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Antonio Gálvez

Publications and source records attributed to Antonio Gálvez.

At least 19 recordsLinked to original sources
PubMed2006

Isolation of bacteriocinogenic Lactobacillus plantarum strains from ben saalga, a traditional fermented gruel from Burkina Faso.

A collection of lactic acid bacteria isolated from ben saalga, a traditional fermented gruel from Burkina Faso, was screened for bacteriocin production. Seven isolates were selected for their broad antimicrobial spectra, which overall included strains of Bacillus cereus, Bacillus licheniformis, Enterococcus faecalis, Listeria innocua, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli and Salmonella enterica. Cluster analysis of RAPD-PCR patterns revealed that six of the isolates represent different strains. The six selected strains were identified as Lactobacillus plantarum by 16S rDNA sequencing, species-specific PCR and multiplex PCR of the recA gene. PCR amplification revealed the presence of genes of the plantaricin cluster described in L. plantarum C11. Among them, strain 5.2.2 carried the largest number of genes from this cluster.

Bacteriocins↗
PubMed2006

Culture-independent analysis of the microbial composition of the African traditional fermented foods poto poto and dégué by using three different DNA extraction methods.

The microbial composition of the traditional fermented foods poto poto (a maize dough from the Rep. of Congo) and dégué (a millet dough from Burkina Faso) was studied by a culture-independent approach using TTGE to separate the amplified target V3 region of the 16S rRNA gene from total microbial community, followed by DNA sequencing and homology search. Three different extraction methods were used. Guanidium thiocyanate-based DNA extraction provided better performance regarding purity and DNA yield, allowing the detection of a higher number of DNA bands by TTGE in poto poto. By contrast, all three methods yielded similar results for dégué samples, indicating that the performance of the DNA extraction method largely depends on the food composition. Sequencing of DNA bands from TTGE gels corresponding to poto poto samples revealed the presence of Lactobacillus gasseri, Enterococcus sp., Escherichia coli, Lactobacillus plantarum/paraplantarum, Lactobacillus acidophilus, Lactobacillus delbrueckii, Bacillus sp., Lactobacillus reuteri and Lactobacillus casei. The following bacteria were identified in dégué: L. gasseri, Enterococcus sp., E. coli, Lactobacillus fermentum, Lactobacillus brevis, and L. casei.

Bacillus↗
PubMed2006

Plasmid profile patterns and properties of pediococci isolated from caper fermentations.

A collection comprising 14 isolates of Pediococcus pentosaceus and one Pediococcus acidilactici from the fermentation of caper fruits was studied. All isolates showed very similar fermentation profiles and produced a limited number of exoenzymes. All isolates carried large plasmids of diverse sizes between 20 and 55 kb, while some also contained smaller plasmids between 10 and 16 kb. Cluster analysis of plasmid profiles revealed four main groups with various degrees of similarities. All amino acid decarboxylation tests were negative, suggesting that pediococci are not involved in generation of biogenic amines. None of the isolates showed hemolytic activity. Antimicrobial resistance tests revealed that all isolates were sensitive to 11 different antimicrobials while being resistant to ciprofloxacin (MIC > or =2 mg/liter) and intrinsically resistant to vancomycin (MIC > or =16 mg/liter) and teicoplanin (MIC > or =16 mg/liter).

Anti-Bacterial Agents↗
PubMed2005

Inhibition of toxicogenic Bacillus cereus in rice-based foods by enterocin AS-48.

The antimicrobial effect of the broad-spectrum bacteriocin enterocin AS-48 against the toxicogenic psychrotrophic strain Bacillus cereus LWL1 has been investigated in a model food system consisting of boiled rice and in a commercial infant rice-based gruel dissolved in whole milk stored at temperatures of 37 degrees C, 15 degrees C and 6 degrees C. In food samples supplemented with enterocin AS-48 (in a concentration range of 20-35 mug/ml), viable cell counts decreased rapidly over incubation time, depending on the bacteriocin concentration, the temperature of incubation and the food sample. Enterotoxin production at 37 degrees C was also inhibited. Heat sensitivity of endospores increased markedly in food samples supplemented with enterocin AS-48: inactivation of endospores was achieved by heating for 1 min at 90 degrees C in boiled rice or at 95 degrees C in rice-based gruel. Activity of enterocin AS-48 in rice gruel was potentiated by sodium lactate in a concentration-dependent way.

Bacillus cereus↗
PubMed2005

Control of Listeria monocytogenes in model sausages by enterocin AS-48.

In this work we describe the control of Listeria monocytogenes CECT 4032 in sausage by adding the enterocin AS-48 producer strains Enterococcus faecalis A-48-32 and Enterococcus faecium S-32-81, and also by adding a semi-purified preparation of the bacteriocin. Addition of preformed AS-48 caused a significant decrease (P<0.01) in the number of viable listeria even at the lowest bacteriocin concentration tested (112 AU/g). At a higher concentration (225 AU/g) listeria were below the detection level (1.99 log units/g) in meat at 3 days of incubation but growth of listeria was observed again after 9 days. For an AS-48 concentration of 450 AU/g, no viable listeria were detected after 6 and 9 days of incubation. When E. faecalis A-48-32 was used as inoculum at approximately 10(7) cfu/g, listeria counts decreased progressively from start of experiment, being below detection level at day 9. The best results were obtained with E. faecium S-32-81, since listeria were undetectable at 6 days of incubation. Bacteriocin concentrations in samples reached concentrations of 60 and 80 AU/g for strains A-48-32 and S-32-81, respectively. These results clearly indicate that AS-48 can be used in the control of L. monocytogenes in sausages.

Animals↗
PubMed2005

Enterocin AS-48RJ: a variant of enterocin AS-48 chromosomally encoded by Enterococcus faecium RJ16 isolated from food.

The bacteriocinogenic strain RJ16 isolated from goat cheese has been identified as Enterococcusfaecium by species-specific PCR, DNA-rRNA hybridization and rDNA sequencing. Purified bacteriocin from strain RJ16 is a carboxypeptidase A-resistant peptide with a molecular mass (7125 Da) very close to the cyclic peptide enterocin AS-48. Bacteriocin from strain RJ16 and AS-48 show identical antibacterial spectra, although the former is slightly less active on strains of Listeria monocytogenes and Bacillus cereus. Producer strains show cross-immunity. PCR amplification of total DNA from strain RJ16 with primers for the AS-48 structural gene and sequencing of the amplified fragment revealed an almost identical sequence (99.5%), except for a single mutation that predicts the change of Glu residue at position 20 of AS-48 to Val. Therefore, bacteriocin produced by E. faecium RJ16 should be considered a variant of AS-48, which we call AS-48RJ. PCR amplification revealed that strain RJ16 contains the complete as-48. gene cluster. Hybridization with probes for as-48 gene cluster revealed a chromosomal location of as-48 genes in strain RJ16, being the first example of a chromosomal location of this bacteriocin trait. Strain RJ16 produced enzymes of interest in food processing (esterase, esterase lipase and phytase activities), and did not decarboxylate amino acids precursors for biogenic amines. Strain RJ16 did not exhibit haemolytic or gelatinase activities, and PCR amplification revealed the lack of genes encoding for known virulence determinants (aggregation substance, collagen adhesin, enterococcal surface protein, endocarditis antigens, as well as haemolysin and gelatinase production). Strain RJ16 was resistant to ciprofloxacin (MIC > 2 mgl(-1)) and levofloxacin (MIC > 4 mgl(-1)) and showed intermediate resistance to nitrofurantoin and erythromycin, but was sensitive to ampicillin, penicillin, streptomycin, gentamicin, rifampicin, chloramphenicol, tetracycline, quinupristin/dalfopristin, vancomycin and teicoplanin. Altogether, results from this study suggest that this broad-spectrum bacteriocin-producing strain may have a potential use in food preservation.

Amino Acid Sequence↗
PubMed2005

Effect of immersion solutions containing enterocin AS-48 on Listeria monocytogenes in vegetable foods.

The effect of immersion solutions containing enterocin AS-48 alone or in combination with chemical preservatives on survival and proliferation of Listeria monocytogenes CECT 4032 inoculated on fresh alfalfa sprouts, soybean sprouts, and green asparagus was tested. Immersion treatments (5 min at room temperature) with AS-48 solutions (25 microg/ml) reduced listeria counts of artificially contaminated alfalfa and soybean sprouts by approximately 2.0 to 2.4 log CFU/g compared to a control immersion treatment in distilled water. The same bacteriocin immersion treatment applied on green asparagus had a very limited effect. During storage of vegetable samples treated with immersion solutions of 12.5 and 25 microg of AS-48/ml, viable listeria counts were reduced below detection limits at days 1 to 7 for alfalfa and soybean sprouts at 6 and 15 degrees C, as well as green asparagus at 15 degrees C. Only a limited inhibition of listeria proliferation was detected during storage of bacteriocin-treated alfalfa sprouts and green asparagus at 22 degrees C. Treatment with solutions containing AS-48 plus lactic acid, sodium lactate, sodium nitrite, sodium nitrate, trisodium phosphate, trisodium trimetaphosphate, sodium thiosulphate, n-propyl p-hydroxybenzoate, p-hydoxybenzoic acid methyl ester, hexadecylpyridinium chloride, peracetic acid, or sodium hypochlorite reduced viable counts of listeria below detection limits (by approximately 2.6 to 2.7 log CFU/g) upon application of the immersion treatment and/or further storage for 24 h, depending of the chemical preservative concentration. Significant increases of antimicrobial activity were also detected for AS-48 plus potassium permanganate and in some combinations with acetic acid, citric acid, sodium propionate, and potassium sorbate.

Bacteriocins↗
PubMed2005

Microbiological study of lactic acid fermentation of Caper berries by molecular and culture-dependent methods.

Fermentation of capers (the fruits of Capparis sp.) was studied by molecular and culture-independent methods. A lactic acid fermentation occurred following immersion of caper berries in water, resulting in fast acidification and development of the organoleptic properties typical of this fermented food. A collection of 133 isolates obtained at different times of fermentation was reduced to 75 after randomly amplified polymorphic DNA (RAPD)-PCR analysis. Isolates were identified by PCR or 16S rRNA gene sequencing as Lactobacillus plantarum (37 isolates), Lactobacillus paraplantarum (1 isolate), Lactobacillus pentosus (5 isolates), Lactobacillus brevis (9 isolates), Lactobacillus fermentum (6 isolates), Pediococcus pentosaceus (14 isolates), Pediococcus acidilactici (1 isolate), and Enterococcus faecium (2 isolates). Cluster analysis of RAPD-PCR patterns revealed a high degree of diversity among lactobacilli (with four major groups and five subgroups), while pediococci clustered in two closely related groups. A culture-independent analysis of fermentation samples by temporal temperature gradient electrophoresis (TTGE) also indicated that L. plantarum is the predominant species in this fermentation, in agreement with culture-dependent results. The distribution of L. brevis and L. fermentum in samples was also determined by TTGE, but identification of Pediococcus at the species level was not possible. TTGE also allowed a more precise estimation of the distribution of E. faecium, and the detection of Enterococcus casseliflavus (which was not revealed by the culture-dependent analysis). Results from this study indicate that complementary data from molecular and culture-dependent analysis provide a more accurate determination of the microbial community dynamics during caper fermentation.

Biodiversity↗
PubMed2004

Quantification of Enterococcus faecalis and Enterococcus faecium in different foods using rRNA-targeted oligonucleotide probes.

The abundance of Enterococcus faecalis and Enterococcus faecium in different Spanish foods was evaluated by using taxon-specific oligonucleotide probes targeted against extracted rRNA. Two satisfactory methods were developed for RNA extraction. Although the yield and purity of total RNA obtained largely depended on the type of food, method 1 should be recommended. The quantitative results obtained with the oligonucleotide probes DB6 for E. faecium and DB8 for E. faecalis showed that these two species accounted for less than 0.5% of the active microflora in all the food samples tested. These results suggest that enterococci form only a minor portion of the microflora of these products.

Animals↗
PubMed2004

Inhibition of Listeria monocytogenes by enterocin EJ97 produced by Enterococcus faecalis EJ97.

Enterocin EJ97 from Enterococcus faecalis EJ97 showed a concentration-dependent antimicrobial activity against Listeria monocytogenes CECT 4032. Activity of enterocin EJ97 against L. monocytogenes CECT 4032 increased slightly at 4 degrees C, and cold-adapted cells did not show any increased resistance. Sensitivity of L. monocytogenes CECT 4032 to enterocin EJ97 was not modified by the addition of sodium benzoate, sodium acetate, NaCl or sodium tripolyphosphate. Anti-listeria activity was enhanced by potassium nitrate, and especially by sodium nitrite at concentrations of 50 microg/ml or above. E. faecalis EJ97 produced bacteriocin activity during cocultivation with L. monocytogenes CECT 4032 at 37 degrees C and also at 15 degrees C, but not at 4 degrees C. Growth of L. monocytogenes CECT 4032 was inhibited by bacteriocin produced during cocultivation at 37 and 15 degrees C, and the degree of inhibition was influenced by the incubation temperature and the initial concentrations of enterococci and listeria. E. faecalis EJ97 also produced bacteriocin during cocultivation in half-skimmed milk, although its capacity to control L. monocytogenes was limited to populations of 10(3) CFU/ml or lower.

Animals↗
PubMed2004

Functional and safety aspects of Enterococci isolated from different Spanish foods.

The incidence and diversity of enterococci in retail food samples of meat, dairy and vegetable origin was investigated. Enterococci were present, at concentrations of 10(1) to 10(4) CFU/g. Fifty selected isolates from food samples grouped in two separate clusters by RAPD analysis. Cluster G1 (72% of the isolates) contained the E. faecium CECT 410T type strain, and also showed a high degree of genetic diversity. Cluster G2 (28% of the isolates) contained the E. faecalis CECT 481T type strain and was genetically more homogeneous. Virulence traits (haemolysin, gelatinase or DNAse activities, or the presence of structural genes cylL, ace, asal and esp) were not detected. All isolates were sensitive to the antibiotics ampicillin, penicillin, gentamicin, streptomycin and chloramphenicol. A high pecentage of isolates were resistant to erythromycin and rifampicin. Many isolates showed intermediate sensitivity to several antibiotics (tetracycline, ciprofloxacin, levofloxacin, or quinupristin/dalfopristin). Vancomycin and teicoplanin resistance was detected in one strain, but vanA, vanB, vanC1, vanC2 or vanC3 genes were not detected. Many of the isolates showed functional properties of food or health relevance. Production of antimicrobial substances was detected in 17 of the isolates, and 14 of them carried structural genes for enterocins A, B and/or P.

Cluster Analysis↗
PubMed2004

Peptide AS-48: prototype of a new class of cyclic bacteriocins.

After the discovery of bacteriocin AS-48, a 70-residue cyclic peptide produced by Enterococcus faecalis subsp. liquefaciens, some naturally-occurring cyclic proteins from bacteria have been reported. AS-48 is encoded by the 68-kb pheromone-responsive plasmid pMB2, and the gene cluster involved in production and immunity has been identified and sequenced. This peptide exerts a bactericidal action on sensitive cells (most of the Gram-positive and some Gram-negative bacteria). Its target is the cytoplasmic membrane, in which it opens pores, leading to the dissipation of the proton motive force and cell death, a mechanism similar to that proposed for the action of defensins or, most generally, cationic antibacterial peptides. This fact, together with its remarkable stability and solubility over a wide pH range, suggest that this bacteriocin could be a good candidate as a natural food preservative. The amino acid composition of purified AS-48 shows the absence of modified or dehydrated residues, making it clearly different from lantibiotics. Bacteriocin AS-48 also differs from defensins in that it does not contain cysteines and consequently no disulfide bridges, which makes is high stability even more remarkable. Composition analysis of AS-48 shows a high proportion of basic to acidic amino acids, conferring to this peptide a strong basic character, with an isoelectric point close to 10.5. Determination of the AS-48 structural gene DNA sequence, together with the sequences of AS-48 protease digestion fragments and mass spectrometry determinations, allowed us to determine unambiguously the cyclic structure of the molecule, being the first example of a posttranslational modification in which a cyclic structure arises from a "head-to-tail" linkage. We have solved the three-dimensional structure of AS-48 in solution, and it consists of a globular arrangement of five alpha-helices enclosing a compact hydrophobic core. Interestingly, the head-to-tail peptide link between Trp-70 and Met-1 lies in the middle of alpha-helix 5, which is shown to have a pronounced effect on the stability of the three-dimensional structure. Analysis of structure-function relationship allowed us to propose models to understand the aspects of the molecular function of AS-48. The purpose of this work is to review recent developments in our understanding about the biochemical and biological characteristics and structure of this unusual type of bacteriocin.

Animals↗
PubMed2004

Biocontrol of psychrotrophic enterotoxigenic Bacillus cereus in a nonfat hard cheese by an enterococcal strain-producing enterocin AS-48.

Bacillus cereus is a food poisoning bacterium of great concern, especially in milk products. In this study, we describe the efficient control of the psychrotrophic and toxigenic strain B. cereus LWL1 in milk and in a nonfat hard cow's cheese by the enterocin AS-48 producer strain Enterococcus faecalis A-48-32 (Bac+). No viable B. cereus cells were detected after 72 h incubation in milk coinoculated with the AS-48-producing strain and B. cereus. Diarrheic toxin production was also markedly inhibited by the Bac+ strain to eightfold lower levels compared with control cultures of B. cereus. In cheeses manufactured by inoculation with a commercial starter (about 6.8 log CFU/ml) and B. cereus (about 4 log CFU/ml), the latter reached 6.27 log CFU/g after 5 days of maturation, and approximately 8 log CFU/g after 15 days. However, in cheeses made from milk inoculated with the starter along with a mixture of E. faecalis-B. cereus (2/1 ratio), counts of B. cereus decreased by approximately 1.0, 2.0, 4.32, and 5.6 log units with respect to control cheeses after 5, 10, 15, and 30 days of ripening, respectively. Growth of E. faecalis A-48-32 was associated with enterocin AS-48 production and persistence in cheese. Interestingly, growth of starter cultures was not affected by the Bac+ strain, and neither was lactic acid production. These results clearly indicate that E. faecalis A-48-32 produced satisfactory amounts of bacteriocin in cheese and support the potential use of AS-48-producing strains as culture adjuncts to inhibit B. cereus during cheese manufacture and ripening.

Animals↗
PubMed2003

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.

Bacteriocins↗
PubMed2003

The genes coding for enterocin EJ97 production by Enterococcus faecalis EJ97 are located on a conjugative plasmid.

Enterococcus faecalis EJ97 produces a cationic bacteriocin (enterocin EJ97) of low molecular mass (5,327.7 Da). The complete amino acid sequence of enterocin EJ97 was elucidated after automated microsequencing of oligopeptides generated by endoproteinase GluC digestion and cyanogen bromide treatment. Transfer of the 60-kb conjugative plasmid pEJ97 from the bacteriocinogenic strain E. faecalis EJ97 to E. faecalis OG1X conferred bacteriocin production and resistance on the recipient. The genetic determinants of enterocin EJ97 were located in an 11.3-kb EcoRI-BglII DNA fragment of pEJ97. This region was cloned and sequenced. It contains the ej97A structural gene plus three open reading frames (ORFs) (ej97B, ej97C, and ej97D) and three putative ORFs transcribed in the opposite direction (orfA, orfB, and orfC). The gene ej97A translated as a 44-amino-acid residue mature protein lacking a leader peptide with no homology to other bacteriocins described so far. The product of ej97B (Ej97B) shows strong homology in its C-terminal domain to the superfamily of bacterial ATP-binding cassette transporters. The products of ej97C (Ej97C) and ej97D (Ej97D) could be proteins with 71 and 64 residues, respectively, of unknown functions and with no significant similarity to known proteins. There are two additional ORFs (ORF1 and ORF6) flanking the ej97 module, which have been identified as a transposon-like structure (tnp). ORF1 shows similarities to transposase of the Lactococcus lactis element ISS1 and is up to 50% identical to IS1216. This is flanked by two 18-bp inverted repeats (IRs) that are almost identical to those of ISS1 and IS1216. ORF6 (resEJ97) shows strong homology to the resolvase of plasmid pAM373 and up to 40 to 50% homology with the recombinase of several multiresistant plasmids and transposons from Staphylococcus aureus and E. faecalis. These data suggest that EJ97 could represent a new class of bacteriocins with a novel secretion mechanism and that the whole structure could be a composite transposon. Furthermore, two additional gene clusters were found: one cluster is probably related to the region responsible for the replication of plasmid pEJ97, and the second cluster is related to the sex pheromone response. These regions showed a high homology to the corresponding regions of the conjugative plasmids pAM373, pPD1, and pAD1 of E. faecalis, suggesting that they have a common origin.

Amino Acid Sequence↗
PubMed2002

The denaturation of circular enterocin AS-48 by urea and guanidinium hydrochloride.

The unfolding thermodynamics of the circular enterocin protein AS-48, produced by Enterococcus faecalis, has been studied. The native structure of the 70-amino-acid-long protein turned out to be extremely stable against heat and denaturant-induced unfolding. At pH 2.5 and low ionic strength, it denatures at 102 degrees C, while at 25 degrees C, the structure only unfolds in 6.3 M guanidinium hydrochloride (GuHCl) and does not unfold even in 8 M urea. A comparison of its thermal unfolding in water and in the presence of urea shows a good correspondence between the two deltaGw(298) values, which are about 30 kJ mol(-1) at pH 2.5 and low ionic strength. The stability of the structure is highly dependent upon ionic strength and so GuHCl acts both as a denaturant and a stabilising agent. This seems to be why the deltaGw(298) value calculated from the unfolding data in GuHCl is twice as high as in the absence of this salt. At least part of the high stability of native AS-48 can almost certainly be put down to its circular organization since other structural features are quite normal for a protein of this size.

Bacteriocins↗
PubMed2002

Inhibition of bacterial growth, enterotoxin production, and spore outgrowth in strains of Bacillus cereus by bacteriocin AS-48.

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 microg/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.

Anti-Bacterial Agents↗