Ligilactobacillus salivarius functionalities, applications, and manufacturing challenges.

Ligilactobacillus salivarius is a lactic acid bacteria that has been gaining attention as a promising probiotic. Numerous strains exhibit functional properties with health benefits such as antimicrobial activity, immunological effects, and the ability to modulate the intestinal microbiota. However, just a small number of them are manufactured at an industrial scale and included in commercial products. The under exploitation of L. salivarius strains that remain in the freezer of companies is due to their incapacity to overcome the environmental stresses induced by production and stabilization processes.The present study summarizes the functionalities and applications of L. salivarius reported to date. It aims also at providing a critical evaluation of the literature available on the manufacturing steps of L. salivarius concentrates, the bacterial quality after each step of the process, and the putative degradation and preservation mechanisms. Here, we highlight the principal issues and future research challenges for improving the production and long-term preservation at the industrial scale of this microorganism, and probably of other probiotics.Key points• L. salivarius beneficial properties and commercialized products.• Production conditions and viability of L. salivarius after stabilization processes.• Prospects for identifying preservation mechanisms to improve L. salivarius stability.

Biological control of mites by xerophile Eurotium species isolated from the surface of dry cured ham and dry beef cecina.

Some meat dry products, including dry cured ham and dry beef cecina, are cured in cellars at moderately cold temperature allowing the growth of a lawn of fungi on their surface. During the curing process, frequently these products became contaminated with fungivore mites of the Acaridae family that feed on fungal mycelium and spores. AIMS: The aim of this article is to study the possible biological control of mites by fungi that form part of the normal microbiota of these meat products. METHODS AND RESULTS: Some yellow/orange pigmented fungi growing on the ham surface decreased the proliferation of mites; therefore, we isolated from ham and cecina xerophilic yellow/orange coloured fungal strains that were identified as members of the genus Eurotium (recently reclassified as Aspergillus section Aspergillus). Using molecular genetic tools, we have identified 158 strains as Eurotium rubrum (Aspergillus ruber), Eurotium repens (Aspergillus pseudoglaucus) and Eurotium chevalieri (Aspergillus chevalieri). Two strains, E. rubrum C47 and E. rubrum C49, showed strong miticidal activity. The toxic compound(s) are associated with the formation of cleistothecia. In synchronized mite development experiments, we observed that all stages of the mite lifecycle were inhibited by the E. rubrum C47 strain. In addition, we searched for miticidal activity in 13 culture collection Eurotium strains isolated from different habitats, and found that only one, Eurotium cristatum NRRL 4222 (Aspergillus cristatus) has a strong miticidal activity. CONCLUSIONS: These fungal strains have proliferated on the surface of ham and cecina for decades, and possibly have acquired miticidal activity as a resistance mechanism against fungivores. SIGNIFICANCE AND IMPACT OF THE STUDY: Biological control of infecting mites by favouring growth of E. rubrun C47, in place of the normal mixed population of Aspergillus and Penicillium, is an attractive approach to control mite infestations.

Towards Q-PCR of pathogenic bacteria with improved electrochemical double-tagged genosensing detection.

A very sensitive assay for the rapid detection of pathogenic bacteria based on electrochemical genosensing has been designed. The assay was performed by the PCR specific amplification of the eaeA gene, related with the pathogenic activity of Escherichia coli O157:H7. The efficiency and selectivity of the selected primers were firstly studied by using standard Quantitative PCR (Q-PCR) based on TaqMan fluorescent strategy. The bacteria amplicon was detected by using two different electrochemical genosensing strategies, a highly selective biosensor based on a bulk-modified avidin biocomposite (Av-GEB) and a highly sensitive magneto sensor (m-GEC). The electrochemical detection was achieved in both cases by the enzyme marker HRP. The assay showed to be very sensitive, being able to detect 4.5 ng microl(-1) and 0.45 ng microl(-1) of the original bacterial genome after only 10 cycles of PCR amplification, when the first and the second strategies were used, respectively. Moreover, the electrochemical strategies for the detection of the amplicon showed to be more sensitive compared with Q-PCR strategies based on fluorescent labels such as TaqMan probes.

In situ DNA amplification with magnetic primers for the electrochemical detection of food pathogens.

A sensitive and selective genomagnetic assay for the electrochemical detection of food pathogens based on in situ DNA amplification with magnetic primers has been designed. The performance of the genomagnetic assay was firstly demonstrated for a DNA synthetic target by its double-hybridization with both a digoxigenin probe and a biotinylated capture probe, and further binding to streptavidin-modified magnetic beads. The DNA sandwiched target bound on the magnetic beads is then separated by using a magneto electrode based on graphite-epoxy composite. The electrochemical detection is finally achieved by an enzyme marker, anti-digoxigenin horseradish peroxidase (HRP). The novel strategy was used for the rapid and sensitive detection of polymerase chain reaction (PCR) amplified samples. Promising resultants were also achieved for the DNA amplification directly performed on magnetic beads by using a novel magnetic primer, i.e., the up PCR primer bound to magnetic beads. Moreover, the magneto DNA biosensing assay was able to detect changes at single nucleotide polymorphism (SNP) level, when stringent hybridization conditions were used. The reliability of the assay was tested for Salmonella spp., the most important pathogen affecting food safety.

Comparing nucleic acid lateral flow and electrochemical genosensing for the simultaneous detection of foodborne pathogens.

Due to the increasing need of rapid tests for application in low resource settings, WHO summarized their ideal features under the acronym ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, Delivered to those who need it). In this work, two different platforms for the rapid and simultaneous testing of the foodborne pathogens E. coli O157:H7 and Salmonella enterica, in detail a nucleic acid lateral flow and an electrochemical magneto-genosensor are presented and compared in terms of their analytical performance. The DNA of the bacteria was amplified by polymerase chain reaction using a quadruple-tagging set of primers specific for E. coli eaeA (151bp) and Salmonella enterica yfiR (375bp) genes. During the amplification, the amplicons were labelled at the same time with biotin/digoxigenin or biotin/fluorescein tags, respectively. The nucleic acid lateral flow assay was based on the use of streptavidin gold nanoparticles for the labelling of the tagged amplicon from E. coli and Salmonella. The visual readout was achieved when the gold-modified amplicons were captured by the specific antibodies. The features of this approach are discussed and compared with an electrochemical magneto-genosensor. Although nucleic acid lateral flow showed higher limit of detection, this strategy was able to clearly distinguish positive and negative samples of both bacteria being considered as a rapid and promising detection tool for bacteria screening.

Virulence and mutation rates of Salmonella typhimurium strains with increased mutagenic strength in a mouse model.

Two strains of Salmonella typhimurium presenting increased mutation rates, either spontaneous or mediated by DNA damage, have been constructed. One of the strains carries a null mutS mutation, while the other harbors plasmid pRW30, which contains the Escherichia coli umuDC operon. The virulence of these strains has been determined by inoculating BALB/c or Swiss mice. The 50% lethal dose of both strains is identical to that obtained for the wild-type. Likewise, the two strains and the wild-type contribute equally to animal death in mixed infections. The frequency of Nal(R) mutants recovered from animals inoculated with either wild-type or MutS(-) cells was not affected by the presence of pRW30. These results indicate that the DNA damage which S. typhimurium cells can suffer during the infectious process by host cell metabolites does not cause induction of the SOS response at levels able to trigger the error-prone DNA repair pathway.

Importance of the galE gene on the virulence of Pasteurella multocida.

The galE gene of Pasteurella multocida has been isolated by complementing galE-defective mutants of Salmonella typhimurium with a plasmid library of this organism. The complete nucleotide sequence of the P. multocida galE gene consists of 1017 nucleotides, encoding a predicted polypeptide of 339 amino acids. The deduced amino acid sequence displayed the highest identity (85%) to the GalE protein of Haemophilus influenzae. However, the gene organization surrounding the galE locus was different from that of H. influenzae. A galE-defective mutant of P. multocida was obtained by replacement of the active galE gene by a copy inactivated in vitro. The resulting galE mutant was highly attenuated as seen in a biological test carried out in a mouse model.

Expression of the Pasteurella multocida ompH gene is negatively regulated by the Fur protein.

The fur gene of Pasteurella multocida has been cloned by complementation of an Escherichia coli fur mutant. The P. multocida fur gene, which encodes a predicted protein of 147 amino acids, displaying the highest identity (89%) with the same protein of Haemophilus influenzae, is negatively regulated by its own product. By construction of a P. multocida fur mutant, it has been demonstrated that the ompH gene, encoding a major structural protein of the outer membrane, presenting high antigenicity power, is negatively regulated by iron and glucose. Furthermore, wild-type and fur-defective cells of P. multocida show the same level of virulence.

Virulence of Pasteurella multocida recA mutants.

In order to determine the role of the RecA protein in the virulence of Pasteurella multocida, a recA mutant was constructed and used in studies of virulence and competition in relation to wild-type strain. To achieve this, firstly, the recA gene was isolated and sequenced, showing an Escherichia coli-like SOS box and encoding a protein of 354 amino acids which has the closest identity with the Haemophilus influenzae RecA protein. Further, the recA mutant was constructed, by inactivating this gene by single recombination of a suicide plasmid containing an internal region of the P. multocida recA gene, and shown to be more sensitive to UV radiation than the parental strain. The P. multocida mutant was slightly attenuated in virulence, as indicated by the LD(50), the time of death of infected animals, and a failure to compete with the wild-type strain in mixed infections. Compared to the parent strain, the mutant had a similar growth rate but a longer lag phase. These data suggest that the diminished virulence of the recA mutant as well as its failure in competition were more a consequence of the long lag phase rather than a direct effect of the inactivation of the recA gene on genes involved in virulence.

Lysine is catabolized to 2-aminoadipic acid in Penicillium chrysogenum by an omega-aminotransferase and to saccharopine by a lysine 2-ketoglutarate reductase. Characterization of the omega-aminotransferase.

The biosynthesis and catabolism of lysine in Penicillium chrysogenum is of great interest because these pathways provide 2-aminoadipic acid, a precursor of the tripeptide delta-L-2-aminoadipyl-L-cysteinyl-D-valine that is an intermediate in penicillin biosynthesis. In vivo conversion of labelled L-lysine into two different intermediates was demonstrated by HPLC analysis of the intracellular amino acid pool. L-lysine is catabolized to 2-aminoadipic acid by an omega-aminotransferase and to saccharopine by a lysine-2-ketoglutarate reductase. In lysine-containing medium both activities were expressed at high levels, but the omega-aminotransferase activity, in particular, decreased sharply when ammonium was used as the nitrogen source. The omega-aminotransferase was partially purified, and found to accept L-lysine, L-ornithine and, to a lesser extent, N-acetyl-L-lysine as amino-group donors. 2-Ketoglutarate, 2-ketoadipate and, to a lesser extent, pyruvate served as amino group acceptors. This pattern suggests that this enzyme, previously designated as a lysine-6-aminotransferase, is actually an omega-aminotransferase. When 2-ketoadipate is used as substrate, the reaction product is 2-aminoadipic acid, which contributes to the pool of this intermediate available for penicillin biosynthesis. The N-terminal end of the purified 45-kDa omega-aminotransferase was sequenced and was found to be similar to the corresponding segment of the OAT1 protein of Emericella (Aspergillus) nidulans. This information was used to clone the gene encoding this enzyme.

Analysis of the expression of the Rhodobacter sphaeroides lexA gene.

The regulation of the Rhodobacter sphaeroides lexA gene has been analyzed using both gel-mobility experiments and lacZ gene fusions. PCR-mediated mutagenesis demonstrated that the second GAAC motif in the sequence GAACN7GAACN7GAAC located upstream of the R. sphaeroides lexA gene is absolutely necessary for its DNA damage-mediated induction. Moreover, mutagenesis of either the first or the third GAAC motif in this sequence reduced, but did not abolish, the inducibility of the R. sphaeroides lexA gene. A R. sphaeroides lexA-defective (Def) mutant has also been constructed by replacing the active lexA gene with an inactivated gene copy constructed in vitro. Crude extracts of the R. sphaeroides lexA(Def) strain are unable to form any protein-DNA complex when added to the wild-type lexA promoter of R. sphaeroides. Likewise, the R. sphaeroides lexA(Def) cells constitutively express the recA and lexA genes. All these data clearly indicate that the lexA gene product is the negative regulator of the R. sphaeroides SOS response. Furthermore, the morphology, growth and viability of R. sphaeroides lexA(Def) cultures do not show any significant change relative to those of the wild-type strain. Hence, R. sphaeroides is so far the only bacterial species whose viability is known not to be affected by the presence of a lexA(Def) mutation.

Molecular characterization of the Acremonium chrysogenum cefG gene product: the native deacetylcephalosporin C acetyltransferase is not processed into subunits.

Constructions starting at each of the three in-frame ATG codons of the Acremonium chrysogenum cefG gene (Met1, Met46 and Met60) were expressed in Escherichia coli, obtaining proteins of 49, 44 and 43 kDa, respectively. All three proteins showed deacetylcephalosporin C (DAC) acetyltransferase activity. The native A. chrysogenum DAC acetyltransferase was purified to electrophoretic homogeneity by immunoaffinity chromatography. It showed a molecular mass of 50 kDa by filtration in calibrated Sephadex G-75 SF or Superose 12 (FPLC) columns. The N-terminal end of the pure DAC acetyltransferase was Met-Leu-Pro-Ser-Ala-Gln-Val-Ala-Arg-Leu, which matched perfectly the deduced amino acid sequence starting at Met1. The putative alpha- and beta-subunits of DAC acetyltransferase were also obtained in E. coli but showed no enzymic activity either separately or in combination. Immunoblotting (Western) analysis revealed that the 50 kDa DAC acetyltransferase showed high protein levels in A. chrysogenum cultures at 72 and 96 h and decreased sharply thereafter, but in all cases no detectable processing of the enzyme into subunits was found. Three different A. chrysogenum strains (including the wild-type Brotzu strain and two high-cephalosporin-producing mutants) showed the same unprocessed 50 kDa DAC acetyltransferase. The non-producer mutant ATCC 20371 showed no DAC acetyltransferase protein band but formed a normal transcript of 1.4 kb.

Production of Paracoccidioides brasiliensis exoantigens for immunodiffusion tests.

Growth curves of the yeast form of Paracoccidioides brasiliensis B-339 based on total and viable cell counts were determined. Crude culture filtrate antigens were obtained after 7, 10, 15, 20, 25, and 30 days of incubation. Different patterns of proteins were obtained by affinity chromatography on Sepharose 4B-immunoglobulin G complex made with immunoglobulin G from patients with paracoccidioidomycosis, with subsequent analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scanning densitometry. Three major proteins were excreted during the time course of a 30-day culture: a doublet at 20 to 21 kilodaltons (kDa) and molecules of 43 and 52 kDa. The 43-kDa antigen was present throughout the growth period, and its level reached a peak on days 15 to 20 and then decreased considerably toward day 30. The antigenic preparations collected on days 7, 10, 15, and 20 gave better reactions in immunodiffusion tests than those collected on days 25 and 30. The 7-day exoantigen gave a sensitivity of 97.1% and specificity of 100% on immunodiffusion. The main line of precipitation had a very high intensity, showing a total identity with that of a previously purified glycoprotein of 43 kDa. A 7-day crude exoantigen displayed a high level of sensitivity and specificity, being reproducible from batch to batch and retaining its activity for years when kept lyophilized. A protocol is recommended for the production of a stable diagnostic antigen to be used in immunodiffusion tests for paracoccidioidomycosis.

Cloning and characterization of the gene cahB encoding a cephalosporin C acetylhydrolase from Acremonium chrysogenum.

An important problem during the production of cephalosporin C by Acremonium chrysogenum is the hydrolysis of cephalosporin C to deacetylcephalosporin C, since the latter compound has no commercial value and represents an unwanted side-product. Characterization of the enzymatic process that gives rise to deacetylcephalosporin C will help to avoid the accumulation of this side-product. An extracellular cephalosporin C acetylhydrolase (CPC-AH) from Acremonium chrysogenum C10 was purified to near homogeneity. This enzyme had a molecular mass of 31 kDa, a pl of 4.0, and showed relatively little affinity for cephalosporin C (Km 33.7 mM). We sequenced twenty amino acids at the amino-terminal end; a probe based on this sequence was then used to clone the cephalosporin acetylhydrolase (cahB) gene. cahB encodes a pre-protein of 383 amino acids with a deduced molecular mass of 38,228 Da. The sequenced 20 amino acids of the purified protein corresponded to amino acids 107-127 deduced from the cahB gene, suggesting that mature CPC-AH results from processing of the pre-protein after Gln-106. cahB is located on chromosome VIII of A. chrysogenum C10 and is not linked to the cephalosporin early or late gene clusters. It is expressed as a single 1.4-kb transcript after 72 h of cultivation. Expression declined in batch cultures after 120 h even though CPC-AH activity was observed until 144 h. The CPC-AH protein resembles other wide-spectrum substrate fungal esterases that are functionally related to serine proteases. The cahB gene does not seem to be related to the cephalosporin biosynthesis genes and encodes an esterase active on several substrates in addition to cephalosporin C.

LexA-binding sequences in Gram-positive and cyanobacteria are closely related.

The lexA gene of the cyanobacterium Anabaena sp. strain PCC7120 has been cloned by PCR amplification with primers designed after TBLASTN analysis of its genome sequence using the Escherichia coli LexA sequence as a probe. After over-expression in E. coli and subsequent purification, footprinting experiments demonstrated that the Anabaena LexA protein binds to the sequence TAGTACTAATGTTCTA, which is found upstream of its own coding gene. Directed mutagenesis and sequence comparison of promoters of other Anabaena genes, as well as those of several cyanobacteria, allowed us to define the motif RGTACNNNDGTWCB as the LexA box in this bacterial phylum. Substitution of a single nucleotide in this motif present in the Anabena lexA promoter is sufficient to enable it to bind the Bacillus subtilis LexA protein. These data indicate that Cyanobacteria and Gram-positive bacteria are phylogenetically closely related.

Peritonitis with continuous ambulatory peritoneal dialysis and continuous cycling peritoneal dialysis.

The frequency and cause of peritonitis in 18 children receiving continuous ambulatory peritoneal dialysis (CAPD) and nine children receiving continuous cycling peritoneal dialysis (CCPD) are described. Cumulative CAPD and CCPD experience demonstrated 58 episodes of peritonitis in 294 patient treatment months (one case per 5.1 patient treatment months). Total hospitalization for the treatment of peritonitis was 0.18 days per patient treatment month. Life table analysis revealed no significant difference in the peritonitis-free "survival" between the two modalities. Gram-negative organisms accounted for a significantly increased percentage of the peritonitis in CAPD compared with CCPD (65% vs 17%) (P less than 0.001). Thirty-seven percent of the gram-negative infections in the CAPD population were in children with nephrostomies. Factors predisposing to peritonitis were identified in 76% of cases occurring with CAPD. Peritonitis remains the major contributor to the morbidity associated with peritoneal dialysis, regardless of the technique. The resultant frequency of hospitalization is not prohibitive. Attention to the "high-risk" pediatric patient and education directed at several well-recognized predisposing factors may yield improved results.