Response of sensitive and resistant Listeria monocytogenes strains against bacteriocins produced by different Enterococcus spp. strains.

Listeria monocytogenes is a relevant foodborne pathogen causing invasive listeriosis in humans, a disease with high mortality rates. Its ubiquity and growth characteristics enable this pathogen to survive harsh food processing environments. The addition of bacteriocins, antimicrobial peptides ribosomally synthesized by certain bacteria, appears as a natural alternative to control this pathogen in food. However, the emergence of L. monocytogenes strains resistant to the inhibitory action of bacteriocins has been detected. In order to analyse the development of this resistance, different properties of L. monocytogenes strains susceptible to bacteriocins (strains 01/155, 99/287 and 99/267) and their respective resistant isolates (strains 01/155B6R, 99/287B6R, 99/286C1R, 99/287 Mo1R, 99/287 M1bR, 99/287 M2dR, 99/267B6R), were compared in this work. Differences were analysed in: a) growth of the pathogen strains in direct contact with bacteriocin solution, in co-cultures with the producing strain, or with different sugars; b) response to antibiotics typically used against listeriosis; c) changes in cell morphology, observed by transmission or scanning electron microscopy; d) expression of mobility and haemolysin activity, two of L. monocytogenes main virulence factors; and e) biofilm formation ability. For all the isolates, the acquired resistance was permanent and crossed between the different bacteriocins under study. An inhibitory effect was observed for resistant strains only when they were grown in mixed culture with any of the bacteriocin-producing strains, with an acidified medium as additional growth stress. In all cases, the decrease in viability was lower for resistant strains and followed a particular profile for each strain. The variation of sugar substrate influenced resistant variants growth ability, with a more pronounced difference in the medium supplemented with glucose. Susceptibility to antibiotics was similar or higher for resistant variants, while neither the mobility nor the haemolytic activity presented differences among resistant or susceptible strains. Finally, the resistant variants showed a greater capacity to form biofilms, although this effect was reversed when grown in the presence of bacteriocins. Each resistant isolate had a particular behaviour pattern, and the acquisition of resistance appeared to be strain and bacteriocin dependent. These results contribute to the knowledge of L. monocytogenes bacteriocin-resistance development, which is essential to favour the use of these peptides as biopreservatives.

Immobilization of glucosyltransferase from Erwinia sp. using two different techniques.

Two different techniques of glucosyltransferase immobilization were studied for the conversion of sucrose into isomaltulose. The optimum conditions for immobilization of Erwinia sp. glucosyltransferase onto Celite 545, determined using response surface methodology, was pH 4.0 and 170 U of glucosyltransferase/g of Celite 545. Using this conditions more than 60% conversion of sucrose into isomaltulose can be obtained. The immobilization of glucosyltransferase was also studied by its entrapment in microcapsules of low-methoxyl pectin and fat (butter and oleic acid). The non-lyophilized microcapsules of pectin, containing the enzyme and fat, showed higher glucosyltransferase activity, compared with lyophilized microcapsules containing enzyme plus fat, and also lyophilized microcapsules containing enzyme without fat addition. The non-lyophilized microcapsules of pectin containing the glucosyltransferase and fat, converted 30% of sucrose into isomaltulose in the first batch. However the conversion decreased to 5% at the 10th batch, indicating inactivation of the enzyme.

Properties of different lactic acid bacteria isolated from Apis mellifera L. bee-gut.

Eight strains belonging to Lactobacillus spp. and five to Enterococcus spp. were isolated from the gut of worker Apis mellifera L. bees. Studies based on 16S rRNA sequencing revealed that AJ5, IG9, A15 and CRL1647 strains had a 99% identity with Lactobacillus johnsonii, while SM21 showed a 99% similarity with Enterococcus faecium. L. johnsonii CRL1647, AJ5 and IG9 were high lactic acid producers (values were between 177 and 275 mM), and in vitro they inhibited different human food-borne pathogens and Paenibacillus larvae, the American foulbrood agent. This bacterium was the most sensitive to the lactic acid effect being inhibited by 44 mM of this metabolite. L. johnsonii CRL1647, AJ5 and IG9 also presented important surface properties. These cells showed between 77% and 93% of auto-aggregation. The preliminary study of the chemical nature of the aggregating factors revealed that the molecules involved in the surface of each L. johnsonii strain were quite complex; and something of a peptidic nature was mainly involved. E. faecium SM21 produced bacteriocin-like compounds with anti-Listeria effects. Furthermore, a band close to 6.0-7.5 kDA was detected by SDS-PAGE studies, and the entA, B and P structural genes were amplified by PCR reactions. For the first time, bee-gut associated L. johnsonii and E. faecium strains have been isolated, identified, cultivated and some of their functional properties reported.

Effect of bacterial metabolites on microsporidian Nosema ceranae and on its host Apis mellifera.

Nosemosis, a disease caused by a microsporidian infection, is one of the most frequently observed parasitic pathologies affecting adult honeybees. Presently, Nosema ceranae seems to be the main microsporidian infection in Apis mellifera. The antibiotic fumagillin is the only compound available to treat Nosema diseases; however, it is no longer licensed in most EU member states; therefore, the need to identify new molecules/substances prevails. The intent of this paper is to test bacterial metabolites by Bacillus and Enterococcus strains, isolated from bee midgut and honey. The toxicity on bees and the antiparasitic activity on N. ceranae were assessed under laboratory conditions. Results did not yield toxicity for the administered surfactin or bacteriocin concentrations. Spores exposed to direct contact with a particular surfactin revealed a significant infectivity reduction when inoculated on bees. This surfactin, administered ad libitum from the individuals' emergence, led to a significant reduction in parasitosis development when bees were infected with untreated spores 7 days postemergence. Based on the results obtained, one of the surfactins is herein postulated as a molecule capable of reducing N. ceranae development, acting either by direct exposure to purified spores or incorporated into the digestive tract of the bee.

Enterococcus faecium isolated from honey synthesized bacteriocin-like substances active against different Listeria monocytogenes strains.

Four Enterococcus faecium strains, isolated from honeycombs (C1 and M2d strains) and feral combs (Mori1 and M1b strains) secreted antimicrobial substances active against fourteen different Listeria spp. strains. The antimicrobial compound(s) present in the cell free supernatant were highly thermostable (121 degrees C for 15 min) and inactivated by proteolytic enzymes, but not by alpha-amylase and lipase, thus suggesting a peptidic nature. Since the structural bacteriocin gene determinants of enterocins A and B were PCR amplified from the four E. faecium isolates, only the bacteriocin produced by strain C1 was further characterized: it showed a broad band of approximately 4.0-7.0 kDa in SDS-PAGE and was bactericidal (4 log decrease) against L. monocytogenes 99/287. L. monocytogenes 99/287R, a clone spontaneously resistant to the enterocin produced by E. avium DSMZ17511 (ex PA1), was not inhibited by the enterocin-like compounds produced by strain C1. However, it was inhibited in mixed culture fermentations by E. faecium C1 and a bacteriostatic effect was observed. The bacteriocin-producer Enterococcus strains were not haemolytic; gelatinase negative and sensitive to vancomycin and other clinically relevant antibiotics.