Cell density and extracellular matrix composition mitigate bacterial biofilm sensitivity to UV-C LED irradiation.

Ultraviolet-C light-emitting diodes (UV-C LEDs) are an emerging technology for decontamination applications in different sectors. In this study, the inactivation of bacterial biofilms was investigated by applying an UV-C LED emitting at 280 nm and by measuring both the influence of the initial cell density (load) and presence of an extracellular matrix (biofilm). Two bacterial strains exposing diverging matrix structures and biochemical compositions were used: Pseudomonas aeruginosa and Leuconostoc citreum. UV-C LED irradiation was applied at three UV doses (171 to 684 mJ/cm2) on both surface-spread cells and on 24-h biofilms and under controlled cell loads, and bacterial survival was determined. All surface-spread bacteria, between 105 and 109 CFU/cm2, and biofilms at 108 CFU/cm2 showed that bacterial response to irradiation was dose-dependent. The treatment efficacy decreased significantly for L. citreum surface-spread cells when the initial cell load was high, while no load effect was observed for P. aeruginosa. Inactivation was also reduced when bacteria were grown under a biofilm form, especially for P. aeruginosa: a protective effect could be attributed to abundant extracellular DNA and proteins in the matrix of P. aeruginosa biofilms, as revealed by Confocal Laser Scanning Microscopy observations. This study showed that initial cell load and exopolymeric substances are major factors influencing UV-C LED antibiofilm treatment efficacy. KEY POINTS: • Bacterial cell load (CFU/cm2) could impact UV-C LED irradiation efficiency • Characteristics of the biofilm matrix have a paramount importance on inactivation • The dose to be applied can be predicted based on biofilm properties.

Response of Controlled Cell Load Biofilms to Cold Atmospheric Plasma Jet: Evidence of Extracellular Matrix Contribution.

AIM: Study of the biocidal effect of a cold atmospheric-pressure plasma in ambient air on single-species bacterial biofilms with controlled cell density, characterized by different extracellular matrices. METHODS AND RESULTS: Two bacterial strains were chosen to present different Gram properties and contrasted extracellular matrices: Pseudomonas aeruginosa ATCC 15442 (Gram-negative), and Leuconostoc citreum NRRL B-1299 (Gram-positive). P. aeruginosa biofilm exhibits a complex matrix, rich in proteins while L. citreum presents the specificity to produce glucan-type exopolysaccharides when grown in the presence of sucrose. Plasma was applied on both surface-spread cells and 24-h grown biofilms with controlled cell loads over 5, 10, or 20 min. Surface-spread bacteria showed a time dependent response, with a maximal bacterial reduction of 2.5 log after 20 min of treatment. On the other hand, in our experimental conditions, no bactericidal effect could be observed when treating biofilms of P. aeruginosa and glucan-rich L. citreum. CONCLUSIONS: For biofilms presenting equivalent cell loads, the response to plasma treatment seemed to depend on the properties of the extracellular matrix characterized by infrared spectroscopy, scanning electron microscopy, or dry weight. SIGNIFICANCE AND IMPACT OF STUDY: Both cell load standardization and biofilm characterization are paramount factors to consider the biocide effect of plasma treatments. The extracellular matrix could affect the plasma efficacy by physical and/or chemical protective effects.

A dynamic resazurin microassay allowing accurate quantification of cells and suitable for acid-forming bacteria.

A resazurin micro-assay was developed to quantify acidifying bacteria. The resorufin fluorescent signal was measured over time and the determined time to reach the max slope (TMS) was plotted against CFU (colony forming unit) counts. This dynamic assay enabled to quantify nine lactic acid bacteria and a Bacillus licheniformis strain despite the increasing acidity of the medium.

Complete Genome Sequence of Lactococcus lactis subsp. lactis A12, a Strain Isolated from Wheat Sourdough.

We report here the complete genome sequence of Lactococcus lactis subsp. lactis strain A12, a strain isolated from sourdough. The circular chromosome and the four plasmids reveal genes involved in carbohydrate metabolism that are potentially required for the persistence of this strain in such a complex ecosystem.

Inventory of the GH70 enzymes encoded by Leuconostoc citreum NRRL B-1299 - identification of three novel α-transglucosylases.

Leuconostoc citreum NRRL B-1299 has long been known to produce α-glucans containing both α-(1→6) and α-(1→2) linkages, which are synthesized by α-transglucosylases of the GH70 family. We sequenced the genome of Leuconostoc citreum NRRL B-1299 to identify the full inventory of GH70 enzymes in this strain. Three new genes (brsA, dsrM and dsrDP) putatively encoding GH70 enzymes were identified. The corresponding recombinant enzymes were characterized. Branching sucrase A (BRS-A) grafts linear α-(1→6) dextran with α-(1→2)-linked glucosyl units, and is probably involved in the α-(1→2) branching of L. citreum NRRL B-1299 dextran. This is the first report of a naturally occurring α-(1→2) branching sucrase. DSR-M and DSR-DP are dextransucrases that are specific for α-(1→6) linkage synthesis and mainly produce oligomers or short dextrans with molar masses between 580 and 27 000 g·mol(-1) . In addition, DSR-DP contains sequences that diverge from the consensus sequences that are typically present in enzymes that synthesize linear dextran. Comparison of the genome with five other L. citreum genomes further revealed that dsrDP is unique to L. citreum NRRL B-1299. The presence of this gene in a prophage represents the first evidence of phage-mediated horizontal transfer of genes encoding such enzymes in lactic acid bacteria. Finally, brsA and dsrM are located in a chromosomal region in which genes encoding strain-specific GH70 enzymes are consistently located. This region may be a good target on which to focus in order to rapidly evaluate the diversity of GH70 enzymes in L. citreum strains.

Overview of the glucansucrase equipment of Leuconostoc citreum LBAE-E16 and LBAE-C11, two strains isolated from sourdough.

The whole set of putative glucansucrases from Leuconostoc citreum LBAE-E16 and LBAE-C11 was retrieved from the draft genome sequence of these two sourdough strains previously suggested as alternan producers. Four and five putative glycoside hydrolase family 70 (GH70) encoding genes were identified in the genome sequence of strain C11 and E16, respectively. Some putative genes have high sequence identity to known Leuconostoc dextransucrases. Molecular and biochemical data confirmed that L. citreum C11 could be considered as a new alternan-producing strain, unlike strain E16. In the latter, two new putative glucansucrases with unusual structural features were retrieved. In particular, the GSE16-5 gene encodes for a protein of 2063 amino acids with a theoretical molecular mass of 229 kDa that shares 61% identity with the alternansucrase (ASR) of L. citreum NRRL B-1355, due to the presence of seven APY repeats identified in the C-terminal peptide sequence. Cloning and expression of the corresponding coding sequence revealed synthesis of a low molecular weight (10(4) Da) linear dextran polymer with glucosyl residues only linked by α-1,6 linkages. This novel GH70 enzyme may thus be viewed as a natural chimeric enzyme resulting from the addition of the ASR C-terminal region in a dextransucrase.

Complete Genome Sequence of Leuconostoc citreum Strain NRRL B-742.

Leuconostoc citreum belongs to the group of lactic acid bacteria and plays an important role in fermented foods of plant origin. Here, we report the complete genome of the Leuconostoc citreum strain NRRL B-742, isolated in 1954 for its capacity to produce dextran.

The carbohydrate metabolism signature of lactococcus lactis strain A12 reveals its sourdough ecosystem origin.

Lactococcus lactis subsp. lactis strain A12 was isolated from sourdough. Combined genomic, transcriptomic, and phenotypic analyses were performed to understand its survival capacity in the complex sourdough ecosystem and its role in the microbial community. The genome sequence comparison of strain A12 with strain IL1403 (a derivative of an industrial dairy strain) revealed 78 strain-specific regions representing 23% of the total genome size. Most of the strain-specific genes were involved in carbohydrate metabolism and are potentially required for its persistence in sourdough. Phenotype microarray, growth tests, and analysis of glycoside hydrolase content showed that strain A12 fermented plant-derived carbohydrates, such as arabinose and α-galactosides. Strain A12 exhibited specific growth rates on raffinose that were as high as they were on glucose and was able to release sucrose and galactose outside the cell, providing soluble carbohydrates for sourdough microflora. Transcriptomic analysis identified genes specifically induced during growth on raffinose and arabinose and reveals an alternative pathway for raffinose assimilation to that used by other lactococci.

Characterization of glucansucrase and dextran from Weissella sp. TN610 with potential as safe food additives.

Pear-derived Weissella sp. TN610 produced extracellular glycosyltransferase activity responsible for the synthesis of soluble exopolysaccharide from sucrose. Acid and dextranase-catalyzed hydrolysis revealed that the synthesized polymer was a glucan. According to (1)H and (13)C NMR analysis, the glucan produced by TN610 was a linear dextran made of 96% α-(1→6) and 4% α-(1→3) linkages. Zymogram analysis confirmed the presence of a unique glucansucrase of approximately 180 kDa in the cell-free supernatant from TN610. The crude enzyme, optimally active at 37°C and pH 5, has promising potential for application as a food additive since it catalyzes dextran synthesis in sucrose-supplemented milk, allowing its solidification. A 4257-bp product corresponding to the mature glucansucrase gene was amplified by PCR from TN610. It encoded a polypeptide of 1418 residues having a calculated molecular mass of 156.089 kDa and exhibiting 96% and 95% identity with glucansucrases from Lactobacillus fermentum Kg3 and Weissella cibaria CMU, respectively.

Characterization of a novel dextransucrase from Weissella confusa isolated from sourdough.

Weissella confusa and Weissella cibaria isolated from wheat sourdoughs produce, from sucrose, linear dextrans due to a single soluble dextransucrase. In this study, the first complete gene sequence encoding dextransucrase from a W. confusa strain (LBAE C39-2) along with the one from a W. cibaria strain (LBAE K39) were reported. Corresponding gene cloning was achieved using specific primers designed on the basis of the draft genome sequence of these species. Deduced amino acid sequence of W. confusa and W. cibaria dextransucrase revealed common structural features of the glycoside hydrolase family 70. Notably, the regions located in the vicinity of the catalytic triad (D, E, D) are highly conserved. However, comparison analysis also revealed that Weissella dextransucrases form a distinct phylogenetic group within glucansucrases of other lactic acid bacteria. We then cloned the W. confusa C39-2 dextransucrase gene and successfully expressed the mature corresponding enzyme in Escherichia coli. The purified recombinant enzyme rDSRC39-2 catalyzed dextran synthesis from sucrose with a K m of 8.6 mM and a V max of 20 µmol/mg/min. According to (1)H and (13)C NMR analysis, the polymer is a linear class 1 dextran with 97.2 % α-(1→6) linkages and 2.8 % α-(1→3) branch linkages, similar to the one produced by W. confusa C39-2 strain. The enzyme exhibited optimum catalytic activity for temperatures ranging from 35 to 40 °C and a pH of 5.4 in 20 mM sodium acetate buffer. This novel dextransucrase is responsible for production of dextran with predominant α-(1→6) linkages that could find applications as food hydrocolloids.

Characterization of glycosyltransferase activity of wild-type Leuconostoc mesenteroides strains from Bulgarian fermented vegetables.

Glycosyltransferase activity of 13 Leuconostoc mesenteroides strains isolated from Bulgarian fermented vegetables was investigated. All the strains displayed a mucoid phenotype on sucrose-containing agar media. Strains were characterized according to carbohydrate fermentation, species-specific multiple PCR using several primers, repetitive element-PCR fingerprinting using (GTG)(5) primers and glycosyltransferase activity. Level of activity and cellular localization (soluble or cell-associated) were variable among strains. Precipitation of exopolysaccharides produced from sucrose by the soluble fractions from these strains allowed recovery of only glucans and further characterization by (1)H and (13)C NMR analysis and enzymatic digestion with dextranase revealed dextran production. However, levans could be detected in presence of raffinose as fructosyl donor. Both fructosyltransferase and glucosyltransferase encoding genes were detected by PCR and both active enzymes were detected after functional characterization by SDS-PAGE electrophoresis and in situ polymer production after incubation with sucrose. This work therefore showed that concomitant production of glucosyltransferase and fructosyltransferase is widespread in L. mesenteroides strains.

Genome sequence of Weissella confusa LBAE C39-2, isolated from a wheat sourdough.

Weissella confusa is a rod-shaped heterofermentative lactic acid bacterium from the family of Leuconostocaceae. Here we report the draft genome sequence of the strain W. confusa LBAE C39-2 isolated from a traditional French wheat sourdough.

Genome sequences of three Leuconostoc citreum strains, LBAE C10, LBAE C11, and LBAE E16, isolated from wheat sourdoughs.

Leuconostoc citreum is a key microorganism in fermented foods of plant origin. Here we report the draft genome sequence for three strains of Leuconostoc citreum, LBAE C10, LBAE C11, and LBAE E16, which have been isolated from traditional French wheat sourdoughs.

Characterization of dextran-producing Weissella strains isolated from sourdoughs and evidence of constitutive dextransucrase expression.

The study of exopolysaccharide production by heterofermentative sourdough lactic acid bacteria has shown that Weissella strains isolated from sourdoughs produce linear dextrans containing α-(1→6) glucose residues with few α-(1→3) linkages from sucrose. In this study, several dextran-producing strains, Weissella cibaria and Weissella confusa, isolated from sourdough, were characterized according to carbohydrate fermentation, repetitive element-PCR fingerprinting using (GTG)(5) primers and glucansucrase activity (soluble or cell-associated). This study reports, for the first time, the characterization of dextransucrase from Weissella strains using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in situ polymer production (after incubation with sucrose) from enzymatic fractions harvested from both sucrose and glucose culture media. Results demonstrate that dextransucrase activity was mainly soluble and associated with a constitutive 180-kDa protein. In addition, microsequencing of the active dextransucrase from W. cibaria LBAE-K39 allowed the design of specific primers that could detect the presence of glucansucrase encoding genes similar to GTFKg3 of Lactobacillus fermentum Kg3 and to DSRWC of W. cibaria CMU. This study hence indicates that sourdough Weissella strains synthesize original dextransucrase.

Characterization of glucan-producing Leuconostoc strains isolated from sourdough.

Sourdough was previously demonstrated to be a fruitful biotope for isolation of lactic acid bacteria producing exopolysaccharides and more accurately diverse glycan polymers which have interesting applications as texturing agents or prebiotics. Characterization of polymers by (1)H and (13)C NMR spectroscopy analysis demonstrated that these strains could synthesize glucans of high structural variety and containing different amounts of α-(1→2), α-(1→3) and α-(1→6) linkages. In this study, fifteen glucan-producing Leuconostoc mesenteroides and L. citreum strains from sourdoughs were characterized according to carbohydrate fermentation, rep-PCR fingerprinting using (GTG)(5) primers and glycansucrase activity (soluble or cell-associated). Enzyme characterization using SDS-PAGE and in situ polymer production after incubation with sucrose correlated with synthesis of classical or α-(1→2) branched dextrans, alternan and levan. In addition, the presence of genes coding for alternansucrase was detected by PCR and partially characterized by sequence analysis. We thus provide new information on the biodiversity of glucan production by sourdough Leuconostoc strains.

Biodiversity of exopolysaccharides produced from sucrose by sourdough lactic acid bacteria.

The distribution and diversity of natural exopolysaccharides (EPS) produced from sucrose by thirty heterofermentative lactic acid bacteria strains from French traditional sourdoughs was investigated. The EPS production was found to be related to glucansucrase and fructansucrase extracellular activities. Depending on the strain, soluble and/or cell-associated glycansucrases were secreted. Structural characterization of the polymers by 1H and 13C NMR spectroscopy analysis further demonstrated a high diversity of EPS structures. Notably, we detected strains that synthesize glucans showing amazing variations in the amount of alpha-(1-->2), alpha-(1-->3) and alpha-(1-->6) linkages. The representation of Leuconostoc strains which produce putative alternan polymers and alpha-(1-->2) branched polymers was particularly high. The existence of glucan- and fructansucrase encoding genes was also confirmed by PCR detection. Sourdough was thus demonstrated to be a very attractive biotope for the isolation of lactic acid bacteria producing novel polymers which could find interesting applications such as texturing agent or prebiotics.

Biodiversity of lactic acid bacteria in French wheat sourdough as determined by molecular characterization using species-specific PCR.

The lactic acid microflora of nine traditional wheat sourdoughs from the Midi-Pyrénées area (South western France) was previously isolated and preliminary characterized using conventional morphological and biochemical analysis. However, such phenotypic methods alone are not always reliable and have a low taxonomic resolution for identification of lactic acid bacteria species. In the present study, a total of 290 LAB isolates were identified by PCR amplification using different sets of specific primers in order to provide a thorough characterization of the lactic flora from these traditional French sourdoughs. Overall, the LAB isolates belonged to 6 genera: Lactobacillus (39%, 8 species), Pediococcus (38%, 1 species), Leuconostoc (17%, 2 species), Weissella (4%, 2 species), Lactococcus (1%, 1 species) and Enterococcus (<1%, 1 species) and 15 different species were detected: L. plantarum, L. curvatus, L. paracasei, L. sanfranciscensis, L. pentosus, L. paraplantarum, L. sakei, L. brevis, P. pentosaceus, L. mesenteroides, L. citreum, W. cibaria, W. confusa, L. lactis and E. hirae. Facultative heterofermentative LAB represent more than 76% of the total isolates, the main species isolated herein correspond to L. plantarum and P. pentosaceus. Obligate heterofermentative lactobacilli (L. sanfranciscencis, L. brevis) represent less than 3% of the total isolates whereas Leuconostoc and Weissella species represent 21% of the total isolates and have been detected in eight of the nine samples. Detection of some LAB species was preferentially observed depending on the isolation culture medium. The number of different species within a sourdough varies from 3 to 7 and original associations of hetero- and homofermentative LAB species have been revealed. Results from this study clearly confirm the diversity encountered in the microbial community of traditional sourdough and highlight the importance of LAB cocci in the sourdough ecosystem, along with lactobacilli.