Manuel Martínez-Bueno

A simple method for semi-preparative-scale production and recovery of enterocin AS-48 derived from Enterococcus faecalis subsp. liquefaciens A-48-32

Production of enterocin AS-48 by Enterococcus faecalis A-48-32 was compared between standard and high-cell density batch fermentations. In high-cell density cultures, bacteriocin production was 2.47-fold higher, provided that the pH was controlled during the fermentation. A two-step procedure for recovery of milligram quantities of purified bacteriocin was developed, based on adsorption of the bacteriocin on Carboxymethyl Sephadex CM-25 followed by reversed-phase chromatography on a semi-preparative column. The purified bacteriocin was active on all the Gram-positive bacteria tested (for example, species of Bacillus, Paenibacillus, Staphylococcus, and Listeria). Strains E. coli U-9, E. coli CECT 102, E. coli CECT 104, E. coli CECT 432, E. coli CECT 543, E. coli CECT 877 and Shigella sonnei CECT 542 were sensitive, while seven other E. coli strains as well as Salmonella choleraesuis CECT 722, S. choleraesuis CECT 916, Enterobacter cloacae CECT 194 and Aeromonas hydrophila CECT 398 were resistant.

Characterization of antimicrobial substances produced by Enterococcus faecalis MRR 10-3, isolated from the uropygial gland of the hoopoe (Upupa epops).

ABSTRACT The uropygial gland (preen gland) is a holocrine secretory gland situated at the base of the tail in birds which produces a hydrophobic fatty secretion. In certain birds, such as the hoopoe, Upupa epops, the composition of this secretion is influenced by both seasonal and sexual factors, becoming darker and more malodorous in females and in their nestlings during the nesting phase. The secretion is spread throughout the plumage when the bird preens itself, leaving its feathers flexible and waterproof. It is also thought to play a role in defending the bird against predators and parasites. We have isolated from the uropygial secretion of a nestling a bacterium that grows in monospecific culture which we have identified unambiguously by phenotypic and genotypic means as Enterococcus faecalis. The strain in question produces antibacterial substances that are active against all gram-positive bacteria assayed and also against some gram-negative strains. Its peptide nature identifies it as a bacteriocin within the group known as enterocins. Two peptides were purified to homogeneity (MR10A and MR10B), and matrix-assisted laser desorption ionization-time of flight (mass spectrometry) analysis showed masses of 5201.58 and 5207.7 Da, respectively. Amino acid sequencing of both peptides revealed high similarity with enterocin L50A and L50B (L. M. Cintas, P. Casaus, H. Holo, P. E. Hernández, I. F. Nes, and L. S. Håvarstein, J. Bacteriol. 180:1988-1994, 1998). PCR amplification of total DNA from strain MRR10-3 with primers for the L50A/B structural genes and sequencing of the amplified fragment revealed almost identical sequences, except for a single conservative change in residue 38 (Glu→Asp) in MR10A and two changes in residues 9 (Thr→Ala) and 15 (Leu→Phe) in MR10B. This is the first time that the production of bacteriocins by a bacterium isolated from the uropygial gland has been described. The production of these broad-spectrum antibacterial substances by an enterococcal strain living in the uropygial gland may be important to the hygiene of the nest and thus to the health of the eggs and chicks.

Analysis of the gene cluster involved in production and immunity of the peptide antibiotic AS‐48 in Enterococcus faecalis

A region of 7.8 kb of the plasmid pMB2 from Enterococcus faecalis S‐48 carrying the information necessary for production and immunity of the peptide antibiotic AS‐48 has been cloned and sequenced. It contains the as‐48A structural gene plus five open reading frames (as‐48B, as‐48C, as‐48C1, as‐48D and as‐48D1 ). Besides As‐48D, all the predicted gene products are basic hydrophobic proteins with potential membrane‐spanning domains (MSDs). None of them shows any homology with protein sequences stored in databanks, except for As‐48D, which shows similarity to the C‐terminal domain of ABC transporters and contains a highly conserved ATP‐binding site. The gene products of as‐48B, as‐48C, as‐48C1 and as‐48D are thought to be involved in AS‐48 production and secretion. The only gene able to provide resistance to AS‐48 by itself is as‐48D1. Immunity also seems to be enhanced at least by the products of as‐48B, as‐48C1 and as‐48D genes. Transcription analysis using probes derived from the different ORFs revealed two large (3.5 and 2.7 kb) mRNAs, suggesting that the different genes are organized in two constitutive operons.

Isolation and characterization of enterocin EJ97, a bacteriocin produced by Enterococcus faecalis EJ97

Abstract The bacteriocinogenic strain of Enterococcus faecalis EJ97 has been isolated from municipal waste water. It produces a cationic bacteriocin (enterocin EJ97) of low molecular mass (5,340 Da) that is very stable under mild heat conditions and is sensitive to proteolytic enzymes. The amino acid sequence of the first 18 N-terminal residues of enterocin EJ97 indicates that it is different from other known protein sequences. Enterocin EJ97 is active on several gram-positive bacteria including enterococci, several species of Bacillus, Listeria, and Staphylococcus aureus. The producer strain is immune to bacteriocin. Enterocin EJ97 has a concentration-dependent bactericidal and bacteriolytic effect on E. faecalis S-47.

Bactericidal and bacteriolytic action of peptide antibiotic AS-48 against gram-positive and gram-negative bacteria and other organisms

A purified peptide antibiotic AS-48 from Streptococcus faecalis spp liquiefaciens S-48 exerted a bactericidal mode of action against most Gram-positive and many Gram-negative bacteria tested. In many Gram-positive bacteria and the two Myxococcus species assayed, a bacteriolytic effect, as a consequence of primary lesions, was also observed. In general, the Gram-negative bacteria were more resistant to AS-48. Escherichia coli protoplasts showed increased sensitivity and those of a resistant yeast. Saccharomyces cerevisiae 3.2, became sensitive. These data suggest that resistance is related to the cell wall structure. AS-48 adsorbed rapidly to cell walls and cytoplasmic membranes of sensitive and resistant cells. Adsorption to cytoplasmic membranes involved complete neutralization of AS-48.

The cyclic structure of the enterococcal peptide antibiotic AS‐48

The complete primary structure of the peptide antibiotic AS‐48 produced by Enterococcus faecalis has been determined by chemical degradation analysis. The cyclic nature of this 70 residues containing peptide was demonstrated by plasma desorption mass analysis of the generated peptides and electrospray ionisation mass analysis of the native polypeptide. As far as we know, this is the first example of an antibiotic protein cyclised by a tail—head peptide bond formation and not by branching of the polypeptide side chains.

Inhibition of Bacterial Growth, Enterotoxin Production, and Spore Outgrowth in Strains of Bacillus cereus by Bacteriocin AS-48

ABSTRACT Bacteriocin AS-48 showed high bactericidal activity for mesophilic and psychrotrophic strains of Bacillus cereus over a broad pH range. AS-48 inhibition of the enterotoxin-producing strain LWL1 was enhanced by sodium nitrite, sodium lactate, and sodium chloride. The latter also enhanced AS-48 activity against strain CECT 131. Bacterial growth and enterotoxin production by strain LWL1 were completely inhibited at bacteriocin concentrations of 7.5 μg/ml. At subinhibitory bacteriocin concentrations, enterotoxin production decreased markedly and sporulation was delayed. Intact spores were resistant to AS-48 but became gradually sensitive to AS-48 during the course of germination.

Symbiotic bacteria living in the hoopoe's uropygial gland prevent feather degradation.

SUMMARY Among potential agents that might damage bird feathers are certain microorganisms which secrete enzymes that digest keratin, as is the case of the ubiquitous bacterium Bacillus licheniformis, present in both the feathers and skin of wild birds. It is therefore a good candidate for testing the effects of bird defences against feather-degrading microorganisms. One of these defences is the oil secreted by the uropygial gland, which birds use to protect their feathers against parasites. In previous studies we have shown how Enterococcus faecalis strains isolated from nestling hoopoes exert antagonistic effects against B. licheniformis, mediated by the production of bacteriocins. Consequently we hypothesized that this enterococcus and the bacteriocins it engenders might act as a defence against feather-degrading microorganisms in hoopoes. We investigated this hypothesis in a series of laboratory experiments and evaluated the extent to which the keratinolytic effects caused by B. licheniformis were reduced by the E. faecalis MRR10-3 strain, isolated from hoopoes, and its bacteriocins. In different treatments, feathers or pure keratin was incubated with B. licheniformis, B. licheniformis together with E. faecalis MRR10-3, and B. licheniformis together with the bacteriocins produced by E. faecalis MRR10-3. Our results were in accordance with the predicted effects on hoopoe feathers. There was a significant decrease both in pure keratin loss and in feather degradation in the presence of the symbiotic bacterium or its bacteriocin. These results suggest that by preening their feathers hoopoes benefit from their symbiotic relationship with bacteriocin-producing enterococci, which constitute a chemical defence against feather degradation.

Control of Staphylococcus aureus in sausages by enterocin AS-48

Results presented here are the first contribution on the anti-staphylococcal activity of bacteriocin AS-48 in a model meat sausage system. We have examined bacteriocin application, by inoculation with the enterocin AS-48 producer strain Enterococcus faecalis A-48-32 or by adding a semi-purified bacteriocin preparation. AS-48 inhibits proliferation of Staphylococcus aureus in sausages when added at concentrations of 30 or 40μg/g, achieving a significant reduction of 2 and 5.31 log units, respectively, in viable counts (CFU/g) of staphylococci with respect to the untreated control. The presence of bacteriocin also had a moderate negative effect on total lactic acid bacteria. AS-48(+) strain was developed well in the meat mixture, producing sufficient amounts of AS-48 (to a maximum of 76-88 arbitrary units/g) to control growth of staphylococci. The best result was achieved with a bacteriocinogenic strain inoculum of 10(7)CFU/g.

Synergistic effect of enterocin AS-48 in combination with outer membrane permeabilizing treatments against Escherichia coli O157:H7.

Aims:  To determine the effects of outer membrane (OM) permeabilizing agents on the antimicrobial activity of enterocin AS‐48 against Escherichia coli O157:H7 CECT 4783 strain in buffer and apple juice.