Rapid Identification of new isolates of Acidipropionibacterium acidipropionici by fluorescence in situ hybridization (FISH)

Abstract Acidipropionibacterium acidipropionici is widely used for many applications, suchas propionic acid production, cereal silage, and also as probiotic. Due to this plethora of appli-cations, new isolates of A. acidipropionici with improved features are being searched for. Thesenew isolates must be accurately identified, however, most approaches become expensive andtime-consuming when the number of isolates is high. On the contrary, fluorescence in situhybridization allows the affordable, reliable, and rapid identification of microorganisms in purecultures and environmental and medical samples. Therefore, the aim of this work was to applya fluorescent in situ hybridization probe for the reliable identification of new A. acidipropioniciisolates. To this end, probe Pap446, specific for A. acidipropionici, was validated by hybridiza-tion assays with strains of this species from different origins, other species of the same genusor family, and unrelated genera. Eight isolates with propionibacterium characteristics wereobtained from milk and feces of cows. Probe Pap446, hybridized only with isolates III and VI.The identity of these isolates was further confirmed by PCR using group and species-specificprimers for propionibacteria and 16S rDNA sequencing.

Resumen Acidipropionibacterium acidipropionici es ampliamente usada para diversas aplicaciones, como producción de ácido propiónico, ensilado de cereales y probiótico. Debido a esta variedad de aplicaciones, continuamente se buscan nuevos aislamientos de A. acidipropionici con características nuevas. Estos nuevos aislamientos deben ser identificados correctamente, pero la mayoría de las técnicas disponibles resultan costosas e insumen mucho tiempo cuando el número de aislamientos es elevado. Por el contrario, la hibridación fluorescente in situ permite una identificación barata, confiable y rápida de microorganismos en cultivos puros y en muestras ambientales y médicas. Por lo tanto, el objetivo de este trabajo fue la aplicación de una sonda oligonucleotídica en un protocolo de hibridación fluorescente in situ para la identificación confiable de nuevos aislamientos de A. acidipropionici. Con este fin, se validó la sonda Pap446, específica de A. acidipropionici mediante ensayos de hibridación con cepas de esta especie de diferente origen, otras especies del mismo género o familia, y géneros no relacionados. Se obtuvieron ocho aislamientos con características de propionibacterias a partir de leche y heces de vacas. La sonda Pap446, hibridó únicamente con los aislamientos III y VI. La identidad de estos aislamientos fue confirmada a través de PCR con cebadores específicos para propionibacterias y para A. acidipropionici, y mediante secuenciación del ADNr 16S.

Growth studies of dominant lactic acid bacteria in orange juice and selection of strains to ferment citric fruit juices with probiotic potential.

The study aimed to evaluate the ability of dominant lactic acid bacteria (LAB) in orange juice to growth on N-depleted MRS medium supplemented or not with cysteine (mMRS), then to select the most nutritionally promising strains for growth assays in the food matrix and evaluation of beneficial attributes for fruit juice fermentation. Levilactobacillus brevis and Lactiplantibacillus plantarum were dominant species among the total of 103 LAB isolates as confirmed by multiplex PCR and/or 16 s rDNA sequence analysis. Based on growing lower than 20% and higher than 70% in mMRS (1.0 g/l meat extract, without peptone and yeast extract) with and without cysteine requirement, one L. brevis (JNB23) and two L. plantarum (JNB21 and JNB25) were selected. These bacteria and the L. plantarum strains N4 and N8 (previously isolated from oranges peel) when inoculated in orange juice grew up to 1.0 log cfu/ml for 24 h incubation at 30 °C and mainly produced lactic acid, with strains JNB25 and JNB23 reaching the highest and lowest cell densities in agreement with their nutritional exigency. In addition, all L. plantarum strains exhibited antagonistic activity against the majority of tested bacterial pathogens (in opposition to L. brevis), ability to grow or survive to pH 3.0 for 3 h, to grow with 0.5% sodium taurocholate, and a decrease after simulated gastrointestinal digestion assay which did not exceed 1.0 or 2.0 log units, depending on the strain. Thus, autochthonous L. plantarum strains with ability for overcoming nutritional limitations and beneficial attributes are promising candidates for further investigations as novel probiotic and/or preservative starters to ferment citric fruit juices.

Rapid identification of new isolates of Acidipropionibacterium acidipropionici by fluorescence in situ hybridization (FISH).

Acidipropionibacterium acidipropionici is widely used for many applications, such as propionic acid production, cereal silage, and also as probiotic. Due to this plethora of applications, new isolates of A. acidipropionici with improved features are being searched for. These new isolates must be accurately identified, however, most approaches become expensive and time-consuming when the number of isolates is high. On the contrary, fluorescence in situ hybridization allows the affordable, reliable, and rapid identification of microorganisms in pure cultures and environmental and medical samples. Therefore, the aim of this work was to apply a fluorescent in situ hybridization probe for the reliable identification of new A. acidipropionici isolates. To this end, probe Pap446, specific for A. acidipropionici, was validated by hybridization assays with strains of this species from different origins, other species of the same genus or family, and unrelated genera. Eight isolates with propionibacterium characteristics were obtained from milk and feces of cows. Probe Pap446, hybridized only with isolates III and VI. The identity of these isolates was further confirmed by PCR using group and species-specific primers for propionibacteria and 16S rDNA sequencing.

Cytotoxic damage of soybean agglutinin on intestinal epithelial cells of broiler chicks: in vitro protection by Bifidobacterium infantis CRL1395.

Plant lectins, which are proteins/glycoproteins present in a wide range of vegetables, fruits, cereals and beans, are resistant to digestive enzymes and food cooking temperatures. They bind reversibly to specific glycosidic residues expressed on the membrane of intestinal epithelial cells (IEC) and cause anti-nutritional effects in humans and animals. Soybean lectin (SBA) has been detected in poultry diets, and its ability to bind to the intestinal epithelium has been reported. The development of new methods for removing SBA from feeds or to prevent interaction with the intestinal mucosa is of interest. In this study, the in vitro cytotoxicity of SBA on IEC of chicks was demonstrated for the first time. The LD50, assessed after 2 h exposure of IEC to SBA, was 6.13 µg mL(-1) The ability of Bifidobacterium infantis CRL1395 to bind SBA on the bacterial envelope was confirmed, and prevention of IEC cytotoxicity by lectin removal was demonstrated. Safety of B. infantis CRL1395, resistance to gastrointestinal stress and adhesion were also determined. It was concluded that the early administration of B. infantis CRL1395 to chicks would effectively reduce the toxicity of SBA. Besides, it would favour the colonization of the gut with a beneficial microbiota.

Selection of indigenous lactic acid bacteria to reinforce the intestinal microbiota of newly hatched chicken: relevance of in vitro and ex vivo methods for strains characterization.

Based on the natural benefits of the indigenous microbiota, lactic acid bacteria (LAB) from poultry origin were isolated from hens and broilers intestine, and their probiotic potential was further studied. The tolerance to digestion, adhesion, capture of a mannose-binding lectin, absence of virulent factors and antibiotic resistances were studied. Different in vitro and ex vivo assays were performed to select tolerant and adherent strains because standardized protocols have not been defined. Fourteen strains highly tolerant to gastrointestinal digestion were genetically identified. Hydrophobic surfaces were not required for the bacterial adhesion and only nine strains adhered ex vivo to the intestinal mucosa. Three strains captured a lectin of the same specificity of Type-1 fimbriae. Virulence factors were absent but some strains evidenced multiple antibiotic resistances. These results provide bases for a future standardization of methods for the selection of probiotic strains intended to reinforce the microbiota of newly hatched chickens.

Physiological and functional characteristics of Propionibacterium strains of the poultry microbiota and relevance for the development of probiotic products.

The prevention and control of pathogens colonization through probiotics administration in poultry feeding is of increasing interest. The genus Propionibacterium is an attractive candidate for the development of probiotic cultures as they produce short chain fatty acids (SCFA) by carbohydrates fermentation. The presence of strains of this genus in hens of conventional production systems and backyard hens was investigated. Propionibacteria were isolated from the intestine and identified by physiological and biochemical tests. PCR amplification of the 16S rRNA gene of the isolates was performed and products were compared with sequences from databases. The presence of the genus Propionibacterium was demonstrated in 26% of hens and Propionibacterium acidipropionici and Propionibacterium avidum were the identified species. A comparative study of their physiological and functional characteristics was performed. P. acidipropionici strains were the most resistant to in vitro gastrointestinal digestion, but the adhesion to intestinal tissue was strain dependent. Some differences were found between both species with respect to their growth and SCFA production in an in vitro cecal water model, but all the strains were metabolically active. The production of SCFA in cecal slurries inoculated with the strain P. acidipropionici LET 105 was 30% higher than in non-inoculated samples. SCFA concentrations obtained were high enough to inhibit Salmonella enterica serovar Enteritidis when assayed in a cecal water model. P. acidipropionici LET 105 was also able to compete with Salmonella for adhesion sites on the intestinal mucosa in ex vivo assays. Results contribute to the knowledge of the species diversity of the genus Propionibacterium in the intestine of poultry and provide evidence of their potential for probiotics products development.

Fluorescence in situ hybridization for detection of classical propionibacteria with specific 16S rRNA-targeted probes and its application to enumeration in Gruyère cheese.

The classical or dairy propionibacteria have well-documented industrial applications and have been proposed for probiotic applications. Given their industrial importance it is necessary to employ fast and reliable techniques to monitor the growth during products elaboration, industrial fermentations or the intestinal transit. Therefore, the aim of this investigation was to design oligonucleotide probes targeting the 16S rRNA of dairy propionibacteria and optimise the fluorescence in situ hybridization (FISH) protocol to detect these bacteria. Two specific probes were in silico designed to detect Propionibacterium freudenreichii and P. jensenii, named Pfr435 and Pj446 respectively. The FISH protocol was optimised for the hybridisation of propionibacteria cells with the universal probe Eub338 and the designed probes. These probes were assayed in situ for their specificity to hybridise species of propionibacteria by observation using fluorescence microscopy and results were compared with the probe Pap446 previously designed for P. acidipropionici. Probes Pap446, Pfr435 and Pj446 were also evaluated by fluorescence spectrophotometry to assess the influence of cells physiological state during growth in batch culture in the fluorescence intensity. The maximum fluorescence intensity was observed at the onset of the stationary phase of growth and was then reduced. However, changes on the cells permeability did not reduce the efficiency of 16S rRNA hybridisation with the fluorescence-labelled probes. Propionibacteria counts obtained by FISH and plate count methods were compared in a commercial Gruyère cheese. The results showed that this method can be used as a rapid technique for the enumeration of these bacteria in cheese samples.