Multiplex PCR for the detection of Lactobacillus pontis and two related species in a sourdough fermentation

A specific multiplex PCR assay based on the amplification of parts of the 16S rRNA molecule was designed. Primers derived from variable regions of the 16S rRNA provided a means of easily differentiating the species Lactobacillus pontis and Lactobacillus panis. They could be clearly discriminated from the phylogenetically related species Lactobacillus vaginalis, Lactobacillus oris, and Lactobacillus reuteri and from other lactobacilli commonly known to be present in sourdough. Other strains isolated together with L. pontis from an industrial sourdough fermentation could be clearly separated from these species by comparative sequence analysis and construction of a specific PCR primer. For a fast identification a DNA isolation protocol based on the ultrasonic lysis of cells from single colonies was developed. To demonstrate the potential of such techniques for tracking these organisms in a laboratory-scale fermentation, we combined the specific PCR assay with direct DNA extraction from the organisms in the sourdough without previous cultivation.     

Rapid Differentiation and In Situ Detection of 16 Sourdough Lactobacillus Species by Multiplex PCR

A two-step multiplex PCR-based method was designed for the rapid detection of 16 species of lactobacilli known to be commonly present in sourdough. The first step of multiplex PCR was developed with a mixture of group-specific primers, while the second step included three multiplex PCR assays with a mixture of species-specific primers. Primers were derived from sequences that specify the 16S rRNA, the 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene. The primer pairs designed were shown to exclusively amplify the targeted rrn operon fragment of the corresponding species. Due to the reliability of simultaneously identifying Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum, a previously described multiplex PCR method employing recA gene-derived primers was included in the multiplex PCR system. The combination of a newly developed, quick bacterial DNA extraction method from sourdough and this multiplex PCR assay allows the rapid in situ detection of several sourdough-associated lactobacilli, including the recently described species Lactobacillus rossii, and thus represents a very useful alternative to culture-based methodologies.     

Diversity and Stability of Lactic Acid Bacteria in Rye Sourdoughs of Four Bakeries with Different Propagation Parameters

We identified the lactic acid bacteria within rye sourdoughs and starters from four bakeries with different propagation parameters and tracked their dynamics for between 5–28 months after renewal. Evaluation of bacterial communities was performed using plating, denaturing gradient gel electrophoresis, and pyrosequencing of 16S rRNA gene amplicons. Lactobacillus amylovorus and Lactobacillus frumenti or Lactobacillus helveticus, Lactobacillus pontis and Lactobacillus panis prevailed in sourdoughs propagated at higher temperature, while ambient temperature combined with a short fermentation cycle selected for Lactobacillus sanfranciscensis, Lactobacillus pontis, and Lactobacillus zymae or Lactobacillus helveticus, Lactobacillus pontis and Lactobacillus zymae. The ratio of species in bakeries employing room-temperature propagation displayed a seasonal dependence. Introduction of different and controlled propagation parameters at one bakery (higher fermentation temperature, reduced inoculum size, and extended fermentation time) resulted in stabilization of the microbial community with an increased proportion of L. helveticus and L. pontis. Despite these new propagation parameters no new species were detected.     

Rapid differentiation and in situ detection of 16 sourdough lactobacillus species by multiplex PCR

A two-step multiplex PCR-based method was designed for the rapid detection of 16 species of lactobacilli known to be commonly present in sourdough. The first step of multiplex PCR was developed with a mixture of group-specific primers, while the second step included three multiplex PCR assays with a mixture of species-specific primers. Primers were derived from sequences that specify the 16S rRNA, the 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene. The primer pairs designed were shown to exclusively amplify the targeted rrn operon fragment of the corresponding species. Due to the reliability of simultaneously identifying Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum, a previously described multiplex PCR method employing recA gene-derived primers was included in the multiplex PCR system. The combination of a newly developed, quick bacterial DNA extraction method from sourdough and this multiplex PCR assay allows the rapid in situ detection of several sourdough-associated lactobacilli, including the recently described species Lactobacillus rossii, and thus represents a very useful alternative to culture-based methodologies.     

Growth characterization of individual rye sourdough bacteria by isothermal microcalorimetry

Aims: The present work tests the feasibility of the isothermal microcalorimetry method to study the performance of individual lactic acid bacteria during solid‐state fermentation in rye sourdough. Another aim was to elucidate the key factors leading to the formation of different microbial consortia in laboratory and industrial sourdough during continuous backslopping propagation. Methods and Results: Strains of the individual LAB isolated from industrial and laboratory sourdough cycle were grown in 10 kGy irradiated rye dough in vials of an isothermal calorimeter and the power–time curves were obtained. Sugars, organic acids and free amino acids in the sourdough were measured. The OD–time curves of the LAB strains during growth in flour extract or MRS (De Man, Rogosa and Sharpe) broth were also determined. The maximum specific growth rates of Lactobacillus sakei, Lactobacillus brevis, Lactobacillus curvatus and Leuconostoc citreum strains that dominated in backslopped laboratory sourdough were higher than those of Lactobacillus helveticus, Lactobacillus panis, Lactobacillus vaginalis, Lactobacillus casei and Lactobacillus pontis strains originating from industrial sourdough. Industrial strains had higher specific growth rates below pH 4·8. It was supposed that during long‐run industrial backslopping processes, the oxygen sensitive species start to dominate because of the O₂ protective effect of rye sourdough. Conclusions: Measurements of the power–time curves revealed that the LAB strains dominating in the industrial sourdough cycle had better acid tolerance but lower maximum growth rate and oxygen tolerance than species isolated from a laboratory sourdough cycle. Significance and Impact of the Study: Isothermal microcalorimetry combined with chemical analysis is a powerful method for characterization of sourdough fermentation process and determination of growth characteristics of individual bacteria in sourdough.     

Microbiological Characterization of Wet Wheat Distillers' Grain, with Focus on Isolation of Lactobacilli with Potential as Probiotics

Wet wheat distillers' grain (WWDG), a residue from ethanol fermentation, was examined from a microbiological perspective. After storage, WWDG was characterized by a high content of lactobacilli, nondetectable levels of other bacteria, occasional occurrence of yeasts, and a pH of about 3.6 and contained a mixture of lactic acid, acetic acid, and ethanol. The composition of lactobacilli in WWDG was simple, including primarily the species Lactobacillus amylolyticus, Lactobacillus panis, and Lactobacillus pontis, as determined by 16S rRNA gene sequencing. Since the use of WWDG as pig feed has indicated a health-promoting function, some relevant characteristics of three strains of each of these species were examined together with basal physiological parameters, such as carbohydrate utilization and growth temperature. Seven of the strains were isolated from WWDG, and two strains from pig feces were included for comparison. It was clear that all three species could grow at temperatures of 45 to 50°C, with L. amylolyticus being able to grow at temperatures as high as 54°C. This finding could be the explanation for the simple microflora of WWDG, where a low pH together with a high temperature during storage would select for these organisms. Some strains of L. panis and L. pontis showed prolonged survival at pH 2.5 in synthetic stomach juice and good growth in the presence of porcine bile salt. In addition, members of all three species were able to bind to immobilized mucus material in vitro. Especially the isolates from pig feces but, interestingly, some isolates from WWDG as well possessed properties that might be of importance for colonization of the gastrointestinal tracts of pigs.     

Development and Assessment of a Real-Time PCR Assay for Rapid and Sensitive Detection of a Novel Thermotolerant Bacterium, Lactobacillus thermotolerans, in Chicken Feces

A new real-time PCR assay was successfully developed using a TaqMan fluorescence probe for specific detection and enumeration of a novel bacterium, Lactobacillus thermotolerans, in chicken feces. The specific primers and probe were designed based on the L. thermotolerans 16S rRNA gene sequences, and these sequences were compared to those of all available 16S rRNA genes in the GenBank database. The assay, targeting 16S rRNA gene, was evaluated using DNA from a pure culture of L. thermotolerans, DNA from the closely related bacteria Lactobacillus mucosae DSM 13345^T and Lactobacillus fermentum JCM 1173^T, and DNA from other lactic acid bacteria in quantitative experiments. Serial dilutions of L. thermotolerans DNA were used as external standards for calibration. The minimum detection limit of this technique was 1.84 × 10³ cells/ml of an L. thermotolerans pure culture. The assay was then applied to chicken feces in two different trials. In the first trial, the cell population was 10⁴ cells/g feces on day 4 and 10⁵ cells/g feces on days 11 to 18. However, cell populations of 10⁶ to 10⁷ cells/g feces were detected in the second trial. The total bacterial count, measured by 4′,6-diamidino-2-phenylindole (DAPI) staining, was approximately 10¹¹ cells/g feces. These results suggest that in general, L. thermotolerans is a normal member of the chicken gut microbiota, although it is present at relatively low levels in the feces.     

Spelt and emmer flours: characterization of the lactic acid bacteria microbiota and selection of mixed starters for bread making

Aims: This study aimed at characterizing the lactic acid bacteria microbiota and selecting mixed endogenous starters to be used for sourdough fermentation of spelt or emmer flours. Methods and Results: Identification of lactic acid bacteria was carried out by partial sequencing of the 16S rRNA, recA, 16S/23S rRNA spacer region and pheS genes. Spelt flour showed the largest biodiversity, while Lactobacillus plantarum dominated in emmer flour. Isolates were subjected to RAPD‐PCR analysis and screened based on the kinetics of growth and acidification, quotient of fermentation and liberation of free amino acids (FAA) during sourdough fermentation. After selection, mixed starters were used according to a two‐step fermentation process. Wheat flour was fermented by the same starters. Spelt and emmer sourdoughs had slightly higher pH than wheat sourdoughs but titratable acidity, concentration of FAA and phytase activity were higher. Specific volume and crumb grain of emmer and, especially, spelt breads approached those of wheat breads. Sensory analysis confirmed the suitability of spelt and emmer for bread making. Conclusions: The sourdough biotechnology was indispensable to completely exploit the potential of spelt and emmer flours. Significance and Impact of the Study: Results filled up the lack of knowledge on the lactic acid bacteria microbiota and technological performances of spelt and emmer flours.     

Application of a novel polyphasic approach to study the lactobacilli composition of sourdoughs from the Abruzzo region (central Italy)

AIMS: To characterize the lactobacilli community of 20 sourdoughs using a novel polyphasic approach. METHODS AND RESULTS: A polyphasic approach, consisting of a two-step multiplex polymerase chain reaction (PCR) system, 16S rRNA gene sequence analysis and physiological features, was applied to identify 127 isolates, representing about 37% of the presumptive lactobacilli collected from sourdough samples. Multiplex PCR successfully identified 111 isolates, while 16S rRNA gene sequencing was applied for the other 16 isolates, two of which could not be associated with any previously described lactic acid bacteria (LAB) species. Strain diversity was evaluated by phenotypic and random amplified polymorphic DNA-PCR analysis. Molecular detection of Lactobacillus group species was also performed on total DNA extracted from the doughs. CONCLUSIONS: Abruzzo region sourdough lactobacilli biodiversity, reflected in both Lactobacillus species composition and strain polymorphism, is similar to that of other Italian regions and is a source of novel LAB species. SIGNIFICANCE AND IMPACT OF THE STUDY: Within culture-independent methods, multiplex PCR is a rapid tool to study the lactobacilli population of sourdoughs.     

Simultaneous Detection and Identification of Common Cell Culture Contaminant and Pathogenic Mollicutes Strains by Reverse Line Blot Hybridization

We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with “universal” primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.     

High-Resolution Melting Curve Analysis for Identification of Pasteurellaceae Species in Experimental Animal Facilities

Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes “Jawetz” and “Heyl”, Actinobacillus muris, and Haemophilus influenzaemurium. We used a set of six reference strains for assay development, with the melting profiles of these strains clearly distinguishable due to DNA sequence variations in the amplicon. For evaluation, we used real-time PCR/HRM to test 25 unknown Pasteurellaceae isolates obtained from an external diagnostic laboratory and found the results to be consistent with those of partial 16S rRNA sequencing. The real-time PCR/HRM method provides a sensitive, rapid, and closed-tube approach for Pasteurellaceae species identification for health monitoring of laboratory mice.     

Influence of Geographical Origin and Flour Type on Diversity of Lactic Acid Bacteria in Traditional Belgian Sourdoughs

A culture-based approach was used to investigate the diversity of lactic acid bacteria (LAB) in Belgian traditional sourdoughs and to assess the influence of flour type, bakery environment, geographical origin, and technological characteristics on the taxonomic composition of these LAB communities. For this purpose, a total of 714 LAB from 21 sourdoughs sampled at 11 artisan bakeries throughout Belgium were subjected to a polyphasic identification approach. The microbial composition of the traditional sourdoughs was characterized by bacteriological culture in combination with genotypic identification methods, including repetitive element sequence-based PCR fingerprinting and phenylalanyl-tRNA synthase (pheS) gene sequence analysis. LAB from Belgian sourdoughs belonged to the genera Lactobacillus, Pediococcus, Leuconostoc, Weissella, and Enterococcus, with the heterofermentative species Lactobacillus paralimentarius, Lactobacillus sanfranciscensis, Lactobacillus plantarum, and Lactobacillus pontis as the most frequently isolated taxa. Statistical analysis of the identification data indicated that the microbial composition of the sourdoughs is mainly affected by the bakery environment rather than the flour type (wheat, rye, spelt, or a mixture of these) used. In conclusion, the polyphasic approach, based on rapid genotypic screening and high-resolution, sequence-dependent identification, proved to be a powerful tool for studying the LAB diversity in traditional fermented foods such as sourdough.     

A one-step reaction for the rapid identification of Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti using oligonucleotide primers designed from the 16S-23S rRNA intergenic sequences

Aims:  Species-specific primers targeting the 16S–23S ribosomal DNA (rDNA) intergenic spacer region (ISR) were designed to rapidly discriminate between Lactobacillus mindensis , Lactobacillus panis , Lactobacillus paralimentarius , Lactobacillus pontis and Lactobacillus frumenti species recently isolated from French sourdough.
Methods and Results:  The 16S–23S ISRs were amplified using primers 16S/p2 and 23S/p7, which anneal to positions 1388–1406 of the 16S rRNA gene and to positions 207–189 of the 23S rRNA gene respectively, Escherichia coli numbering (GenBank accession number V00331 ). Clone libraries of the resulting amplicons were constructed using a pCR2·1 TA cloning kit and sequenced. Species-specific primers were designed based on the sequences obtained and were used to amplify the 16S–23S ISR in the Lactobacillus species considered. For all of them, two PCR amplicons, designated as small ISR (S-ISR) and large ISR (L-ISR), were obtained. The L-ISR is composed of the corresponding S-ISR, interrupted by a sequence containing tRNA^Ile and tRNA^Ala genes. Based on these sequences, species-specific primers were designed and proved to identify accurately the species considered among 30 reference Lactobacillus species tested.
Conclusions:  Designed species-specific primers enable a rapid and accurate identification of L. mindensis , L. paralimentarius , L. panis , L. pontis and L. frumenti species among other lactobacilli.
Significance and Impact of the Study:  The proposed method provides a powerful and convenient means of rapidly identifying some sourdough lactobacilli, which could be of help in large starter culture surveys.     

Identification and Population Dynamics of Yeasts in Sourdough Fermentation Processes by PCR-Denaturing Gradient Gel Electrophoresis

Four sourdoughs (A to D) were produced under practical conditions, using a starter obtained from a mixture of three commercially available sourdough starters and baker's yeast. The doughs were continuously propagated until the composition of the microbiota remained stable. A fungi-specific PCR-denaturing gradient gel electrophoresis (DGGE) system was established to monitor the development of the yeast biota. The analysis of the starter mixture revealed the presence of Candida humilis, Debaryomyces hansenii, Saccharomyces cerevisiae, and Saccharomyces uvarum. In sourdough A (traditional process with rye flour), C. humilis dominated under the prevailing fermentation conditions. In rye flour sourdoughs B and C, fermented at 30 and 40°C, respectively, S. cerevisiae became predominant in sourdough B, whereas in sourdough C the yeast counts decreased within a few propagation steps below the detection limit. In sourdough D, which corresponded to sourdough C in temperature but was produced with rye bran, Candida krusei became dominant. Isolates identified as C. humilis and S. cerevisiae were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively. The yeast species isolated from the sourdoughs were also detected by PCR-DGGE. However, in the gel, additional bands were visible. Because sequencing of these PCR fragments from the gel failed, cloning experiments with 28S rRNA amplicons obtained from rye flour were performed, which revealed Cladosporium sp., Saccharomyces servazii, S. uvarum, an unculturable ascomycete, Dekkera bruxellensis, Epicoccum nigrum, and S. cerevisiae. The last four species were also detected in sourdoughs A, B, and C.     

Toward an International Standard for PCR-Based Detection of Food-Borne Thermotolerant Campylobacters: Assay Development and Analytical Validation

As part of a European research project (FOOD-PCR), we developed a standardized and robust PCR detection assay specific for the three most frequently reported food-borne pathogenic Campylobacter species, C. jejuni, C. coli, and C. lari. Fifteen published and unpublished PCR primers targeting the 16S rRNA gene were tested in all possible pairwise combinations, as well as two published primers targeting the 23S rRNA gene. A panel of 150 strains including target and nontarget strains was used in an in-house validation. Only one primer pair, OT1559 plus 18-1, was found to be selective. The inclusivity and exclusivity were 100 and 97%, respectively. In an attempt to find a thermostable DNA polymerase more resistant than Taq to PCR inhibitors present in chicken samples, three DNA polymerases were evaluated. The DNA polymerase Tth was not inhibited at a concentration of 2% (vol/vol) chicken carcass rinse, unlike both Taq DNA polymerase and DyNAzyme. Based on these results, Tth was selected as the most suitable enzyme for the assay. The standardized PCR test described shows potential for use in large-scale screening programs for food-borne Campylobacter species under the assay conditions specified.     

Monitoring the Bacterial Population Dynamics in Sourdough Fermentation Processes by Using PCR-Denaturing Gradient Gel Electrophoresis

Four sourdoughs (A to D) were produced under practical conditions by using a starter mixture of three commercially available sourdough starters and a baker's yeast constitutively containing various species of lactic acid bacteria (LAB). The sourdoughs were continuously propagated until the composition of the LAB flora remained stable. Two LAB-specific PCR-denaturing gradient gel electrophoresis (DGGE) systems were established and used to monitor the development of the microflora. Depending on the prevailing ecological conditions in the different sourdough fermentations, only a few Lactobacillus species were found to be competitive and became dominant. In sourdough A (traditional process with rye flour), Lactobacillus sanfranciscensis and a new species, L. mindensis, were detected. In rye flour sourdoughs B and C, which differed in the process temperature, exclusively L. crispatus and L. pontis became the predominant species in sourdough B and L. crispatus, L. panis, and L. frumenti became the predominant species in sourdough C. On the other hand, in sourdough D (corresponding to sourdough C but produced with rye bran), L. johnsonii and L. reuteri were found. The results of PCR-DGGE were consistent with those obtained by culturing, except for sourdough B, in which L. fermentum was also detected. Isolates of the species L. sanfranciscensis and L. fermentum were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively.     

Specific Ribosomal DNA Sequences from Diverse Environmental Settings Correlate with Experimental Contaminants

Phylogenetic analysis of 16S ribosomal DNA (rDNA) clones obtained by PCR from uncultured bacteria inhabiting a wide range of environments has increased our knowledge of bacterial diversity. One possible problem in the assessment of bacterial diversity based on sequence information is that PCR is exquisitely sensitive to contaminating 16S rDNA. This raises the possibility that some putative environmental rRNA sequences in fact correspond to contaminant sequences. To document potential contaminants, we cloned and sequenced PCR-amplified 16S rDNA fragments obtained at low levels in the absence of added template DNA. 16S rDNA sequences closely related to the genera Duganella (formerly Zoogloea), Acinetobacter, Stenotrophomonas, Escherichia, Leptothrix, and Herbaspirillum were identified in contaminant libraries and in clone libraries from diverse, generally low-biomass habitats. The rRNA sequences detected possibly are common contaminants in reagents used to prepare genomic DNA. Consequently, their detection in processed environmental samples may not reflect environmentally relevant organisms.     

Bacterial Community Structure and Location in Stilton Cheese

The microbial diversity occurring in Stilton cheese was evaluated by 16S ribosomal DNA analysis with PCR-denaturing gradient gel electrophoresis. DNA templates for PCR experiments were directly extracted from the cheese as well as bulk cells harvested from a variety of viable-count media. The variable V3 and V4-V5 regions of the 16S genes were analyzed. Closest relatives of Lactococcus lactis, Enterococcus faecalis, Lactobacillus plantarum, Lactobacillus curvatus, Leuconostoc mesenteroides, Staphylococcus equorum, and Staphylococcus sp. were identified by sequencing of the DGGE fragments. Fluorescently labeled oligonucleotide probes were developed to detect Lactococcus lactis, Lactobacillus plantarum, and Leuconostoc mesenteroides in fluorescence in situ hybridization (FISH) experiments, and their specificity for the species occurring in the community of Stilton cheese was checked in FISH experiments carried out with reference cultures. The combined use of these probes and the bacterial probe Eub338 in FISH experiments on Stilton cheese sections allowed the assessment of the spatial distribution of the different microbial species in the dairy matrix. Microbial colonies of bacteria showed a differential location in the different parts of the cheese examined: the core, the veins, and the crust. Lactococci were found in the internal part of the veins as mixed colonies and as single colonies within the core. Lactobacillus plantarum was detected only underneath the surface, while Leuconostoc microcolonies were homogeneously distributed in all parts observed. The combined molecular approach is shown to be useful to simultaneously describe the structure and location of the bacterial flora in cheese. The differential distribution of species found suggests specific ecological reasons for the establishment of sites of actual microbial growth in the cheese, with implications of significance in understanding the ecology of food systems and with the aim of achieving optimization of the fermentation technologies as well as preservation of traditional products.