Cinzia Caggia

Comparative Genomic and Functional Analysis of 100 Lactobacillus rhamnosus Strains and Their Comparison with Strain GG

Lactobacillus rhamnosus is a lactic acid bacterium that is found in a large variety of ecological habitats, including artisanal and industrial dairy products, the oral cavity, intestinal tract or vagina. To gain insights into the genetic complexity and ecological versatility of the species L. rhamnosus, we examined the genomes and phenotypes of 100 L. rhamnosus strains isolated from diverse sources. The genomes of 100 L. rhamnosus strains were mapped onto the L. rhamnosus GG reference genome. These strains were phenotypically characterized for a wide range of metabolic, antagonistic, signalling and functional properties. Phylogenomic analysis showed multiple groupings of the species that could partly be associated with their ecological niches. We identified 17 highly variable regions that encode functions related to lifestyle, i.e. carbohydrate transport and metabolism, production of mucus-binding pili, bile salt resistance, prophages and CRISPR adaptive immunity. Integration of the phenotypic and genomic data revealed that some L. rhamnosus strains possibly resided in multiple niches, illustrating the dynamics of bacterial habitats. The present study showed two distinctive geno-phenotypes in the L. rhamnosus species. The geno-phenotype A suggests an adaptation to stable nutrient-rich niches, i.e. milk-derivative products, reflected by the alteration or loss of biological functions associated with antimicrobial activity spectrum, stress resistance, adaptability and fitness to a distinctive range of habitats. In contrast, the geno-phenotype B displays adequate traits to a variable environment, such as the intestinal tract, in terms of nutrient resources, bacterial population density and host effects.

Lactobacillus casei, dominant species in naturally fermented Sicilian green olives.

This study investigated the phenotypic and genotypic characteristics of lactic acid bacteria in naturally fermented green olives, collected from different areas of Sicily. Both classical biochemical tests and PCR/Restriction Fragments Length Polymorphism (RFLP) of 16S rDNA were used to characterize the isolates. The identity of the isolates was obtained by the partial sequencing analysis of the 16S rDNA. The BioMerieux software assigned the 13 heterofermentative strains to the Lactobacillus brevis species; 24 homofermentative strains were classified as Lactobacillus casei and the remaining 11 homofermentative lactobacilli were identified as Lactobacillus plantarum. The rapid ID 32 STREP test identified coccal-shaped strains as Enterococcus faecium species. The PCR/RFLP analysis showed a remarkable bacterial heterogeneity within the isolates. The 16S rDNA partial sequencing did not confirm biochemical identification, revealing a strong dominance of isolates belonging to the L. casei species. It is noteworthy that this species has never been reported as dominant species in fermented vegetables.A combination of molecular and biochemical analysis allowed the identification of species involved in natural food fermentations.

Comparative Genomic and Functional Analysis of Lactobacillus casei and Lactobacillus rhamnosus Strains Marketed as Probiotics

ABSTRACT Four Lactobacillus strains were isolated from marketed probiotic products, including L. rhamnosus strains from Vifit (Friesland Campina) and Idoform (Ferrosan) and L. casei strains from Actimel (Danone) and Yakult (Yakult Honsa Co.). Their genomes and phenotypes were characterized and compared in detail with L. casei strain BL23 and L. rhamnosus strain GG. Phenotypic analysis of the new isolates indicated differences in carbohydrate utilization between L. casei and L. rhamnosus strains, which could be linked to their genotypes. The two isolated L. rhamnosus strains had genomes that were virtually identical to that of L. rhamnosus GG, testifying to their genomic stability and integrity in food products. The L. casei strains showed much greater genomic heterogeneity. Remarkably, all strains contained an intact spaCBA pilus gene cluster. However, only the L. rhamnosus strains produced mucus-binding SpaCBA pili under the conditions tested. Transcription initiation mapping demonstrated that the insertion of an iso-IS30 element upstream of the pilus gene cluster in L. rhamnosus strains but absent in L. casei strains had constituted a functional promoter driving pilus gene expression. All L. rhamnosus strains triggered an NF-κB response via Toll-like receptor 2 (TLR2) in a reporter cell line, whereas the L. casei strains did not or did so to a much lesser extent. This study demonstrates that the two L. rhamnosus strains isolated from probiotic products are virtually identical to L. rhamnosus GG and further highlights the differences between these and L. casei strains widely marketed as probiotics, in terms of genome content, mucus-binding and metabolic capacities, and host signaling capabilities.

Pecorino Crotonese cheese: study of bacterial population and flavour compounds.

The diversity and dynamics of the dominant bacterial population during the manufacture and the ripening of two artisanal Pecorino Crotonese cheeses, provided by different farms, were investigated by the combination of culture-dependent and -independent approaches. Three hundred and thirty-three strains were isolated from selective culture media, clustered using Restriction Fragment Length Polymorphism and were identified by 16S rRNA gene sequencing. The results indicate a decrease in biodiversity during ripening, revealing the presence of Lactococcus lactis and Streptococcus thermophilus species in the curd and in aged cheese samples and the occurrence of several lactobacilli throughout cheese ripening, with the dominance of Lactobacillus rhamnosus species. Bacterial dynamics determined by Denaturant Gradient Gel Electrophoresis provided a more precise description of the distribution of bacteria, highlighting differences in the bacterial community among cheese samples, and allowed to detect Lactobacillus plantarum, Lactobacillus buchneri and Leuconostoc mesenteroides species, which were not isolated. Moreover, the concentration of flavour compounds produced throughout cheese ripening was investigated and related to lactic acid bacteria presence. Fifty-seven compounds were identified in the volatile fraction of Pecorino Crotonese cheeses by Gas Chromatography-Mass Spectrometry. Esters, alcohols and free fatty acids were the most abundant compounds, while aldehydes and hydrocarbons were present at low levels.

Survival of Lactobacillus rhamnosus strains in the upper gastrointestinal tract

In the present study six probiotic Lactobacillus rhamnosus strains were investigated for their ability to survive in the human upper gastrointestinal tract through a dynamic gastric model of digestion. MRS broth was used as delivery vehicle and survival was investigated during in vitro gastric and gastric plus duodenal digestion. Results highlighted that all tested strains showed good survival rate during both gastric and duodenal digestion. In particular, three strains exhibited a great survival showing a recovery percentage in the range between 117 and 276%. In agreement with survival data, high lactic acid production was detected for all strains, confirming their metabolic activity during digestion.

Bacterial population in traditional sourdough evaluated by molecular methods

Aims:  To study the microbial communities in artisanal sourdoughs, manufactured by traditional procedure in different areas of Sicily, and to evaluate the lactic acid bacteria (LAB) population by classical and culture‐independent approaches.

Effect of wild strains used as starter cultures and adjunct cultures on the volatile compounds of the Pecorino Siciliano cheese

The effect of six wild strains on the volatile profile of the PS cheese was investigated and compared to that generated from industrial starters generally used to produce PS cheese. All cheeses were subjected to microbiological, physicochemical, and volatile compounds analyses. The DGGE of the 16S rDNA analysis was also applied. The volatile compounds generated during ripening were studied through the SPME and the GC-MS methods. No difference was detected between the experimental and control cheeses throughout chemical and microbiological analyses, while the DGGE results showed the presence of Streptococcus thermophilus in all cheeses, and the dominance of Enterococcus durans, Lactobacillus rhamnosus, and Lactobacillus casei in most of the experimental cheeses. Moreover, the presence of Lactococcus lactis species as in the control and in the experimental P2 and P4 cheeses was also revealed. The SPME results showed more pronounced volatile compounds in the experimental cheese samples than in the control ones.

Occurrence of Listeria monocytogenes in Green Table Olives

Microbiological safety of green table olives from different cultivars, prepared by both the Spanish-style and biological methods and fermented with starter cultures of lactic acid bacteria (Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus pentosus), was investigated. The fermentation process was monitored by measuring pH values, titratable acidities, and growth of lactic acid bacteria over time. During fermentation, lactic acid bacteria and yeasts were major microbial populations. Microbiological safety was evaluated by analysis for Listeria monocytogenes with the use of an enrichment method during storage (from 55 days to 18 months). Results demonstrated that L. monocytogenes can survive and grow in green table olives. L. monocytogenes was found in one of the commercial (thermally treated) samples analyzed and in all samples older than 2 months, irrespective of olive cultivar, lactic acid bacteria starter used, pH and titratable acidity of brine samples, or treatment applied.

Karyotyping of Saccharomyces strains with different temperature profiles

This study examined the karyotype, the fermentation performance and the optimum growth temperature (Topt) of 28 yeast strains all identified as species belonging to Saccharomyces sensu stricto. The strains were isolated from fermented musts, which had not been inoculated, at two temperature ranges: 20–40 °C and approximately 0–6 °C. The results demonstrated a correlation between the Topt and the chromosome organization. In particular, strains with Topt of less than 30 °C showed only two bands in the region between 365 and 225 kb, while those with a Topt greater than 30 °C had three bands in this size range. From a taxonomic viewpoint, the Topt is a better indicator for the Saccharomyces sp. than the ceiling temperature of 37 °C currently used to differentiate cryotolerant Saccharomyces bayanus and S. pastorianus from non‐cryotolerant S. cerevisiae and S. paradoxus strains.

Yeast dynamics during the fermentation of brined green olives treated in the field with kaolin and Bordeaux mixture to control the olive fruit fly.

The yeast microbiota associated with naturally fermented and inoculated green table olives, differently treated in the field with non-conventional repellent and antiovipositional products in the control of Bactrocera oleae, was analysed using a combination of culture-dependent and -independent molecular fingerprinting. The routine yeast isolation gave rise to 118 strains, whose identification was performed by PCR-RFLP of the internal transcribed spacer (ITS) regions. Total DNA was extracted directly from the brine throughout fermentation by means of an experimental protocol that included the removal of Taq polymerase inhibitors. Denaturing Gradient Gel Electrophoresis (DGGE) of 26S rRNA gene PCR amplicons highlighted the yeast community. Comparison of both culture-dependent and independent methods indicated that the yeast species Saccharomyces cerevisiae, Wickerhamomyces anomalus, Candida diddensiae and Issatchenkia orientalis were dominant during fermentation despite the addition of the Lactobacillus plantarum starter used in brining. The resultant isolated species were unaffected by treatments in field, except for C. diddensiae whose growth was delayed by kaolin.