Adaptation to aerobic environment of Lactobacillus johnsonii/gasseri strains

Oxygen is considered one of the main factors affecting probiotic bacteria survival due to the induction of oxidative damages caused by the action of reactive oxygen species (ROS). It has been shown that oxidative stress resistance in lactic acid bacteria is strongly dependent on the type of cell metabolism. Shift from fermentative to respiratory metabolism (through the addition of heme and menaquinone and in presence of oxygen) was associated to increase in biomass, long-term survival, and production of antioxidant enzymes. The aim of this work was to investigate the effect of aerobic (presence of oxygen) and respiratory (presence of oxygen, heme, and menaquinone) cultivation on the growth kinetic, catalase production, oxygen uptake, and oxidative stress response of Lactobacillus johnsonii/gasseri strains previously isolated from infant feces. Seven strains showed to consume oxygen under aerobic and respiratory conditions. The strain AL5 showed a catalase activity in both growth conditions, while AL3 showed this activity only in respiratory condition. Respiratory condition improved their tolerance to oxidative compounds (hydrogen peroxide and ROS generators) and further they showed promising probiotic features. The exploration of respiratory competent phenotypes with probiotic features may be extremely useful for the development of competitive starter or probiotic cultures

Temperature and respiration affect the growth and stress resistance of Lactobacillus plantarum C17

Aims: The aim of the study is to gain further insight on the respiratory behaviour of Lactobacillus plantarum and its consequences on stress tolerance. Methods and Results: We investigated the effect of temperature and respiration on the growth and stress (heat, oxidative, freezing, freeze-drying) response of Lact. plantarum C17 during batch cultivations. Temperature as well as respiration clearly affected the physiological state of cells, and generally, cultures grown under respiratory conditions exhibited improved tolerance of some stresses (heat, oxidative, freezing) compared to those obtained in anaerobiosis. Our results revealed that the activities in cell-free extracts of the main enzymes related to aerobic metabolism, POX (pyruvate oxidase) and NPR (NADH peroxidase), were significantly affected by temperature. POX was completely inhibited at 37°C, while the activity of NPR slightly increased at 25°C, indicating that in Lact. plantarum, the temperature of growth may be involved in the activation and modulation of aerobic/respiratory metabolism. Conclusions: We confirmed that respiration confers robustness to Lact. plantarum cells, allowing a greater stress tolerance and advantages in the production of starter and probiotic cultures. Significance and Impact of the Study: This is the first study on respiratory metabolism on a strain other than the model strains WCFS1; novel information on the role of temperature in the modulation of aerobic/ respiratory metabolism in Lact. plantarum is presented

Effect of Respiratory Growth on the Metabolite Production and Stress Robustness of Lactobacillus casei N87 Cultivated in Cheese Whey Permeate Medium

Cheese whey permeate (WP) is a low-cost feedstock used for the production of biomass and metabolites from several lactic acid bacteria (LAB) strains. In this study, Lactobacillus casei N87 was cultivated in an optimized WP medium (WPM) to evaluate the effect of anaerobic and respiratory conditions on the growth performances (kinetics, biomass yield), consumption of sugars (lactose, galactose, glucose) and citrate, metabolite production [organic acids, volatile organic compounds (VOCs)] and stress survival (oxidative, heat, freezing, freeze-drying). The transcription of genes involved in the main pathways for pyruvate conversion was quantified through Real Time-PCR to elucidate the metabolic shifts due to respiratory state. Cultivation in WPM induced a diauxic growth in both anaerobic and respiratory conditions, and L. casei N87 effectively consumed the lactose and galactose present in WPM. Genomic information suggested that membrane PTS system and tagatose-6-P pathway mediated the metabolism of lactose and galactose in L. casei N87. Respiration did not affect specific growth rate and biomass production, but significantly altered the pyruvate conversion pathways, reducing lactate accumulation and promoting the formation of acetate, acetoin and diacetyl to ensure the redox balance. Ethanol was not produced under either cultivation. Pyruvate oxidase (pox), acetate kinase (ack), alpha-acetolactate decarboxylase (ald), acetolactate synthase (als) and oxaloacetate decarboxylase (oad) genes were up-regulated under respiration, while L-lactate dehydrogenase (ldh), pyruvate formate lyase (pfl), pyruvate carboxylase (pyc), and phosphate acetyltransferase (pta) were down regulated by oxygen. Transcription analysis was consistent with metabolite production, confirming that POX-ACK and ALS-ALD were the alternative pathways activated under aerobic cultivation. Respiratory growth affected the production of volatile compounds useful for the development of aroma profile in several fermented foods, and promoted the survival of L. casei N87 to oxidative stresses and long-term storage. This study confirmed that the respiration-based technology coupled with cultivation on low-cost medium may be effectively exploited to produce competitive and functional starter and/or adjunct cultures. Our results, additionally, provided further information on the activation and regulation of metabolic pathways in homofermentative LAB grown under respiratory promoting conditions

Evaluation of a differential medium for the preliminary identification of members of the Lactobacillus plantarum and Lactobacillus casei groups

A medium originally developed for differential enumeration of probiotic species in fermented milk (mMRS-BPB, Lee and Lee 2008) was evaluated for its ability to correctly discriminate members of the Lactobacillus plantarum (L. plantarum) and L. casei groups from other species. The medium was tested on 461 strains of lactic acid bacteria (LAB) belonging to eight genera and thirty-five species. Colony morphology was relatively consistent for L. plantarum, L. paraplantarum, L. pentosus, L. paracasei, L. casei, and L. rhamnosus, but, when used alone, was not always sufficient to discriminate these species from other species potentially present in cheese. A procedure based on tree classification was developed to obtain preliminary identification on the basis of colony morphology, cell morphology, and CO2 production from glucose. By combining results of the tree classification procedure and heuristic rules, correct preliminary identification at the species or group level could be obtained in 74.4 % of cases overall, and the percentage of correct identifications was as high as 88-100 % for members of the L. plantarum and L. casei groups. When species belonging to groups that can be easily discriminated by rapid molecular methods were combined, the decision tree allowed to correct identification at the group level for the 95 % of the strains. Logistic regression was used to evaluate the effect of strain, operator, light source, and incubation temperature. Although all factors significantly affected one or more of the characters used for identification, the classification procedure proved to be quite robust. It may be difficult to use mMRS-BPB in the differential enumeration of LAB in cheese, except when species composition is relatively simple; however, it can be used as a simple tool to guide molecular identification in studies focused on the isolation of new strains from cheese

High resolution melting analysis (HRM) as a new tool for the identification of species belonging to the Lactobacillus casei group and comparison with species-specific PCRs and multiplex PCR

The correct identification and characterisation of bacteria is essential for several reasons: the classification of lactic acid bacteria (LAB) has changed significantly over the years, and it is important to distinguish and define them correctly, according to the current nomenclature, avoiding problems in the interpretation of literature, as well as mislabelling when probiotic are used in food products. In this study, species-specific PCR and HRM (high-resolution melting) analysis were developed to identify strains belonging to the Lactobacillus casei group and to classify them into L. casei, Lactobacillus paracasei and Lactobacillus rhamnosus. HRM analysis confirmed to be a potent, simple, fast and economic tool for microbial identification. In particular, 201 strains, collected from International collections and attributed to the L. casei group, were examined using these techniques and the results were compared with consolidated molecular methods, already published. Seven of the tested strains don't belong to the L. casei group. Among the remaining 194 strains, 6 showed inconsistent results, leaving identification undetermined. All the applied techniques were congruent for the identification of the vast majority of the tested strains (188). Notably, for 46 of the strains, the identification differed from the previous attribution

Functional properties of Lactobacillus plantarum strains: A multivariate screening study

Abstract Thirty-two Lactobacillus plantarum strains isolated from different sources were genetically characterized at subspecies level with recA gene based multiplex PCR and pulsed-field electrophoresis. All the strains were tested in vitro for functional properties (ability to form biofilms, agglutination of yeast cells, bile salt hydrolase activity, β-galactosidase activity, surface hydrophobicity, resistance to lysozyme, gastric juice and bile salts), for antimicrobial activity and for antibiotic resistance. The presence of bsh and msa genes and of the pln bacteriocin loci were also evaluated. Hierarchical cluster analysis was used to identify eight different plantaritypes sharing similar patterns of pln loci. A global functional score was calculated by transforming values for continuous in vitro functional properties in an ordinal scale by cluster analysis, while a nominal scale was used for the other properties. Multidimensional scaling was used to evaluate the similarity in functional properties among the isolates and to evaluate the relationships between source of isolation and functional properties. Nine strains showed the best in vitro functional potential and a significant relationship was found between source of isolation and functional score. This study confirmed a high heterogeneity in functional properties among L. plantarum strains and provides insight for optimal screening strategies

Polymorphisms in stress response genes in Lactobacillus plantarum: implications for classification and heat stress response

The polymorphism of 5 stress response genes (hrcA, ctsR, clpP, ftsH, dnaK) in 32 Lactobacillus plantarum strains was evaluated by multilocus restriction typing (MLRT) and by sequence analysis of ctsR, hrcA and clpP genes. Both these approaches allowed the discrimination of the subspecies L. plantarum ssp. plantarum and L. plantarum ssp. argentoratensis. HrcA sequence analysis also allowed discrimination at the species and subspecies level of several species of lactic acid bacteria, thus confirming that it can be used as a valuable taxonomic marker. No significant relationship was found between stress response gene polymorphism and resistance to heat treatments. The effect of temperature on growth kinetics and the protein expression were investigated for selected strains carrying different mutations in hrcA. L. plantarum ssp. argentoratensis NCIMB12120 and L. plantarum ssp. plantarum DPC2159, both of which had mutations in domains of HrcA which are important for the repressor functionality, had a reduced growth rate at all temperatures tested (25, 30, 37, 40, and 42 °C) compared to L. plantarum WCFS1. In L. plantarum DPC2159, protein expression upon temperature shifts from 25 to 40 °C or growth at 40 °C was altered compared to L. plantarum WCFS1, but further study is needed to unequivocally confirm the relationship with mutations in hrcA

Relevance of Bacteriophage 933W in the development of the Hemolytic Uremic Syndrome (HUS)

Hemolytic uremic syndrome (HUS), principally caused by shiga toxins (Stxs), is associated with Shiga toxin-producing Escherichia coli (STEC) infections. We previously reported Stx2 expression by host cells in vitro and in vivo. As the genes encoding the two Stx subunits are located in bacteriophage genomes, the aim of the current study was to evaluate the role of bacteriophage induction in HUS development in absence of an E. coli O157:H7 genomic background. Mice were inoculated with a non-pathogenic E. coli strain carrying the lysogenic bacteriophage 933W (C600Φ933W), and bacteriophage excision was induced by an antibiotic. The mice died 72 h after inoculation, having developed pathogenic damage typical of STEC infection. As well as renal and intestinal damage, markers of central nervous system (CNS) injury were observed, including aberrant immunolocalization of neuronal nuclei (NeuN) and increased expression of glial fibrillary acidic protein (GFAP). These results show that bacteriophage 933W without an E. coli O157:H7 background is capable of inducing the pathogenic damage associated with STEC infection. In addition, a novel mouse model was developed to evaluate therapeutic approaches focused on the bacteriophage as a new target