N strain has good immunogenicity, genetic stability, and safety. PMID:25573932

Permissiveness of freshly isolated environmental strains of amoebae for growth of Legionella pneumophila.

PubMed

Dupuy, Mathieu; Binet, Marie; Bouteleux, Celine; Herbelin, Pascaline; Soreau, Sylvie; Héchard, Yann

2016-03-01

Legionella pneumophila is a pathogenic bacterium commonly found in water and responsible for severe pneumonia. Free-living amoebae are protozoa also found in water, which feed on bacteria by phagocytosis. Under favorable conditions, some L. pneumophila are able to resist phagocytic digestion and even multiply within amoebae. However, it is not clear whether L. pneumophila could infect at a same rate a large range of amoebae or if there is some selectivity towards specific amoebal genera or strains. Also, most studies have been performed using collection strains and not with freshly isolated strains. In our study, we assess the permissiveness of freshly isolated environmental strains of amoebae, belonging to three common genera (i.e. Acanthamoeba, Naegleria and Vermamoeba), for growth of L. pneumophila at three different temperatures. Our results indicated that all the tested strains of amoebae were permissive to L. pneumophila Lens and that there was no significant difference between the strains. Intracellular proliferation was more efficient at a temperature of 40°C. In conclusion, our work suggests that, under favorable conditions, virulent strains of L. pneumophila could equally infect a large number of isolates of common freshwater amoeba genera. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Isolation, identification and utilization of thermophilic strains in aerobic digestion of sewage sludge.

PubMed

Liu, Shugen; Zhu, Nanwen; Li, Loretta Y; Yuan, Haiping

2011-11-15

Two representative thermophilic bacterial strains (T1 and T2) were isolated from a one-stage autothermal thermophilic aerobic digestion pilot-scale reactor. 16S rRNA gene analysis indicated that they were Hydrogenophilaceae and Xanthomonodaceae. These isolated strains were inoculated separately and/or jointly in sewage sludge, to investigate their effects on sludge stabilization under thermophilic aerobic digestion condition. Four digestion conditions were tested for 480 h. Digestion without inoculation and inoculation with strain T2, as well as joint- inoculation with strains T1 and T2, achieved 32.6%, 43.0%, and 38.2% volatile solids (VS) removal, respectively. Removal in a digester inoculated with stain T1 only reached 27.2%. For the first 144 h, the three inoculated digesters all experienced higher VS removal than the digester without inoculations. Both specific thermophilic strains and micro-environment significantly affected the VS removal. DGGE profiles revealed that the isolated strains T1 and T2 can successfully establish in the thermophilic digesters. Other viable bacteria (including anaerobic or facultative microbes) also appeared in the digestion system, enhancing the microbial activity. Copyright © 2011. Published by Elsevier Ltd.

Molecular typing of Vibrio parahaemolyticus strains isolated from the Philippines by PCR-based methods.

PubMed

Maluping, R P; Ravelo, C; Lavilla-Pitogo, C R; Krovacek, K; Romalde, J L

2005-01-01

The main aim of the present study was to use three PCR-based techniques for the analysis of genetic variability among Vibrio parahaemolyticus strains isolated from the Philippines. Seventeen strains of V. parahaemolyticus isolated from shrimps (Penaeus monodon) and from the environments where these shrimps are being cultivated were analysed by random amplified polymorphic DNA PCR (RAPD-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and repetitive extragenic palindromic PCR (REP-PCR). The results of this work have demonstrated genetic variability within the V. parahaemolyticus strains that were isolated from the Philippines. In addition, RAPD, ERIC and REP-PCR are suitable rapid typing methods for V. parahaemolyticus. All three methods have good discriminative ability and can be used as a rapid means of comparing V. parahaemolyticus strains for epidemiological investigation. Based on the results of this study, we could say that REP-PCR is inferior to RAPD and ERIC-PCR owing to the fact that it is less reproducible. Moreover, the REP-PCR analysis yielded a relatively small number of products. This may suggests that the REP sequences may not be widely distributed in the V. parahaemolyticus genome. Genetic variability within V. parahaemolyticus strains isolated in the Philippines has been demonstrated. The presence of ERIC and REP sequences in the genome of this bacterial species was confirmed. The RAPD, ERIC and REP-PCR techniques are useful methods for molecular typing of V. parahaemolyticus strains. To our knowledge this is the first study of this kind carried out on V. parahaemolyticus strains isolated from the Philippines.

Molecular Characteristics of Mycobacterium tuberculosis Strains Isolated from Cutaneous Tuberculosis Patients in China.

PubMed

Jiang, Haiqin; Jin, Yali; Vissa, Varalakshmi; Zhang, Liangfen; Liu, Weijun; Qin, Lianhua; Wan, Kanglin; Wu, Xiaocui; Wang, Hongsheng; Liu, Weida; Wang, Baoxi

2017-04-06

Cutaneous tuberculosis (CTB) is probably underreported due to difficulties in detection and diagnosis. To address this issue, genotypes of Mycobacterium tuberculosis strains isolated from 30 patients with CTB were mapped at multiple loci, namely, RD105 deletions, spacer oligonucleotides, and Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeats (MIRU-VNTRs). Fifty-eight strains of pulmonary tuberculosis (PTB) were mapped as experimental controls. Drug resistance-associated gene mutations were determined by amplicon sequencing of target regions within 7 genes. Beijing family isolates were the most prevalent strains in CTB and PTB. MIRU-VNTR typing separated the Beijing strains from the non-Beijing strains, and the majority of CTB could be separated from PTB counterparts. Drug resistance determining regions showed only one CTB strain expressing isomazid resistance. Thus, while the CTB strains belonged to the same phylogenetic lineages and sub-lineages as the PTB strains, they differed at the level of several MIRU-VNTRs and in the proportion of drug resistance.

Isolation and Characterization of Hydrocarbon-Degrading Yeast Strains from Petroleum Contaminated Industrial Wastewater

PubMed Central

Gargouri, Boutheina; Mhiri, Najla; Karray, Fatma; Aloui, Fathi; Sayadi, Sami

2015-01-01

Two yeast strains are enriched and isolated from industrial refinery wastewater. These strains were observed for their ability to utilize several classes of petroleum hydrocarbons substrates, such as n-alkanes and aromatic hydrocarbons as a sole carbon source. Phylogenetic analysis based on the D1/D2 variable domain and the ITS-region sequences indicated that strains HC1 and HC4 were members of the genera Candida and Trichosporon, respectively. The mechanism of hydrocarbon uptaking by yeast, Candida, and Trichosporon has been studied by means of the kinetic analysis of hydrocarbons-degrading yeasts growth and substrate assimilation. Biodegradation capacity and biomass quantity were daily measured during twelve days by gravimetric analysis and gas chromatography coupled with mass spectrometry techniques. Removal of n-alkanes indicated a strong ability of hydrocarbon biodegradation by the isolated yeast strains. These two strains grew on long-chain n-alkane, diesel oil, and crude oil but failed to grow on short-chain n-alkane and aromatic hydrocarbons. Growth measurement attributes of the isolates, using n-hexadecane, diesel oil, and crude oil as substrates, showed that strain HC1 had better degradation for hydrocarbon substrates than strain HC4. In conclusion, these yeast strains can be useful for the bioremediation process and decreasing petroleum pollution in wastewater contaminated with petroleum hydrocarbons. PMID:26339653

Phenotypic and genotypic characterization of lactic acid bacteria isolated from raw goat milk and effect of farming practices on the dominant species of lactic acid bacteria.

PubMed

Tormo, Hélène; Ali Haimoud Lekhal, Djamila; Roques, C

2015-10-01

Lactic acid bacteria, in particular Lactococcus lactis, play a decisive role in the cheese making process and more particularly in lactic cheeses which are primarily produced on goat dairy farms. The objective of this study was therefore to identify the main lactic acid bacteria found in raw goats' milk from three different regions in France and evaluate if certain farming practices have an effect on the distribution of species of lactic acid bacteria in the various milk samples. Identification at genus or species level was carried out using phenotypic tests and genotypic methods including repetitive element REP-PCR, species-specific PCR and 16S rRNA gene sequencing. The distribution of the main bacterial species in the milk samples varied depending on farms and their characteristics. Out of the 146 strains identified, L. lactis was the dominant species (60% of strains), followed by Enterococcus (38%) of which Enterococcus faecalis and Enterococcus faecium. Within the species L. lactis, L. lactis subsp lactis was detected more frequently than L. lactis subsp cremoris (74% vs. 26%). The predominance of L. lactis subsp cremoris was linked to geographical area studied. It appears that the animals' environment plays a role in the balance between the dominance of L. lactis and enterococci in raw goats' milk. The separation between the milking parlor and the goat shed (vs no separation) and only straw in the bedding (vs straw and hay) seems to promote L. lactis in the milk (vs enterococci). Copyright © 2015 Elsevier B.V. All rights reserved.

Cyclopropanation of Membrane Unsaturated Fatty Acids Is Not Essential to the Acid Stress Response of Lactococcus lactis subsp. cremoris ▿

PubMed Central

To, Thi Mai Huong; Grandvalet, Cosette; Tourdot-Maréchal, Raphaëlle

2011-01-01

Cyclopropane fatty acids (CFAs) are synthetized in situ by the transfer of a methylene group from S-adenosyl-l-methionine to a double bond of unsaturated fatty acid chains of membrane phospholipids. This conversion, catalyzed by the Cfa synthase enzyme, occurs in many bacteria and is recognized to play a key role in the adaptation of bacteria in response to a drastic perturbation of the environment. The role of CFAs in the acid tolerance response was investigated in the lactic acid bacterium Lactococcus lactis MG1363. A mutant of the cfa gene was constructed by allelic exchange. The cfa gene encoding the Cfa synthase was cloned and introduced into the mutant to obtain the complemented strain for homologous system studies. Data obtained by gas chromatography (GC) and GC-mass spectrometry (GC-MS) validated that the mutant could not produce CFA. The CFA levels in both the wild-type and complemented strains increased upon their entry to stationary phase, especially with acid-adapted cells or, more surprisingly, with ethanol-adapted cells. The results obtained by performing quantitative reverse transcription-PCR (qRT-PCR) experiments showed that transcription of the cfa gene was highly induced by acidity (by 10-fold with cells grown at pH 5.0) and by ethanol (by 9-fold with cells grown with 6% ethanol) in comparison with that in stationary phase. Cell viability experiments were performed after an acidic shock on the mutant strain, the wild-type strain, and the complemented strain, as a control. The higher viability level of the acid-adapted cells of the three strains after 3 h of shock proved that the cyclopropanation of unsaturated fatty acids is not essential for L. lactis subsp. cremoris survival under acidic conditions. Moreover, fluorescence anisotropy data showed that CFA itself could not maintain the membrane fluidity level, particularly with ethanol-grown cells. PMID:21421775

Nutritional Requirements of Acinetobacter Strains Isolated from Soil, Water, and Sewage

PubMed Central

Warskow, Alice L.; Juni, Elliot

1972-01-01

One hundred five strains of Acinetobacter were isolated from water, soil, and sewage on nonselective complex media, and their nutritional properties were studied. Only one of these strains requires growth factors in order to grow in a mineral medium containing a single carbon source. PMID:4563966

Complete genome sequence of porcine parvovirus N strain isolated from guangxi, china.

PubMed

Su, Qian-Lian; Li, Bin; Zhao, Wu; Liang, Jia-Xing; He, Ying; Qin, Yi-Bin; Lu, Bing-Xia

2015-01-08

We report here the complete genomic sequence of the porcine parvovirus (PPV) N strain, isolated in 1989 from the viscera of a stillborn fetus farrowed by a gilt in Guangxi, southern China. Phylogenetic analyses suggest that the PPV-N strain is closely related to attenuated PPV NADL-2 strains. The PPV-N strain has good immunogenicity, genetic stability, and safety. Copyright © 2015 Su et al.

Probiotic potential of lactobacillus strains isolated from sorghum-based traditional fermented food.

PubMed

Rao, K Poornachandra; Chennappa, G; Suraj, U; Nagaraja, H; Raj, A P Charith; Sreenivasa, M Y

2015-06-01

Sorghum-based traditional fermented food was screened for potential probiotic lactic acid bacteria. The isolates were identified by biochemical, physiological and genetic methods. Species identification was done by 16s rRNA sequence analysis. The functional probiotic potential of the two Lactobacillus species viz., Lactobacillus plantarum (Lact. plantarum) and Lactobacillus pentosus (Lact. pentosus) was assessed by different standard parameters. The strains were tolerant to pH 2 for 1 h and resistant to methicillin, kanamycin, vancomycin and norfloxacin. Two (Lact. plantarum COORG-3 and Lact. pentosus COORG-8) out of eight isolates recorded the cell surface hydrophobicity to be 59.12 and 64.06%, respectively. All the strains showed tolerance to artificial duodenum juice (pH 2) for 3 h, positive for bile salt hydrolase test and negative for haemolytic test. The neutralized cell-free supernatant of the strains Lact. pentosus COORG-4, Lact. plantarum COORG-1, Lact. plantarum COORG-7, Lact. pentosus COORG-8 and Lact. plantarum COORG-3 showed good antibiofilm activity. Lact. pentosus COORG-8 exhibited 74% activity against Pseudomonas aeruginosa-MTCC 7903 and Lact. plantarum COORG-7 showed 68% inhibition of biofilm against Klebsiella pneumonia MTCC 7407. Three (Lact. plantarum COORG-7, Lact. pentosus COORG-5 and Lact. pentosus COORG 8) out of eight isolates exhibited a good antimicrobial activity against Listeria monocytogenes and five isolates (Lact. pentosus COORG 2, Lact. plantarum COORG 1, Lact. plantarum COORG 4, Lact. pentosus COORG 3 and Lact. plantarum COORG 6) are active against Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Enterobacter aerogenes, Klebsiella pneumonia, Enterococcus faecalis. The study also evaluated the cholesterol lowering property of the Lactobacillus strains using hen egg yolk as the cholesterol source. The cholesterol in hen egg yolk was assimilated by 74.12 and 68.26% by Lact. plantarum COORG 4 and Lact. pentosus COORG 7

[Molecular-biological properties of the rubella virus strains isolated in St. Petersburg].

PubMed

Buzitskaia, Zh V; Sirotkin, A K; Gudkova, T M; Prochukhanova, A P; Karpov, A V; Tsybalova, L M; Kiselev, O I

2012-01-01

In the surveillance of rubella in the northwest region of Russia samples of nasopharyngeal swabs from 37 patients with rubella, which were treated in the 442nd district military hospital named after Z.P. Solovyov in autumn 2007 were screened for the rubella virus using RK-13 cell line, 22 strains of rubella virus were isolated. Gene sequencing of E1 region of rubella virus isolates was carried out. Rubella virus strains isolated in St. Petersburg during the 2007 outbreak belonged to rubella virus genotype 1E. The morphogenesis of RK-13 cells with formation of replication complexes and enveloped virions of rubella virus was shown.

Mycotoxin production and cytotoxicity of Fusarium strains isolated from Norwegian cereals.

PubMed

Langseth, W; Bernhoft, A; Rundberget, T; Kosiak, B; Gareis, M

Thirty-four isolates of the eight most common Fusarium species isolated from Norwegian cereals; F. avenaceum, F. culmorum, F. equiseti, F. graminearum, F. poae, F. sporotrichioides, F. torulosum and F. tricinctum were studied for their cytotoxicity and ability to produce mycotoxins. The strains were cultivated on rice, and analysed for trichothecenes (all species), zearalenone (all species), fusarochromanone (F. equiseti), wortmannin (F. torulosum), moniliformin and enniatins (F. avenaceum, F. tricinctum and F. torulosum). The cytotoxicity of the extracts were examined with an (in vitro) MTT-cell culture assay. All F. graminearum and five of seven F. culmorum isolates belonged to chemotype IA, producing deoxynivalenol and 3-acetyl-deoxynivalenol, while the two other F. culmorum strains were nivalenol producers (chemotype II). The F. equiseti isolates and one of the F. poae isolates produced both type A and B trichothecenes, and relatively large quantities of fusarochromanone were detected in the F. equiseti cultures. All Fusarium species studied showed significant cytotoxicity, but with a large variation between species, and also within each species. F. sporotrichioides and F. equiseti showed the highest average cytotoxicity.

Molecular characterization of isoniazid-resistant Mycobacterium tuberculosis clinical strains isolated in the Philippines.

PubMed

Herrera, Laura; Valverde, Azucena; Saiz, Pilar; Sáez-Nieto, Juan A; Portero, José L; Jiménez, M Soledad

2004-06-01

The prevalence of mutations in the katG, inhA and oxyR-ahpC genes of isoniazid (INH)-resistant Mycobacterium tuberculosis isolates in the Philippines were determined. Of 306 M. tuberculosis isolates studied, 81 (26.5%) exhibited INH-resistance. Forty-four strains (54.3%) had mutations in the katG gene, eighteen strains (22.2%) had mutations in the putative inhA locus region, seven had mutations in both regions and five strains had mutations in the oxyR-ahpC operon. Only seven strains had no mutations. A total of 71 of the 81 (87.6%) resistant strains and 65 of the 72 (90.3%) INH sensitive randomly selected strains showed amino acid substitution in codon 463 (Arg to Leu) (88.9%). This fact supports the hypothesis that mutations at codon 463 are independent of INH-resistance and are linked to the geographical origins of the strains. Copyright 2004 Elsevier B.V.

Genetic characterization of Streptococcus phocae strains isolated from Atlantic salmon, Salmo salar L., in Chile.

PubMed

Valdés, I; Jaureguiberry, B; Romalde, J L; Toranzo, A E; Magariños, B; Avendaño-Herrera, R

2009-04-01

Streptococcus phocae is a beta-haemolytic bacterium frequently involved in disease outbreaks in seals causing pneumonia or respiratory infection. Since 1999, this pathogen has been isolated from diseased Atlantic salmon, Salmo salar, causing serious economic losses in the salmon industry in Chile. In this study, we used different molecular typing methods, such as pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), repetitive extragenic palindromic PCR (REP-PCR) and restriction of 16S-23S rDNA intergenic spacer regions to evaluate the genetic diversity in S. phocae. Thirty-four strains isolated in different years were analysed. The S. phocae type strain ATCC 51973(T) was included for comparative purposes. The results demonstrated genetic homogeneity within the S. phocae strains isolated in Chile over several years, suggesting the existence of clonal relationships among S. phocae isolated from Atlantic salmon. The type strain ATCC 51973(T) presented a different genetic pattern with the PFGE, RAPD, ERIC-PCR and REP-PCR methods. However, the fingerprint patterns of two seal isolates were distinct from those of the type strain.

Occurrence of mannose resistant hemagglutinins in Escherichia coli strains isolated from porcine colibacillosis.

PubMed

Truszczyński, M; Osek, J

1987-01-01

Three-hundred and fifty-eight E. coli strains isolated from piglets were tested for the presence of hemagglutinins by the use of the active hemagglutination test with or without mannose. Additionally 86 strains from the mentioned number of strains were investigated for the presence of common fimbriae using the same method but growing the strains in media especially suited for the development of this kind of fimbriae. These 358 strains and additionally 202 E. coli strains were tested using antisera for 987P and K88 antigens. It was found, using the active hemagglutination test, that 51.4% of the strains were hemagglutinating. The hemagglutinating strains carried the K88 antigen. All these strains were isolated from new-born and weaned piglets with enterotoxic form of colibacillosis, called also E. coli diarrhea. From cases of this form of colibacillosis originated also 26.7% of the strains in which common fimbriae (type 1) were detected. This result was obtained when the BHI medium was used for cultivation. In case of TSA medium only 2.3% of strains were positive. No specific or common fimbriae were found in strains recovered from septic form of colibacillosis and oedema disease (called also enterotoxaemic form of colibacillosis). No strain of 560 examined showed the presence of fimbrial 987P antigen.

Rifaximin-resistant Clostridium difficile strains isolated from symptomatic patients.

PubMed

Reigadas, E; Muñoz-Pacheco, P; Vázquez-Cuesta, S; Alcalá, L; Marín, M; Martin, A; Bouza, E

2017-12-01

Rifaximin has been proposed as an alternative treatment for specific cases of Clostridium difficile infection (CDI) and intestinal decontamination. Rifaximin-resistant C. difficile has occasionally been reported. Antibiotic susceptibility testing relies on anaerobic agar dilution (reference method), which is cumbersome and not routinely used. There is no commercial test for detection of resistance to rifaximin. To assess resistance to rifaximin by C. difficile and to evaluate the correlation between the results of the rifampicin E-test and susceptibility to rifaximin. We compared the in vitro susceptibility of clinical CDI isolates to rifaximin over a 6-month period using the agar dilution method with susceptibility to rifampicin using the E-test. All isolates were characterized using PCR-ribotyping. Clinical data were recorded prospectively. We recovered 276 consecutive C. difficile isolates and found that 32.2% of episodes were caused by rifaximin-resistant strains. The MICs for rifaximin ranged from <0.0009-256 mg/L, with a geometric mean (GM) of 0.256 mg/L, an MIC 50/90 of 0.015/>256 mg/L. Rifaximin and rifampicin MICs were comparable, and all strains classed as resistant by agar dilution were correctly classified as resistant by E-test. The most common ribotypes were 001 (37.2%), 078/126 (14.3%), and 014 (12.0%). Ribotype 001 exhibited the highest MICs for rifaximin. Resistance to rifaximin was common; resistance rates were higher in ribotype 001 strains. Susceptibility to rifaximin determined by agar dilution correlated with susceptibility to rifampicin determined using the E-test, including rifaximin-resistant strains. Our results suggest that the rifampicin E-test is a valid method for the prediction of rifaximin-resistant C. difficile. Copyright © 2017 Elsevier Ltd. All rights reserved.

Three draft genomes of Vibrio coralliilyticus strains isolated from bivalve hatcheries

USDA-ARS?s Scientific Manuscript database

Reported here are the draft genomes of three Vibrio coralliilyticus isolates RE87, AIC-7, and 080116A. Each strain was isolated in association with diseased oyster larvae in commercial aquaculture systems. These draft genomes will be useful for further studies in understanding the genomic features...

Improvement of the respiration efficiency of Lactococcus lactis by decreasing the culture pH.

PubMed

Shi, Weijia; Li, Yu; Gao, Xueling; Fu, Ruiyan

2016-03-01

The growth characteristics and intracellular hemin concentrations of Lactococcus lactis grown under different culture pH and aeration conditions were examined to investigate the effect of culture pH on the respiration efficiency of L. lactis NZ9000 (pZN8148). Cell biomass and biomass yield of L. lactis grown with 4 μg hemin/ml and O2 were higher than those without aeration when the culture pH was controlled at 5-6.5. The culture pH affected the respiratory efficiency in the following order of pH: 5 > 5.5 > 6 > 6.5; the lag phase increased as the culture pH decreased. Hemin accumulation was sensitive to culture pH. Among the four pH conditions, pH 5.5 was optimal for hemin accumulation in the cells. The highest intracellular hemin level in L. lactis resting cells incubated at different pH saline levels (5-6.5) was at pH 5.5. The respiration efficiency of L. lactis under respiration-permissive conditions increases markedly as the culture pH decreases. These results may help develop high cell-density L. lactis cultures. Thus, this microorganism may be used for industrial applications.

Antimicrobial susceptibility of Clostridium perfringens strains isolated from broiler chickens

PubMed Central

Silva, R. O. S.; Salvarani, F.M.; Assis, R.A.; Martins, N.R.S.; Pires, P.S.; Lobato, F.C.F.

2009-01-01

Clostridium perfringens is a normal inhabitant of the intestinal tract of chickens as well as a potential pathogen that causes necrotic enteritis and colangio hepatitis. The minimum inhibitory concentration (MIC) of seven different compounds used for therapy, growth promotion or prevention of coccidiosis was determined by agar dilution method for 55 C. perfringens strains isolated from the intestines of broiler chickens. All strains showed high susceptibility to penicillin, avilamycin, monensin and narasin. Only 7.3% of the strains showed an intermediated sensitivity to lincomycin, and 49 (89.1%) were considered susceptible. For tetracycline and bacitracin, 41.8% and 47.3% of strains, respectively, were considered resistant. PMID:24031355

Spoilage potential of brettanomyces bruxellensis strains isolated from Italian wines.

PubMed

Guzzon, Raffaele; Larcher, Roberto; Guarcello, Rosa; Francesca, Nicola; Settanni, Luca; Moschetti, Giancarlo

2018-03-01

Brettanomyces bruxellensis is an important wine spoilage agent. In this study a population of Brettanomyces strains isolated from Italian wines was thoroughly investigated to evaluate adaptability to wine conditions and spoilage potential. The presumptive isolates of Brettanomyces were identified at species level with 26S rRNA gene sequencing and species-specific PCR, and subsequently subjected to analysis of intra-species variability through the study of intron splice sites (ISS-PCR). Although, some strains were tracked in wines from different regions, extensive genetic biodiversity was observed within the B. bruxellensis population investigated. All strains were evaluated for their growth ability in the presence of ethanol, high sugar content, low pH, different temperatures and sulphur dioxide, using optical density and flow cytometry measurement. The ability of yeasts to produce ethyl phenols in red wines with different chemical compositions was evaluated by means of high performance liquid chromatography with electrochemical detection (HPLC-ECD). The results highlighted wide variability in B. bruxellensis in response to wine limiting factors and in terms of the accumulation of ethyl phenols. As regards this last aspect, the differences found among strains were closely related to chemical composition of wine and strain resistance to environmental stress factors, making a priori evaluation of risk of wine alteration quite difficult. These results suggest that strategies for the control of Brettanomyces should be tailored on the basis of strain distribution and wine characteristics. Copyright © 2017. Published by Elsevier Ltd.

High prevalence of methicillin resistant staphylococci strains isolated from surgical site infections in Kinshasa.

PubMed

Iyamba, Jean-Marie Liesse; Wambale, José Mulwahali; Lukukula, Cyprien Mbundu; za Balega Takaisi-Kikuni, Ntondo

2014-01-01

Surgical site infections (SSIs) after surgery are usually caused by Staphylococcus aureus and coagulase-negative staphylococci (CNS). In low income countries, methicillin resistant Staphylococcus aureus (MRSA) and methicillin resistant coagulase-negative staphylococci (MR-CNS) surgical site infections are particularly associated with high treatment cost and remain a source of mortality and morbidity. This study aimed to determine the prevalence and the sensitivity to antibiotics of MRSA and MR-CNS isolated from SSIs. Wound swabs were collected from 130 hospitalized surgical patients in two major hospitals of Kinshasa. S. aureus and CNS strains were identified by standard microbiological methods and latex agglutination test (Pastorex Staph-Plus). The antibiotic susceptibility of all staphylococcal strains was carried out using disk-diffusion method. Eighty nine staphylococcal strains were isolated. Out of 74 S. aureus and 15 CNS isolated, 47 (63.5%) and 9 (60%) were identified as MRSA and MR-CNS respectively. Among the MRSA strains, 47 strains (100%) were sensitive to imipenem, 39 strains (89%) to amoxycillin-clavulanic acid and 38 strains (81%) to vancomycin. All MR-CNS were sensitive to imipenem, amoxycillin-clavulanic acid and vancomycin. The isolated MRSA and MR-CNS strains showed multidrug resistance. They were both resistant to ampicillin, cotrimoxazole, erythromycin, clindamycin, ciprofloxacin, cefotaxime and ceftazidime. The results of the present study showed a high prevalence of MRSA and MR-CNS. Imipenem, amoxycillin-clavulanic acid and vancomycin were the most active antibiotics. This study suggests that antibiotic surveillance policy should become national priority as MRSA and MR-CNS were found to be multidrug resistant.

Adhesive properties and extracellular enzymatic activity of Staphylococcus aureus strains isolated from oral cavity.

PubMed

Merghni, Abderrahmen; Ben Nejma, Mouna; Hentati, Hajer; Mahjoub, Aouni; Mastouri, Maha

2014-08-01

Staphylococcus aureus is one of prominent bacterial pathogen that occurs in oral region. In this study, 21 strains of S. aureus isolated from the oral cavity of Tunisian patients were investigated for slime production using Congo red agar method (CRA) and adherence assay. Biofilm formation of oral isolates on orthodontic biomaterials (Bis-GMA and PMMA) was also evaluated by MTT reduction assay. In addition, the production of hydrolytic enzymes by S. aureus strains was analyzed and the presence of protease, lipase and β-hemolysin genes (sspA, sspB, geh, hlb) was achieved by polymerase chain reaction (PCR). Qualitative biofilm production tested on CRA revealed that 91% of strains were slime producers. The result of OD570 showed that five strains isolated from the oral cavity were highly biofilm positive. The metabolic activity of S. aureus biofilm formed on Bis-GMA and PMMA did not differ between tested strains. The atomic force micrographs demonstrated that biofilm formed by S. aureus strains was organized in typical cocci cells attached to each other through production of exopolymeric substances. The production of hydrolytic enzymes showed that all S. aureus strains were protease positive. Lipase (77%) and beta hemolytic (59%) activities were also detected. Among the tested strains, 17 were positive for sspA, sspB and hlb genes. While only ten S. aureus strains harbor the geh gene (48%). These data highlight the importance of evaluation of biofilm formation and exoenzyme production in oral S. aureus isolates to investigate the role of this pathogen and its impact in oral pathology. Copyright © 2014 Elsevier Ltd. All rights reserved.

Isolation and identification of symbiotic bacteria from the skin, mouth, and rectum of wild and captive tree shrews.

PubMed

Li, Gui; Lai, Ren; Duan, Gang; Lyu, Long-Bao; Zhang, Zhi-Ye; Liu, Huang; Xiang, Xun

2014-11-18

Endosymbionts influence many aspects of their hosts' health conditions, including physiology, development, immunity, metabolism, etc. Tree shrews (Tupaia belangeri chinensis) have attracted increasing attention in modeling human diseases and therapeutic responses due to their close relationship with primates. To clarify the situation of symbiotic bacteria from their body surface, oral cavity, and anus, 12 wild and 12 the third generation of captive tree shrews were examined. Based on morphological and cultural characteristics, physiological and biochemical tests, as well as the 16S rDNA full sequence analysis, 12 bacteria strains were isolated and identified from the wild tree shrews: body surface: Bacillus subtilis (detection rate 42%), Pseudomonas aeruginosa (25%), Staphlococcus aureus (33%), S. Epidermidis (75%), Micrococcus luteus (25%), Kurthia gibsonii (17%); oral cavity: Neisseria mucosa (58%), Streptococcus pneumonia (17%); anus: Enterococcus faecalis (17%), Lactococus lactis (33%), Escherichia coli (92%), Salmonella typhosa (17%); whereas, four were indentified from the third generation captive tree shrews: body surface: S. epidermidis (75%); oral cavity: N.mucosa (67%); anus: L. lactis (33%), E. coli (100%). These results indicate that S. epidermidis, N. mucosa, L. lactis and E. coli were major bacteria in tree shrews, whereas, S. aureus, M. luteus, K. gibsonii, E. faecalis and S. typhosa were species-specific flora. This study facilitates the future use of tree shrews as a standard experimental animal and improves our understanding of the relationship between endosymbionts and their hosts.

Antibiotic susceptibility of Lactobacillus strains isolated from domestic geese.

PubMed

Dec, M; Wernicki, A; Puchalski, A; Urban-Chmiel, R

2015-01-01

The aim of this study was to determine the antibiotic susceptibility of 93 Lactobacillus strains isolated from domestic geese raised on Polish farms. The minimal inhibitory concentration (MIC) of 13 antimicrobial substances was determined by the broth microdilution method. All strains were sensitive to the cell wall inhibitors ampicillin and amoxicillin (MIC ≤ 8 μg/ml). Resistance to inhibitors of protein synthesis and to fluoroquinolone inhibitors of replication was found in 44.1% and 60.2% of isolates, respectively; 26.9% strains were resistant to neomycin (MIC ≥ 64 μg/ml), 23.6% to tetracycline (MIC ≥ 32 μg/ml), 15% to lincomycin (MIC ≥ 64 μg/ml), 18.3% to doxycycline (MIC ≥ 32 μg/ml), 9.7% to tylosin (MIC ≥ 32 μg/ml), 56% to flumequine (MIC ≥ 256 μg/ml) and 22.6% to enrofloxacin (MIC ≥ 64 μg/ml). Bimodal distribution of MICs indicative of acquired resistance and unimodal distribution of the high MIC values indicative of intrinsic resistance were correlated with Lactobacillus species. Eleven (11.8%) strains displayed multiple resistance for at least three classes of antibiotics. Data derived from this study can be used as a basis for reviewing current microbiological breakpoints for categorisation of susceptible and resistant strains of Lactobacillus genus and help to assess the hazards associated with the occurrence of drug resistance among natural intestinal microflora.

A strain-isolation design for stretchable electronics

NASA Astrophysics Data System (ADS)

Wu, Jian; Li, Ming; Chen, Wei-Qiu; Kim, Dae-Hyeong; Kim, Yun-Soung; Huang, Yong-Gang; Hwang, Keh-Chih; Kang, Zhan; Rogers, John A.

2010-12-01

Stretchable electronics represents a direction of recent development in next-generation semiconductor devices. Such systems have the potential to offer the performance of conventional wafer-based technologies, but they can be stretched like a rubber band, twisted like a rope, bent over a pencil, and folded like a piece of paper. Isolating the active devices from strains associated with such deformations is an important aspect of design. One strategy involves the shielding of the electronics from deformation of the substrate through insertion of a compliant adhesive layer. This paper establishes a simple, analytical model and validates the results by the finite element method. The results show that a relatively thick, compliant adhesive is effective to reduce the strain in the electronics, as is a relatively short film.

Full genome sequences and molecular characterization of tick-borne encephalitis virus strains isolated from human patients.

PubMed

Formanová, Petra; Černý, Jiří; Bolfíková, Barbora Černá; Valdés, James J; Kozlova, Irina; Dzhioev, Yuri; Růžek, Daniel

2015-02-01

Tick-borne encephalitis virus (TBEV) causes tick-borne encephalitis (TBE), one of the most important human neuroinfections across Eurasia. Up to date, only three full genome sequences of human European TBEV isolates are available, mostly due to difficulties with isolation of the virus from human patients. Here we present full genome characterization of an additional five low-passage TBEV strains isolated from human patients with severe forms of TBE. These strains were isolated in 1953 within Central Bohemia in the former Czechoslovakia, and belong to the historically oldest human TBEV isolates in Europe. We demonstrate here that all analyzed isolates are distantly phylogenetically related, indicating that the emergence of TBE in Central Europe was not caused by one predominant strain, but rather a pool of distantly related TBEV strains. Nucleotide identity between individual sequenced TBEV strains ranged from 97.5% to 99.6% and all strains shared large deletions in the 3' non-coding region, which has been recently suggested to be an important determinant of virulence. The number of unique amino acid substitutions varied from 3 to 9 in individual isolates, but no characteristic amino acid substitution typical exclusively for all human TBEV isolates was identified when compared to the isolates from ticks. We did, however, correlate that the exploration of the TBEV envelope glycoprotein by specific antibodies were in close proximity to these unique amino acid substitutions. Taken together, we report here the largest number of patient-derived European TBEV full genome sequences to date and provide a platform for further studies on evolution of TBEV since the first emergence of human TBE in Europe. Copyright © 2014 Elsevier GmbH. All rights reserved.

Biofilm formation by Staphylococcus hominis strains isolated from human clinical specimens.

PubMed

Szczuka, Ewa; Telega, Kinga; Kaznowski, Adam

2015-01-01

Staphylococcus hominis is the third species of coagulase-negative staphylococci (CoNS) most frequently isolated from specimens of patients with hospital-acquired infections. Many infections caused by CoNS appeared to be associated with biofilms. Nevertheless, the knowledge of the ability of S. hominis to form a biofilm is limited. The aim of this study was to analyze the formation of the biofilm by 56 S. hominis strains isolated from clinical cases. The biofilm three-dimensional structure was reconstructed by confocal laser scanning microscopy. We found that most of S. hominis strains carried icaADBC genes encoding polysaccharide intercellular adhesin (PIA), which plays a crucial role in the formation of biofilms in staphylococci strains. However, only a half of the ica-positive strains had an ability to form a biofilm in vitro. In this study, we also accessed the sensitivity of biofilms of S. hominis strains to sodium metaperiodate, proteinase K and DNase. We found that polysaccharides and proteins are the major components of the extracellular matrix of the biofilm formed by S. hominis. DNase did not have a significant effect on biofilms, which suggested that nucleic acid plays a minor role in the mature biofilm.

Molecular typing of environmental and clinical strains of Vibrio vulnificus isolated in the northeastern USA.

PubMed

Reynaud, Yann; Pitchford, Steven; De Decker, Sophie; Wikfors, Gary H; Brown, Christopher L

2013-01-01

Vibrio vulnificus is a ubiquitous marine bacterium that is responsible for infections and some seafood-related illnesses and deaths in the United States, mainly in individuals with compromised health status in the Gulf of Mexico region. Most phylogenetic studies focus on V. vulnificus strains isolated in the southern United States, but almost no genetic data are available on northeastern bacterial isolates of clinical or environmental origin. Our goal in this study was to examine the genetic diversity of environmental strains isolated from commercially-produced oysters and in clinical strains of known pathogenicity in northeastern United States. We conducted analyses of a total of eighty-three strains of V. vulnificus, including 18 clinical strains known to be pathogenic. A polyphasic, molecular-typing approach was carried out, based upon established biotypes, vcg, CPS, 16S rRNA types and three other genes possibly associated with virulence (arylsulfatase A, mtlABC, and nanA). An established Multi Locus Sequence Typing (MLST) method was also performed. Phylogenetic analyses of these markers and MLST results produced similar patterns of clustering of strains into two main lineages (we categorized as 'LI' and 'LII'), with clinical and environmental strains clustering together in both lineages. Lineage LII was comprised primarily but not entirely of clinical bacterial isolates. Putative virulence markers were present in both clinical and environmental strains. These results suggest that some northeastern environmental strains of V. vulnificus are phylogenetically close to clinical strains and probably are capable of virulence. Further studies are necessary to assess the risk of human illness from consuming raw oysters harvested in the northeastern US.

Functional Properties of Lactobacillus mucosae Strains Isolated from Brazilian Goat Milk.

PubMed

de Moraes, Georgia Maciel Dias; de Abreu, Louricélia Rodrigues; do Egito, Antônio Silvio; Salles, Hévila Oliveira; da Silva, Liana Maria Ferreira; Nero, Luís Augusto; Todorov, Svetoslav Dimitrov; Dos Santos, Karina Maria Olbrich

2017-09-01

The search for probiotic candidates among lactic acid bacteria (LAB) isolated from food may uncover new strains with promising health and technological properties. Lactobacillus mucosae strains attracted recent research attention due to their ability to adhere to intestinal mucus and to inhibit pathogens in the gastrointestinal tract, both related to a probiotic potential. Properties of interest and safety aspects of three Lb. mucosae strains (CNPC006, CNPC007, and CNPC009) isolated from goat milk were investigated employing in vitro tests. The presence of genetic factors related to bile salt hydrolase production (bsh), intestinal adhesion properties (msa, map, mub, and ef-tu), virulence, and biogenic amine production were also verified. All strains exhibited the target map, mub, and ef-tu sequences; the msa gene was detected in CNPC006 and CNPC007 strains. Some of the searched sequences for virulence factors were detected, especially in the CNPC009 strain; all strains carried the hyl gene, related to the production of hyaluronidase. Lb. mucosae CNPC007 exhibited a high survival rate in simulated gastric and enteric conditions. Besides, all strains exhibited the bsh sequence, and CNPC006 and CNPC007 were able to deconjugate salts of glycodeoxycholic acid (GDC). Regarding technological properties for dairy product applications, a relatively higher milk acidification and clotting capacity, diacetyl production, and proteolytic activity were registered for CNPC007 in comparison to the other strains. Collectively, the results aim at Lb. mucosae CNPC007 as a promising probiotic candidate for application in dairy products, deserving further studies to confirm and explore its potential.