Lia Rossetti

An evaluation of chelex-based DNA purification protocols for the typing of lactic acid bacteria

An easy and rapid protocol to extract DNA to be used as template for polymerase chain reaction (PCR) fingerprinting experiments from cultivable lactic acid bacteria (LAB) is proposed. Different procedures for rapid extraction of DNA by chelex (iminodiacetid acid) ionic resin were compared. Factors affecting the quality and reproducibility of PCR fingerprinting profiles were also investigated. Two out of three chelex-based protocols allowed to obtain DNA samples which, after PCR amplification, provided electrophoretic patterns comparable with those obtained by classical lysozyme and phenol-chloroform DNA extraction. A good level of reproducibility and consistency of the InstaGene procedure was verified. The procedure is fast, practical, and the DNA is of quality similar to that obtained by phenol-chloroform extraction. Although applied to a little number of LAB strains, chelex-based protocols are potentially applicable to a vast array of organisms and/or biological materials.

Grana Padano cheese whey starters: microbial composition and strain distribution.

The aim of this work was to evaluate the species composition and the genotypic strain heterogeneity of dominant lactic acid bacteria (LAB) isolated from whey starter cultures used to manufacture Grana Padano cheese. Twenty-four Grana Padano cheese whey starters collected from dairies located over a wide geographic production area in the north of Italy were analyzed. Total thermophilic LAB streptococci and lactobacilli were quantified by agar plate counting. Population structure of the dominant and metabolically active LAB species present in the starters was profiled by reverse transcriptase, length heterogeneity-PCR (RT-LH-PCR), a culture-independent technique successfully applied to study whey starter ecosystems. The dominant bacterial species were Lactobacillus helveticus, Lactobacillus delbrueckii subsp. lactis, Streptococcus thermophilus, and Lactobacillus fermentum. Diversity in the species composition allowed the whey cultures to be grouped into four main typologies, the one containing L. helveticus, L. delbrueckii subsp. lactis, and S. thermophilus being the most frequent one (45% of the cultures analyzed), followed by that containing only the two lactobacilli (40%). Only a minor fraction of the cultures contained L. helveticus alone (4%) or all the four LAB species (11%). Five hundred and twelve strains were isolated from the 24 cultures and identified by M13-PCR fingerprinting coupled with 16S rRNA gene sequencing. Most of the strains were L. helveticus (190 strains; 37% of the total), L delbrueckii subsp. lactis (90 strains; 18%) and S. thermophilus (215 strains; 42%). This result was in good agreement with the qualitative whey starter composition observed by RT-LH-PCR. M13-PCR fingerprinting indicated a markedly low infra-species diversity, i.e. the same biotypes were often found in more than one culture. The distribution of the biotypes into the different cultures was mainly dairy plant-specific rather than correlated with the different production areas.

Molecular Diversity within Lactobacillus helveticus as Revealed by Genotypic Characterization

ABSTRACT Lactobacillus helveticus is a homofermentative thermophilic lactic acid bacterium that is used in the manufacture of Swiss type and long-ripened Italian cheeses, such as Emmental, Grana, and Provolone cheeses. Substantial differences in several technologically important characteristics are found among L. helveticus strains isolated from natural dairy starter cultures. In the present study we investigated the genotypic diversity of 74 strains isolated from different dairy cultures used for manufacturing Grana and Provolone cheeses and six collection strains. A restriction fragment length polymorphism analysis of both total genomic DNA and the 16S rRNA gene (ribotyping) was used as genotypic fingerprinting. A multivariate statistical analysis of the data enabled us to identify significant genotypic heterogeneity inL. helveticus. We found that genotypic fingerprinting could be used to distinguish strains; in particular, it was possible to associate the presence of specific strain genotypes with dairy ecosystem sources (e.g., Grana or Provolone cheese). Our data contribute to the description of microbial heterogeneity in L. helveticus and provide a more solid basis for understanding the functional and ecological significance of the presence of differentL. helveticus biotypes in natural dairy starter cultures.

Molecular typing of Lactobacillus delbrueckii of dairy origin by PCR-RFLP of protein-coding genes

Thirty-five strains of Lactobacillus delbrueckii subsp. lactis and subsp. bulgaricus isolated from dairy products were typed by restriction fragment length polymorphism (RFLP) of protein-coding genes. The strains were analysed by RFLP of PCR amplified, infragenic fragments of the following housekeeping genes: beta-galactosidase, lactose permease, and proline dipeptidase. Sequencing of the variable regions of the 16S rDNA was then performed on a reduced number of strains. PCR-RFLP analysis evidenced wide strain heterogeneity. Strains were grouped into genotypes according to both subspecies assignment and infra-species genetic polymorphism. This polymorphism was related to the presence of microbial groups within subspecies populations. The low infra-species sequence polymorphism detected in the variable region of the 16S rRNA gene did not enable to group the strains with the same sensitivity reached by PCR-RFLP of protein-coding genes. PCR-RFLP of protein-coding genes applied to L. delbrueckii seems a promising tool to evaluate microbial diversity within bacterial subpopulations. Differences among bacterial subpopulations based upon molecular heterogeneity in protein-coding genes would enable to better understand the role of strains from different ecological niches.

Quantification of Enterococcus italicus in traditional Italian cheeses by fluorescence whole-cell hybridization

The objective of this work was to investigate the spread of Enterococcus italicus in cheese. For this purpose, a fluorescence whole-cell hybridization protocol (FWCH) with a 16S rRNA probe was optimized to evaluate the presence and abundance of this organism in artisanal Italian cheeses. The FWCH method avoided the quantification problems using classical plate count techniques related to the well-known difficulties to cultivate E. italicus in selective enterococci media. After probe and FWCH optimization, 10 commercially available Italian semi-hard cheeses made with raw ewe or cow milk without starter addition were analyzed. All of them were subjected to FWCH experiments and six of them gave positive results with the probe, i.e. the E. italicus content was >4 log cells/g according to the detection limit of FWCH. Counts showed that E. italicus was present at levels ranging from 5.91+/-0.17 to 7.34+/-0.14 log cells/g; such levels were similar to, or even higher than, the total enterococci counted from the corresponding cheeses using kanamycin aesculin azide agar. The overall reliability of the FWCH method was tested by species-specific PCR. The positive amplification of the expected 323 bp fragment from both a cheese matrix and cell bulks of cheese samples containing high loads of this organism (as determined by FWCH counts) and the successful isolation of E. italicus strains from the above cheeses provided definitive proof of both probe specificity and the presence of this organism in cheeses. Although there is very little available quantitative data on the incidence of E. italicus in cheese, or its role in product quality, this study showed a wide diffusion of this organism in artisanal cheeses, where secondary non-starter lactic acid bacterial microflora, which enterococci belong to, may become dominant during ripening.

Detection and identification of Lactobacillus helveticus bacteriophages by PCR

A PCR protocol for detection of Lactobacillus helveticus bacteriophages was optimized. PCR was designed taking into account the sequence of the lys gene of temperate bacteriophage Phi-0303 and optimized to obtain a fragment of 222 bp using different Lb. helveticus phages from our collection. PCR was applied to total phage DNA extracted from 53 natural whey starters used for the production of Grana cheese and all gave the expected fragment. The presence of actively growing phages in the cultures was verified by traditional tests. Several PCR products of the lys gene were sequenced and aligned. The resulting sequences showed variable heterogeneity between the phages.

Population dynamics of lactobacilli in Grana cheese

A survey on the presence, microbial diversity, and population dynamics of lactobacilli in Grana cheese is presented. Evolution of thermophilic rod lactic acid bacteria within the first two days from cheese making and during ripening was different according to different bacterial groups, which were selectively enumerated and identified by molecular methods. Species-specific microbial counts indicated prevalence ofLactobacillus helveticus in both the whey starter and the cheese at moulding, and ofLactobacillus delbrueckii subsp.lactis in cheese after two months of ripening. In more advanced ripening, a decrease of total thermophilic lactobacilli and an increase of mesophilic lactobacilli (mostly belonging toLactobacillus casei/paracasei andLactobacillus rhamnosus) was observed. PCR fingerprinting of lactobacilli, which was performed by PCR-fingerprinting, indicated a marked microbial heterogeneity within theLactobacillus spp. populations, which enabled strain (or group)-specific fingerprints to be observed.

Biotyping of cultivable lactic acid bacteria isolated from donkey milk

The diversity of lactic acid bacteria (LAB) species in donkeys milk was analysed by culture‐dependent microbial techniques. Dominant strains were isolated on agar media generally used for enumerating LAB. To enrich the number of acidifying LAB present, the milk samples were incubated at 37°C for 24 h (cultured milk samples, CM). One of the CM samples was heat‐treated at 63°C for 10 min before incubation at 37°C (heat‐treated and cultured milk sample, TCM) to select thermophilic LAB. The microflora in these CM and TCM samples was then compared to that of the raw milk samples (RM). Among the 129 LAB isolates, 10 different species (four Enterococcus, five Streptococcus and one Pediococcus) were identified by molecular methods. Although the 10 LAB species were present in the RM samples, only three and two isolates were found in CM and TCM samples, respectively. Despite the selection protocol being set up to favour the isolation of all LAB isolates present in donkey milk, relatively few species and biotypes were isolated. No LAB isolates belonging to the most technologically important dairy starter species were detected. The possible factors related to the limited LAB diversity in donkeys milk have been discussed below.

Heterogeneity of putative surface layer proteins in Lactobacillus helveticus.

ABSTRACT The S-layer-encoding genes of 21 Lactobacillus helveticus strains were characterized. Phylogenetic analysis based on the identified S-layer genes revealed two main clusters, one which includes a sequence similar to that of the slpH1 gene of L. helveticus CNRZ 892 and a second cluster which includes genes similar to that of prtY. These results were further confirmed by Southern blot hybridization. This study demonstrates S-layer gene variability in the species L. helveticus.

Characterization of the Genome of the Dairy Lactobacillus helveticus Bacteriophage ΦAQ113

ABSTRACT The complete genomic sequence of the dairy Lactobacillus helveticus bacteriophage ΦAQ113 was determined. Phage ΦAQ113 is a Myoviridae bacteriophage with an isometric capsid and a contractile tail. The final assembled consensus sequence revealed a linear, circularly permuted, double-stranded DNA genome with a size of 36,566 bp and a G+C content of 37%. Fifty-six open reading frames (ORFs) were predicted, and a putative function was assigned to approximately 90% of them. The ΦAQ113 genome shows functionally related genes clustered together in a genome structure composed of modules for DNA replication/regulation, DNA packaging, head and tail morphogenesis, cell lysis, and lysogeny. The identification of genes involved in the establishment of lysogeny indicates that it may have originated as a temperate phage, even if it was isolated from natural cheese whey starters as a virulent phage, because it is able to propagate in a sensitive host strain. Additionally, we discovered that the ΦAQ113 phage genome is closely related to Lactobacillus gasseri phage KC5a and Lactobacillus johnsonii phage Lj771 genomes. The phylogenetic similarities between L. helveticus phage ΦAQ113 and two phages that belong to gut species confirm a possible common ancestral origin and support the increasing consideration of L. helveticus as a health-promoting organism.