Carol A. van Reenen

Surface-bound proteins of Lactobacillus plantarum 423 that contribute to adhesion of Caco-2 cells and their role in competitive exclusion and displacement of Clostridium sporogenes and Enterococcus faecalis

Elongation factor Tu (EF-Tu), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI) are surface-bound proteins with a role in adhesion of Lactobacillus plantarum 423 to Caco-2 cells. Removal of surface-bound proteins from L. plantarum 423 (treated with 4M guanidine-HCl) reduced adhesion to Caco-2 cells by 40%. In a competitive exclusion experiment where all three strains were given an equal chance to adhere to Caco-2 cells, L. plantarum 423 prevented 71% of cells of Clostridium sporogenes LMG 13570 and 89% of cells of Enterococcus faecalis LMG 13566 from adhering. Cells of L. plantarum 423, from which surface-bound proteins were removed, prevented 49% of cells of C. sporogenes LMG 13570 and 70% of cells of E. faecalis LMG 13566 from adhering to Caco-2 cells, suggesting that factors other than surface-bound proteins are involved in adhesion. Colonization of L. plantarum 423 to Caco-2 cells prevented adhesion of 74% of cells of C. sporogenes LMG 13570 and 62% of cells of E. faecalis LMG 13566. Furthermore, L. plantarum 423 displaced 81% of cells of C. sporogenes LMG 13570 and 91% of cells of E. faecalis LMG 13566 from Caco-2 cells. L. plantarum 423 is a potential probiotic strain.

Horizontal gene transfer amongst probiotic lactic acid bacteria and other intestinal microbiota: what are the possibilities? A review.

Probiotics are live cultures, usually lactic acid bacteria, which are ingested to promote a healthy gastrointestinal tract. These organisms require certain traits to survive and compete in this niche, but these traits may be transferred to other microbiota in the gastrointestinal tract (GIT). Similarly, virulence factors from pathogens may be acquired by probiotic strains. Bacteria have developed a plethora of methods to transfer genetic material between strains, species and genera. In this review, the possible factors that may be exchanged and the methods of exchange are discussed.

Fermentation optimization of plantaricin 423, a bacteriocin produced by Lactobacillus plantarum 423

Abstract More than 15 bacteriocins of Lactobacillus plantarum have been described. However, little information has been published on the fermentation optimization of these peptides. Plantaricin 423, produced by L. plantarum 423, is produced during exponential growth and reaches a maximum activity [6000 activity units (AU)/ml] at the beginning of stationary growth (i.e. after 15 h). This activity level is maintained for 3 h, but declines to 2600 AU/ml towards the end of fermentation (i.e. after 31 h). The increase in plantaricin 423 activity coincided with a decrease in pH from 5.8 to 4.0 during the first 15 h of fermentation. However, when the number of cells are taken into account (ODmax-value), a higher concentration of plantaricin 423 is produced in medium with an initial pH of 4.90 (2961 AU/ml/ODmax) than at pH 5.80 (2368 AU/ml/ODmax). A much lower activity of plantaricin 423 was obtained during the same fermentation period (15 h) when cells were grown in MRS broth (Merck) with an initial pH of 6.9. The production of plantaricin 423 increased from 6000 AU/ml in MRS broth (Merck) to 9600, 12,800 and 19,200 AU/ml when the medium was supplemented with 1.9% (w/v) meat extract (Oxoid), 3.4% (w/v) casaminoacids (Oxoid) or 1.9% (w/v) tryptone (Oxoid), and 1.7% (w/v) bacteriological peptone (Oxoid), respectively. The activity of plantaricin 423 was even further increased with the addition of 1% (w/v) tween 80 to MRS broth (Merck). Low concentrations of MnSO4·H2O (0.014%, w/v) stimulated the growth of strain 423 and increased the activity of plantaricin 423. Although the addition of MgSO4·7H2O had the same stimulating effect on the growth of strain 423, the activity of plantaricin 423 was not increased.

Adhesion of the probiotic strains Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 to Caco-2 cells under conditions simulating the intestinal tract, and in the presence of antibiotics and anti-inflammatory medicaments

Adhesion of Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 to Caco-2 (human carcinoma epithelial) cells was visualized by fluorescent staining. Both strains showed good adhesion compared to L. casei MB1, L. casei Shirota, L. johnsonii La1 and L. rhamnosus GG. No correlation was found between hydrophobicity, aggregation and adhesion to Caco-2 cells. Presence of antibiotics and anti-inflammatory medicaments reduced adhesion of bacterial strains to Caco-2 cells. Proteins sensitive to pepsin, trypsin and pronase are involved in the adhesion of E. mundtii ST4SA and L. plantarum 423 to Caco-2 cells. Adhesion of Listeria monocytogenes ScottA to Caco-2 cells was not prevented by E. mundtii ST4SA and L. plantarum 423. Cell-free culture supernatants of strains ST4SA and 423, containing the antimicrobial peptides plantaricin 423 and peptide ST4SA, prevented the invasion of L. monocytogenes ScottA into Caco-2 cells.

Evaluation of Numerical Analysis of Random Amplified Polymorphic DNA (RAPD)-PCR as a Method to Differentiate Lactobacillus plantarum and Lactobacillus pentosus

Abstract.Lactobacillus plantarum and Lactobacillus pentosus grouped into one protein profile cluster at r ≥ 0.70, separate from Lactobacillus casei, Lactobacillus sake, and Lactobacillus curvatus. Similar sugar fermentation reactions were recorded for representative strains of L. plantarum and L. pentosus. Representative strains, including the type of each species, were selected from the different protein profile clusters and their genetic relatedness determined by using numerical analysis of random amplified polymorphic DNA (RAPD)-PCR. The type strains of L. plantarum (ATCC 14917T) and L. pentosus (NCFB 363T) displayed different RAPD profiles and grouped into two independent clusters, well separated from L. casei, L. curvatus, and L. sake. Numerical analysis of RAPD-PCR proved a reliable and accurate method to distinguish between strains of L. plantarum and L. pentosus.

Evaluation of Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 as probiotics by using a gastro-intestinal model with infant milk formulations as substrate

Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 produce bacteriocins with activity against a number of Gram-positive and Gram-negative bacteria. Both strains survived intestinal conditions simulated in a gastro-intestinal model (GIM) with infant milk formulations as substrate and prevented the growth of Listeria monocytogenes ScottA. The strains are inhibited by the antibiotics amoxicillin, cefadroxil, roxithromycin and doxycycline, anti-inflammatory medicaments containing meloxicam, ibuprofen and sodium diklofenak, and analgesics containing paracetamol, codeine phosphate and promethazine. Strain 423 is sensitive to vancomycin and does not contain genes encoding gelatinase, cell aggregation substance (AS), adhesion to collagen (Ace), enterococcus surface protein (Esp), Enterococcus faecalis endocarditis antigen (EfaAfs), cytolysin and non-cytolysin (beta-hemolysin III). Genes encoding AS, cytolysin and non-cytolysin (beta-hemolysin III) were amplified from the genome of strain ST4SA. Survival of strains ST4SA and 423 improved when used as combined cultures in the GIM and compared well with the survival of commercially available probiotics subjected to the same conditions.

Characterization of a bacteriocin produced by Lactobacillus sakei R1333 isolated from smoked salmon

Strain R1333, isolated from commercially available smoked salmon, was identified as Lactobacillus sakei based on biochemical tests, sugar fermentation reactions (API 50 CHL), PCR with species-specific primers and sequencing of the 16S rRNA gene. Strain R1333 produces a 3811 kDa class IIa bacteriocin, active against Streptococcus caprinus, Streptococcus macedonicus, Streptococcus spp., L. sakei, Lactococcus lactis subsp. lactis, Listeria innocua, Listeria ivanovii subsp. ivanovii and Listeria monocytogenes. The mode of activity against L. innocua 2030C and L. ivanovii subsp. ivanovii ATCC 19119 was bactericidal, resulting in cell lysis and enzyme- and DNA-leakage. The highest level of activity (1600 AU/mL) was recorded when cells were grown at 30°C in MRS broth (initial pH 6.5). Only 800 AU/mL was recorded when strain R1333 was grown in MRS without Tween 80. Lower levels of bacteriocin production were recorded when strain R1333 was grown in MRS at 20°C. Peptide R1333 adsorbs at low levels (200 AU/mL) to producer cells. Purification of bacteriocin R1333 was performed by 60% ammonium sulfate precipitation, followed by separation on a SepPak C(18) column and reverse-phase HPLC on a Nucleosil C(18) column with a linear gradient from 0.1% TFA to 90% acetonitryl. A molecular mass of 3811 kDa was determined by mass spectrometry. Based on mass spectrometry and sequencing of the PCR amplified fragment targeting the sakG gene, L. sakei R1333 is a potential producer of sakacin G. This is the first report of the identification of sakacin G produced by L. sakei isolated from smoked salmon.

Photorhabdus luminescens subsp. noenieputensis subsp. nov., a symbiotic bacterium associated with a novel Heterorhabditis species related to Heterorhabditis indica

The bacterial symbiont AM7(T), isolated from a novel entomopathogenic nematode species of the genus Heterorhabditis, displays the main phenotypic traits of the genus Photorhabdus and is highly pathogenic to Galleria mellonella. Phylogenetic analysis based on a multigene approach (16S rRNA, recA, gyrB, dnaN, gltX and infB) confirmed the classification of isolate AM7(T) within the species Photorhabdus luminescens and revealed its close relatedness to Photorhabdus luminescens subsp. caribbeanensis, P. luminescens subsp. akhurstii and P. luminescens subsp. hainanensis. The five concatenated protein-encoding sequences (4197 nt) of strain AM7(T) revealed 95.8, 95.4 and 94.9 % nucleotide identity to sequences of P. luminescens subsp. caribbeanensis HG29(T), P. luminescens subsp. akhurstii FRG04(T) and P. luminescens subsp. hainanensis C8404(T), respectively. These identity values are less than the threshold of 97 % proposed for classification within one of the existing subspecies of P. luminescens. Unlike other strains described for P. luminescens, strain AM7(T) produces acid from adonitol, sorbitol and xylitol, assimilates xylitol and has no lipase activity on medium containing Tween 20 or 60. Strain AM7(T) is differentiated from P. luminescens subsp. caribbeanensis by the assimilation of N-acetylglucosamine and the absence of haemolytic activity. Unlike P. luminescens subsp. akhurstii, strain AM7(T) does not assimilate mannitol, and it is distinguished from P. luminescens subsp. hainanensis by the assimilation of trehalose and citrate, the inability to produce indole from tryptophan and the presence of acetoin production and urease activity. Strain AM7(T) ( = ATCC BAA-2407(T)  = DSM 25462(T)) belongs to a novel subspecies, and is proposed as the type strain of Photorhabdus luminescens subsp. noenieputensis sp. nov.

Description of Xenorhabdus khoisanae sp. nov., the symbiont of the entomopathogenic nematode Steinernema khoisanae.

Bacterial strain SF87(T), and additional strains SF80, SF362 and 106-C, isolated from the nematode Steinernema khoisanae, are non-bioluminescent Gram-reaction-negative bacteria that share many of the carbohydrate fermentation reactions recorded for the type strains of recognized Xenorhabdus species. Based on 16S rRNA gene sequence data, strain SF87(T) is shown to be closely related (98% similarity) to Xenorhabdus hominickii DSM 17903(T). Nucleotide sequences of strain SF87 obtained from the recA, dnaN, gltX, gyrB and infB genes showed 96-97% similarity with Xenorhabdus miraniensis DSM 17902(T). However, strain SF87 shares only 52.7% DNA-DNA relatedness with the type strain of X. miraniensis, confirming that it belongs to a different species. Strains SF87(T), SF80, SF362 and 106-C are phenotypically similar to X. miraniensis and X. beddingii, except that they do not produce acid from aesculin. These strains are thus considered to represent a novel species of the genus Xenorhabdus, for which the name Xenorhabdus khoisanae sp. nov. is proposed. The type strain is SF87(T) ( =DSM 25463(T) =ATCC BAA-2406(T)).

Characterization of leucocin B-KM432Bz from Leuconostoc pseudomesenteroides isolated from boza, and comparison of its efficiency to pediocin PA-1.

A bacteriocin-producing bacterium was isolated from boza and identified as Leuconostoc pseudomesenteroides KM432Bz. The antimicrobial peptide was purified and shown to be identical to other class IIa bacteriocins: leucocin A from Leuconostoc gelidum UAL-187 and Leuconostoc pseudomesenteroides QU15 and leucocin B from Leuconostoc carnosum Ta11a. The bacteriocin was named leucocin B-KM432Bz. Leucocin B-KM432Bz gene cluster encodes the bacteriocin precursor (lcnB), the immunity protein (lcnI) and the dedicated export machinery (lcnD and lcnE). A gene of unknown and non-essential function (lcnC), which is interrupted by an insertion sequence of the IS30 family, is localized between lcnB and lcnD. The activity of leucocin B-KM432Bz requires subunit C of the EIIt Man mannose permease, which is the receptor for entry into target cells. The determination of the minimum inhibitory concentrations revealed the lowest values for leucocin B-KM432Bz over Listeria strains, with 4 to 32 fold better efficiency than pediocin PA-1.