Long-term safety of early consumption of Lactobacillus fermentum CECT5716: A 3-year follow-up of a randomized controlled trial.

Lactobacillus fermentum CECT5716 is a probiotic strain originally isolated from human breast milk. Previous clinical studies in infants showed that the early administration of a milk formula containing this probiotic strain was safe and may be useful for the prevention of community-acquired infections. This is a 3-year follow-up study aimed at evaluating the long-term effects produced by the early consumption of an infant formula supplemented with L. fermentum CECT5716 (experimental group, EG) compared with a control formula without the probiotic (control group, CG). The infants included in this follow-up study had previously completed a 5-month randomized double-blind controlled trial (from 1 to 6 months of age), where the safety and tolerance of the probiotic formula was evaluated. The main outcome of the follow-up study was the growth of the children. The secondary outcomes included the incidence of infectious and non-infectious diseases, parameters related with intestinal function and faecal microbiota. At 3 years, the mean values of weight, length and head circumference were similar in children of the EG compared with those of the CG. No differences were observed in the incidence of infectious and non-infectious diseases or disorders related with intestinal function. The pattern of faecal microbiota was also similar between both groups. In conclusion, this 3-year study shows that the early administration of the probiotic of L. fermentum CECT5716 in an infant formula is safe and it does not produce measurable differences in children compared with a control formula.

Efficacy of Bifidobacterium breve CECT7263 for infantile colic treatment: an open-label, parallel, randomised, controlled trial.

Infantile colic is a prevalent condition characterised by excessive crying with no effective treatment available. We aimed to evaluate the efficacy of Bifidobacterium breve CECT7263 and a combination of this and Lactobacillus fermentum CECT5716 versus simethicone in reducing the daily time spent crying in colicky infants. A multicentre randomised, open-label, parallel, controlled trial of 28 days was performed in 150 infants who were diagnosed with colic according to the Rome III criteria and who randomly received simethicone (80 mg/day; Simethicone group), B. breve CECT7263 (2×108 cfu/day, Bb group), or a combination of L. fermentum CECT5716 and B. breve CECT7263 (1×108 cfu/day per strain, Bb+Lf group). The main outcomes were minutes of crying per day and the percentage of reduction in daily crying from baseline. Data were analysed per intention to treat. All treatments significantly decreased the daily crying time at the end of the intervention (P-time <0.001). However, the infants in the Bb group had significantly decreased crying time from the first week of the study (P<0.05), whereas the Bb+Lf group and the simethicone group had significantly decreased crying time from the second week (P<0.05). The percentage of reduction in the minutes of crying from baseline in the Bb group was significantly higher than that in the Simethicone group every week of the intervention (-40.3 vs -27.6% at 1-week; -59.2 vs -43.2% at 2-weeks; -64.5 vs -53.5% at 3-week and -68.5 vs -59.5% at 4-weeks, P<0.05). Additionally, in the Bb group, infants had better night sleep, and parents reported a more positive mood at the end of the intervention. All the products used in the study were safe and well tolerated. In conclusion, the breastmilk-isolated probiotic strain B. breve CECT7263 is a safe and effective treatment for infantile colic, presenting an earlier and more robust effect than the reference prescribed drug, simethicone.

Effects of Lactobacillus fermentum CECT5716 Lc40 on infant growth and health: a randomised clinical trial in nursing women.

The breast milk microbiota has been described as a source of bacteria for infant gut colonisation. We studied the effect of Lactobacillus fermentum CECT5716 (Lc40) on growth and infection incidence of the infants, when the probiotic is administrated to the mothers. Moreover, whether such effects might depend on the interaction between the mother or infant microbiota and the probiotic administration. A total of 291 mother-infant pairs were studied for 16 weeks in a randomised double-blinded placebo-controlled multicentre trial. The Lc40 group (n=139) received 1 capsule/day containing 3×109 cfu Lc40; the control group (n=152) received 1 placebo (maltodextrin) capsule/day. A positive and significant correlation of the Staphylococcus load between breast milk and infant faeces was only observed in control group. Additionally, the weight z-score of the infants whose mothers had higher values of Lactobacillus in their breast milk were significantly higher for the Lc40 group. We observed a significant lower incidence of conjunctivitis in the infants whose mothers received Lc40. A higher load of Staphylococcus in infant faeces significantly increased the risk of respiratory infections. Such incidence, under an absent or low Staphylococcus load in the faeces, was significantly 36 times higher in the infants in the control group than in the infants in the Lc40 group. However, the protective effect of Lc40 was gradually reduced as the Staphylococcus load of the milk increased. The administration of Lc40 to nursing women might influence infant growth and health but it seems to depend on its interactions with mother or infant microbiota. Registered in the US Library of Medicine (www.clinicaltrials.gov): NCT02203877.

Characterization and lysine control of expression of the lys1 gene of Penicillium chrysogenum encoding homocitrate synthase.

A 2071-bp DNA fragment, containing a gene (lys1) encoding a protein that showed 71.1% identical amino acids with the Yarrowia lipolytica homocitrate synthase and 71.7% identity with the Saccharomyces cerevisiae homologous enzyme, was cloned from a genomic library of Penicillium chrysogenum. The lys1 gene contained three introns and encoded a protein of 474 amino acids with a deduced molecular mass of 52kDa. lys1 was located in chromosome II (9.6Mb) in the wild-type P. chrysogenum NRRL 1951, whereas it hybridized with chromosome III (7.5Mb) in the high penicillin production strain AS-P-78. The lys1 gene is transcribed as a monocistronic transcript of 2.0kb. Levels of the lys1 transcript were high in P. chrysogenum Wis 54-1255 cultures in defined penicillin production medium at 24 and 48h, coinciding with the rapid growth phase, but clearly decreased during the penicillin production phase, suggesting that alpha-aminoadipic acid formation for penicillin biosynthesis may be limited at the homocitrate synthase level. Expression of lys1 was partially repressed by high concentrations of lysine in the culture medium, but lysine repression seems to be a weak mechanism of control of the lysine pathway as compared to lysine inhibition of homocitrate synthase.

Cloning and characterization of a beta-galactosidase encoding region in Lactobacillus coryniformis CECT 5711.

A chromosomal DNA fragment of 7.8 kb from Lactobacillus coryniformis CECT 5711 was cloned in Escherichia coli K-12 and was found to express a functional beta-galactosidase. Nucleotide sequence analysis showed that this fragment contained two partially overlapping genes, the lacL (1,881 bp) and the lacM (960 bp), that encode the subunits of a heterodimeric beta-galactosidase, with estimated molecular masses of 72,129 and 35,233 Da, respectively. Other three incomplete open reading frames showing homology to another beta-galactosidase, an alpha-galactosidase, and a galactokinase, respectively, were also found. The L. coryniformis beta-galactosidase was overproduced in E. coli by using an isopropyl-beta-D: -thiogalactopyranoside (IPTG) expression system. Two new proteins with an estimated M (r) s of approximately 72,000 and 35,000 appeared upon induction with IPTG, and extracts of the recombinant E. coli strain showed beta-galactosidase activity.

Overexpression of the lys1 gene in Penicillium chrysogenum: homocitrate synthase levels, alpha-aminoadipic acid pool and penicillin production.

Homocitrate synthase activity (encoded by the lys1 gene) catalyzes the first step of the lysine and penicillin pathway and is highly sensitive to feedback regulation by L-lysine. The transcript levels of the lys1 gene and the homocitrate synthase activity are high during the growth phase and decrease during the antibiotic production phase, except in the high penicillin producer strain AS-P-99 which maintained high levels of homocitrate synthase activity in cultures at 96 h and 120 h. The lys1 gene was overexpressed in Penicillium chrysogenum using additional copies of lys1 with its own promoter or under the control of the pcbC promoter in either autonomously replicating or integrative vectors. Transformants containing 3 to 32 additional copies of the lys1 gene were selected. Some of these transformants, particularly Ti-C4 (integrative) and TAR-L9 (with autonomously replicating plasmids) showed very high levels of lys1 transcript and, in the case of TAR-L9, high levels of homocitrate synthase activity in cultures of 120 h. However, these transformants did not show increased alpha-aminoadipate or lysine pools. A mutant P. chrysogenum L-G- disrupted in the lys2 gene (therefore lacking the lysine branch of the pathway) showed increased alpha-aminoadipate levels and produced higher levels of penicillin than non-disrupted control strains. Overexpression of the lys1 gene in the L-G- mutant resulted in high homocitrate synthase levels but no additional increase of the alpha-aminoadipate pool or penicillin production levels. These results suggest that after amplification of the homocitrate synthase levels there are other limiting steps in the common stem of the lysine and penicillin pathways.

Intrachromosomal recombination after targeted monocopy integration in Penicillium chrysogenum: stabilization of the direct repeats to prevent loss of the inserted gene.

Monocopy systems obtained by targeted integration at the pyrG locus of P. chrysogenum led to the formation of unstable direct repeats in the genome. A previously isolated pyrG mutant was sequenced and the mutation was found to be located at nucleotide position 665 of the pyrG gene. A different pyrG mutation was introduced in vitro at the BamHI site of this gene. Recombination products arising from monocopy systems using the bleomycin/phleomycin resistance gene (ble) as a model were studied to elucidate the intrachromosomal recombination mechanisms. Experimental results showed that both gene conversion and deletion events occurred spontaneously at the integration site. Gene conversion products were obtained at a frequency of one in 3.4x10(4) viable transformant spores. When gene conversion occurred, the inserted exogenous gene was retained and was flanked by rearranged direct repeats of the pyrG gene, each containing at least one pyrG mutation. Deletion events resulted in the loss at high frequency of the inserted exogenous gene. Genetic stabilization of a monocopy system was obtained when both pyrG repeats (formed at the targeted integration site) contained at least one identical mutation, since in this case further recombinations can be easily counter-selected.

The specific transport system for lysine is fully inhibited by ammonium in Penicillium chrysogenum: an ammonium-insensitive system allows uptake in carbon-starved cells.

The regulation exerted by ammonium and other nitrogen sources on amino acid utilization was studied in swollen spores of Penicillium chrysogenum. Ammonium prevented the L-lysine, L-arginine and L-ornithine utilization by P. chrysogenum swollen spores seeded in complete media, but not in carbon-deficient media. Transport of L-[14C]lysine into spores incubated in presence of carbon and nitrogen sources was fully inhibited by ammonium ions (35 mM). However, in carbon-derepressed conditions (growth in absence of sugars, with amino acids as the sole carbon source) L-[14C]lysine transport was only partially inhibited. Competition experiments showed that L-lysine (1 mM) inhibits the utilization of L-arginine, and vice versa, L-arginine inhibits the L-lysine uptake. High concentrations of L-ornithine (100 mM) prevented the L-lysine and L-arginine utilization in P. chrysogenum swollen spores. In summary, ammonium seems to prevent the utilization of basic amino acids in P. chrysogenum spores by inhibiting the transport of these amino acids through their specific transport system(s), but not through the general amino acid transport system that is operative under carbon-derepression conditions.

Gene targeting in Penicillium chrysogenum: disruption of the lys2 gene leads to penicillin overproduction.

Two strategies have been used for targeted integration at the lys2 locus of Penicillium chrysogenum. In the first strategy the disruption of lys2 was obtained by a single crossing over between the endogenous lys2 and a fragment of the same gene located in an integrative plasmid. lys2-disrupted mutants were obtained with 1.6% efficiency when the lys2 homologous region was 4.9 kb, but no homologous integration was observed with constructions containing a shorter homologous region. Similarly, lys2-disrupted mutants were obtained by a double crossing over (gene replacement) with an efficiency of 0.14% by using two lys2 homologous regions of 4.3 and 3.0 kb flanking the pyrG marker. No homologous recombination was observed when the selectable marker was flanked by short lys2 homologous DNA fragments. The disruption of lys2 was confirmed by Southern blot analysis of three different lysine auxotrophs obtained by a single crossing over or gene replacement. The lys2-disrupted mutants lacked alpha-aminoadipate reductase activity (encoded by lys2) and showed specific penicillin yields double those of the parental nondisrupted strain, Wis 54-1255. The alpha-aminoadipic acid precursor is channelled to penicillin biosynthesis by blocking the lysine biosynthesis branch at the alpha-aminoadipate reductase level.

Characterization of the lys2 gene of Penicillium chrysogenum encoding alpha-aminoadipic acid reductase.

A DNA fragment containing a gene homologous to LYS2 gene of Saccharomyces cerevisiae was cloned from a genomic DNA library of Penicillium chrysogenum AS-P-78. It encodes a protein of 1409 amino acids (Mr 154859) with strong similarity to the S. cerevisiae (49.9% identity) Schizosaccharomyces pombe (51.3% identity) and Candida albicans (48.12% identity) alpha-aminoadipate reductases and a lesser degree of identity to the amino acid-activating domains of the non-ribosomal peptide synthetases, including the alpha-aminoadipate-activating domain of the alpha-aminoadipyl-cysteinyl-valine synthetase of P. chrysogenum (12.4% identical amino acids). The lys2 gene contained one intron in the 5'-region and other in the 3'-region, as shown by comparing the nucleotide sequences of the cDNA and genomic DNA, and was transcribed as a 4.7-kb monocistronic mRNA. The lys2 gene was localized on chromosome III (7.5 Mb) in P. chrysogenum AS-P-78 and on chromosome IV (5.6 Mb) in strain P2, whereas the penicillin gene cluster is known to be located in chromosome I in both strains. The lys2-encoded protein is a member of the aminoacyladenylate-forming enzyme family with a reductase domain in its C-terminal region.

Intrachromosomal recombination between direct repeats in Penicillium chrysogenum: gene conversion and deletion events.

Recombination between direct repeats has been studied in Penicillium chrysogenum using strain TD7-88 (lys- py+), which contains two inactive copies of the lys2 gene separated by 4.5 kb of DNA (including the pyrG gene) in its genome. Gene conversion leading to products with the lys+ pyr+ phenotype was observed at a frequency of 1 in 3.2x10(3) viable spores. Two types of deletion events giving rise to lys+ pyr- and lys- pyr- phenotypes were obtained with different frequencies. Southern analysis revealed that gene conversion occurs mainly as a result of crossing over events that remove the BamHI frameshift mutation present in one of the repeats. In lys- pyr- recombinants, the deletion events do not affect the frameshift mutation in the BamHI site, while lys+ pyr- recombinants showed repair of the BamHI frameshift mutation and the genotype of the parental non-disrupted strain was restored. In summary, deletion events in P. chrysogenum tend to favor the restoration of the phenotype and genotype characteristic of the parental non-disrupted strain.

Targeted inactivation of the mecB gene, encoding cystathionine-gamma-lyase, shows that the reverse transsulfuration pathway is required for high-level cephalosporin biosynthesis in Acremonium chrysogenum C10 but not for methionine induction of the cephalosporin genes.

Targeted gene disruption efficiency in Acremonium chrysogenum was increased 10-fold by applying the double-marker enrichment technique to this filamentous fungus. Disruption of the mecB gene by the double-marker technique was achieved in 5% of the transformants screened. Mutants T6 and T24, obtained by gene replacement, showed an inactive mecB gene by Southern blot analysis and no cystathionine-gamma-lyase activity. These mutants exhibited lower cephalosporin production than that of the control strain, A. chrysogenum C10, in MDFA medium supplemented with methionine. However, there was no difference in cephalosporin production between parental strain A. chrysogenum C10 and the mutants T6 and T24 in Shen's defined fermentation medium (MDFA) without methionine. These results indicate that the supply of cysteine through the transsulfuration pathway is required for high-level cephalosporin biosynthesis but not for low-level production of this antibiotic in methionine-unsupplemented medium. Therefore, cysteine for cephalosporin biosynthesis in A. chrysogenum derives from the autotrophic (SH(2)) and the reverse transsulfuration pathways. Levels of methionine induction of the cephalosporin biosynthesis gene pcbC were identical in the parental strain and the mecB mutants, indicating that the induction effect is not mediated by cystathionine-gamma-lyase.

Subcellular localization of the homocitrate synthase in Penicillium chrysogenum.

There are conflicting reports regarding the cellular localization in Saccharomyces cerevisiae and filamentous fungi of homocitrate synthase, the first enzyme in the lysine biosynthetic pathway. The homocitrate synthase (HS) gene (lys1) of Penicillium chrysogenum was disrupted in three transformants (HS(-)) of the Wis 54-1255 pyrG strain. The three mutants named HS1(-), HS2(-) and HS3(-) all lacked homocitrate synthase activity and showed lysine auxotrophy, indicating that there is a single gene for homocitrate synthase in P. chrysogenum. The lys1 ORF was fused in frame to the gene for the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria. Homocitrate synthase-deficient mutants transformed with a plasmid containing the lys1-GFP fusion recovered prototrophy and showed similar levels of homocitrate synthase activity to the parental strain Wis 54-1255, indicating that the hybrid protein retains the biological function of wild-type homocitrate synthase. Immunoblotting analysis revealed that the HS-GFP fusion protein is maintained intact and does not release the GFP moiety. Fluorescence microscopy analysis of the transformants showed that homocitrate synthase was mainly located in the cytoplasm in P. chrysogenum; in S. cerevisiae the enzyme is targeted to the nucleus. The control nuclear protein StuA was properly targeted to the nucleus when the StuA (targeting domain)-GFP hybrid protein was expressed in P. chrysogenum. The difference in localization of homocitrate synthase between P. chrysogenum and S. cerevisiae suggests that this protein may play a regulatory function, in addition to its catalytic function, in S. cerevisiae but not in P. chrysogenum.

The cefT gene of Acremonium chrysogenum C10 encodes a putative multidrug efflux pump protein that significantly increases cephalosporin C production.

Transcriptional analysis of the region downstream of the pcbAB gene (which encodes the alpha-aminoadipyl-cysteinyl-valine synthetase involved in cephalosporin synthesis) of Acremonium chrysogenum revealed the presence of two different transcripts corresponding to two new ORFs. ORF3 encodes a putative D-hydroxyacid dehydrogenase and cefT (for transmembrane protein) encodes a multidrug efflux pump belonging to the Major Facilitator Superfamily (MFS) of membrane proteins. The CefT protein has 12 transmembrane segments (TMS) and contains motifs A, B, C, D2 and G characteristic of the Drug:H(+) antiporter 12-TMS group of the major facilitator superfamily. The CefT protein confers resistance to some toxic organic acids, including isovaleric acid and phenylacetic acid. Targeted inactivation of ORF3 and cefT by gene replacement showed that they are not essential for cephalosporin biosynthesis. However, amplification of the cefT gene results in increments of up to 100% in cephalosporin production in the A. chrysogenum C10 strain. Amplification of a truncated form of the cefT insert did not lead to cephalosporin overproduction. It seems that the CefT protein is involved in cephalosporin export from A. chrysogenum or in transmembrane signal transduction, and that there are redundant systems involved in cephalosporin export.

Conversion of pipecolic acid into lysine in Penicillium chrysogenum requires pipecolate oxidase and saccharopine reductase: characterization of the lys7 gene encoding saccharopine reductase.

Pipecolic acid is a component of several secondary metabolites in plants and fungi. This compound is useful as a precursor of nonribosomal peptides with novel pharmacological activities. In Penicillium chrysogenum pipecolic acid is converted into lysine and complements the lysine requirement of three different lysine auxotrophs with mutations in the lys1, lys2, or lys3 genes allowing a slow growth of these auxotrophs. We have isolated two P. chrysogenum mutants, named 7.2 and 10.25, that are unable to convert pipecolic acid into lysine. These mutants lacked, respectively, the pipecolate oxidase that converts pipecolic acid into piperideine-6-carboxylic acid and the saccharopine reductase that catalyzes the transformation of piperideine-6-carboxylic acid into saccharopine. The 10.25 mutant was unable to grow in Czapek medium supplemented with alpha-aminoadipic acid. A DNA fragment complementing the 10.25 mutation has been cloned; sequence analysis of the cloned gene (named lys7) revealed that it encoded a protein with high similarity to the saccharopine reductase from Neurospora crassa, Magnaporthe grisea, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. Complementation of the 10.25 mutant with the cloned gene restored saccharopine reductase activity, confirming that lys7 encodes a functional saccharopine reductase. Our data suggest that in P. chrysogenum the conversion of pipecolic acid into lysine proceeds through the transformation of pipecolic acid into piperideine-6-carboxylic acid, saccharopine, and lysine by the consecutive action of pipecolate oxidase, saccharopine reductase, and saccharopine dehydrogenase.