scholarly journals Use of DNA Quantification To Measure Growth and Autolysis of Lactococcus and Propionibacterium spp. in Mixed Populations

2006 ◽  
Vol 72 (9) ◽  
pp. 6174-6182 ◽  
Author(s):  
Janneke Treimo ◽  
Gerd Vegarud ◽  
Thor Langsrud ◽  
Knut Rudi
Keyword(s):  
Dna Degradation ◽  
Rrna Gene ◽  
Competitive Pcr ◽  
Single Nucleotide ◽  
Bacterial Populations ◽  
Cheese Ripening ◽  
Mixed Populations ◽  
Species Specific ◽  
Cheese Model ◽  
Cheese Production

ABSTRACT Autolysis is self-degradation of the bacterial cell wall that results in the release of enzymes and DNA. Autolysis of starter bacteria, such as lactococci and propionibacteria, is essential for cheese ripening, but our understanding of this important process is limited. This is mainly because the current tools for measuring autolysis cannot readily be used for analysis of bacteria in mixed populations. We have now addressed this problem by species-specific detection and quantification of free DNA released during autolysis. This was done by use of 16S rRNA gene single-nucleotide extension probes in combination with competitive PCR. We analyzed pure and mixed populations of Lactococcus lactis subsp. lactis and three different species of Propionibacterium. Results showed that L. lactis subsp. lactis INF L2 autolyzed first, followed by Propionibacterium acidipropionici ATCC 4965, Propionibacterium freudenreichii ISU P59, and then Propionibacterium jensenii INF P303. We also investigated the autolytic effect of rennet (commonly used in cheese production). We found that the effect was highly strain specific, with all the strains responding differently. Finally, autolysis of L. lactis subsp. lactis INF L2 and P. freudenreichii ISU P59 was analyzed in a liquid cheese model. Autolysis was detected later in this cheese model system than in broth media. A challenge with DNA, however, is DNA degradation. We addressed this challenge by using a DNA degradation marker. We obtained a good correlation between the degradation of the marker and the target in a model experiment. We conclude that our DNA approach will be a valuable tool for use in future analyses and for understanding autolysis in mixed bacterial populations.

scholarly journals Bifidobacterial Distribution Across Italian Cheeses Produced from Raw Milk

2019 ◽  
Vol 7 (12) ◽  
pp. 599 ◽  
Author(s):  
Christian Milani ◽  
Giulia Alessandri ◽  
Leonardo Mancabelli ◽  
Gabriele Andrea Lugli ◽  
Giulia Longhi ◽  
...  
Keyword(s):  
Raw Milk ◽  
Fermented Food ◽  
Species Level ◽  
Rrna Gene ◽  
Bacterial Populations ◽  
Microbial Profiling ◽  
Interactive Network ◽  
Microbe Interactions ◽  
Internally Transcribed Spacer ◽  
Species Specific

Cheese microbiota is of high industrial relevance due to its crucial role in defining the organoleptic features of the final product. Nevertheless, the composition of and possible microbe–microbe interactions between these bacterial populations have never been assessed down to the species-level. For this reason, 16S rRNA gene microbial profiling combined with internally transcribed spacer (ITS)-mediated bifidobacterial profiling analyses of various cheeses produced with raw milk were performed in order to achieve an in-depth view of the bifidobacterial populations present in these microbially fermented food matrices. Moreover, statistical elaboration of the data collected in this study revealed the existence of community state types characterized by the dominance of specific microbial genera that appear to shape the overall cheese microbiota through an interactive network responsible for species-specific modulatory effects on the bifidobacterial population.

scholarly journals Structure of Microbial Communities When Complementary Effluents Are Anaerobically Digested

2021 ◽  
Vol 11 (3) ◽  
pp. 1293
Author(s):  
Ana Eusébio ◽  
André Neves ◽  
Isabel Paula Marques
Keyword(s):  
Anaerobic Digestion ◽  
Microbial Communities ◽  
Volatile Fatty Acids ◽  
Olive Mill Wastewater ◽  
Organic Load ◽  
Rrna Gene ◽  
Bacterial Populations ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Olive Mill

Olive oil and pig productions are important industries in Portugal that generate large volumes of wastewater with high organic load and toxicity, raising environmental concerns. The principal objective of this study is to energetically valorize these organic effluents—piggery effluent and olive mill wastewater—through the anaerobic digestion to the biogas/methane production, by means of the effluent complementarity concept. Several mixtures of piggery effluent were tested, with an increasing percentage of olive mill wastewater. The best performance was obtained for samples of piggery effluent alone and in admixture with 30% of OMW, which provided the same volume of biogas (0.8 L, 70% CH4), 63/75% COD removal, and 434/489 L CH4/kg SVin, respectively. The validation of the process was assessed by molecular evaluation through Next Generation Sequencing (NGS) of the 16S rRNA gene. The structure of the microbial communities for both samples, throughout the anaerobic process, was characterized by the predominance of bacterial populations belonging to the phylum Firmicutes, mainly Clostridiales, with Bacteroidetes being the subdominant populations. Archaea populations belonging to the genus Methanosarcina became predominant throughout anaerobic digestion, confirming the formation of methane mainly from acetate, in line with the greatest removal of volatile fatty acids (VFAs) in these samples.

scholarly journals Development and Validation of a Nested-PCR-Denaturing Gradient Gel Electrophoresis Method for Taxonomic Characterization of Bifidobacterial Communities

2003 ◽  
Vol 69 (11) ◽  
pp. 6380-6385 ◽  
Author(s):  
R. Temmerman ◽  
L. Masco ◽  
T. Vanhoutte ◽  
G. Huys ◽  
J. Swings
Keyword(s):  
Nested Pcr ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Temporal Changes ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Gradient Gel Electrophoresis ◽  
Species Specific ◽  
Taxonomic Characterization

ABSTRACT The taxonomic characterization of a bacterial community is difficult to combine with the monitoring of its temporal changes. None of the currently available identification techniques are able to visualize a “complete” community, whereas techniques designed for analyzing bacterial ecosystems generally display limited or labor-intensive identification potential. This paper describes the optimization and validation of a nested-PCR-denaturing gradient gel electrophoresis (DGGE) approach for the species-specific analysis of bifidobacterial communities from any ecosystem. The method comprises a Bifidobacterium-specific PCR step, followed by purification of the amplicons that serve as template DNA in a second PCR step that amplifies the V3 and V6-V8 regions of the 16S rRNA gene. A mix of both amplicons is analyzed on a DGGE gel, after which the band positions are compared with a previously constructed database of reference strains. The method was validated through the analysis of four artificial mixtures, mimicking the possible bifidobacterial microbiota of the human and chicken intestine, a rumen, and the environment, and of two fecal samples. Except for the species Bifidobacterium coryneforme and B. indicum, all currently known bifidobacteria originating from various ecosystems can be identified in a highly reproducible manner. Because no further cloning and sequencing of the DGGE bands is necessary, this nested-PCR-DGGE technique can be completed within a 24-h span, allowing the species-specific monitoring of temporal changes in the bifidobacterial community.

Species-specific duplex PCR for the detection of Pratylenchus penetrans

Nematology ◽  
2009 ◽  
Vol 11 (6) ◽  
pp. 847-857 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nicole Viaene ◽  
Maurice Moens
Keyword(s):  
Internal Control ◽  
Dna Sequences ◽  
Rrna Gene ◽  
Duplex Pcr ◽  
Specific Primers ◽  
5.8S Rrna Gene ◽  
5.8S Rrna ◽  
Wide Range ◽  
Pcr Products ◽  
Species Specific

Abstract ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.

scholarly journals Phylogenetic Diversity and Molecular Detection of Bacteria in Gull Feces

2008 ◽  
Vol 74 (13) ◽  
pp. 3969-3976 ◽  
Author(s):  
Jingrang Lu ◽  
Jorge W. Santo Domingo ◽  
Regina Lamendella ◽  
Thomas Edge ◽  
Stephen Hill
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Phylogenetic Diversity ◽  
Bacterial Species ◽  
Rrna Gene ◽  
Fecal Dna ◽  
Environmental Waters ◽  
Potential Risks ◽  
Species Specific ◽  
Gut Microbial Community

ABSTRACT In spite of increasing public health concerns about the potential risks associated with swimming in waters contaminated with waterfowl feces, little is known about the composition of the gut microbial community of aquatic birds. To address this, a gull 16S rRNA gene clone library was developed and analyzed to determine the identities of fecal bacteria. Analysis of 282 16S rRNA gene clones demonstrated that the gull gut bacterial community is mostly composed of populations closely related to Bacilli (37%), Clostridia (17%), Gammaproteobacteria (11%), and Bacteriodetes (1%). Interestingly, a considerable number of sequences (i.e., 26%) were closely related to Catellicoccus marimammalium, a gram-positive, catalase-negative bacterium. To determine the occurrence of C. marimammalium in waterfowl, species-specific 16S rRNA gene PCR and real-time assays were developed and used to test fecal DNA extracts from different bird (n = 13) and mammal (n = 26) species. The results showed that both assays were specific to gull fecal DNA and that C. marimammalium was present in gull fecal samples collected from the five locations in North America (California, Georgia, Ohio, Wisconsin, and Toronto, Canada) tested. Additionally, 48 DNA extracts from waters collected from six sites in southern California, Great Lakes in Michigan, Lake Erie in Ohio, and Lake Ontario in Canada presumed to be impacted with gull feces were positive by the C. marimammalium assay. Due to the widespread presence of this species in gulls and environmental waters contaminated with gull feces, targeting this bacterial species might be useful for detecting gull fecal contamination in waterfowl-impacted waters.

scholarly journals Multiplex PCR with 16S rRNA Gene-Targeted Primers of Bifidobacterium spp. To Identify Sources of Fecal Pollution

2004 ◽  
Vol 70 (5) ◽  
pp. 3171-3175 ◽  
Author(s):  
X. Bonjoch ◽  
E. Ballesté ◽  
A. R. Blanch
Keyword(s):  
16S Rrna ◽  
Multiplex Pcr ◽  
Fecal Pollution ◽  
Sensitivity Threshold ◽  
Rrna Gene ◽  
Specific Primers ◽  
Animal Wastewater ◽  
Wastewater Samples ◽  
Species Specific ◽  
Different Origins

ABSTRACT Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals and may be used as indicators of human fecal pollution. The presence of nine human-related Bifidobacterium species was analyzed in human and animal wastewater samples of different origins by using species-specific primers based on 16S rRNA sequences. Only B. adolescentis and B. dentium were found exclusively in human sewage. A multiplex PCR approach with strain-specific primers was developed. The method showed a sensitivity threshold of 10 cells/ml. This new molecular method could provide useful information for the characterization of fecal pollution sources.

scholarly journals Loss of Heterozygosity and Base Mutation Rates Vary Among Saccharomyces cerevisiae Hybrid Strains

2020 ◽  
Vol 10 (9) ◽  
pp. 3309-3319 ◽  
Author(s):  
Ajith V Pankajam ◽  
Suman Dash ◽  
Asma Saifudeen ◽  
Abhishek Dutta ◽  
Koodali T Nishant
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Loss Of Heterozygosity ◽  
Mutation Rates ◽  
Single Nucleotide ◽  
Single Nucleotide Mutation ◽  
Genome Wide ◽  
Nucleotide Mutation ◽  
Specific Variation ◽  
Chromosomal Changes ◽  
Species Specific

Abstract A growing body of evidence suggests that mutation rates exhibit intra-species specific variation. We estimated genome-wide loss of heterozygosity (LOH), gross chromosomal changes, and single nucleotide mutation rates to determine intra-species specific differences in hybrid and homozygous strains of Saccharomyces cerevisiae. The mutation accumulation lines of the S. cerevisiae hybrid backgrounds - S288c/YJM789 (S/Y) and S288c/RM11-1a (S/R) were analyzed along with the homozygous diploids RM11, S288c, and YJM145. LOH was extensive in both S/Y and S/R hybrid backgrounds. The S/Y background also showed longer LOH tracts, gross chromosomal changes, and aneuploidy. Short copy number aberrations were observed in the S/R background. LOH data from the S/Y and S/R hybrids were used to construct a LOH map for S288c to identify hotspots. Further, we observe up to a sixfold difference in single nucleotide mutation rates among the S. cerevisiae S/Y and S/R genetic backgrounds. Our results demonstrate LOH is common during mitotic divisions in S. cerevisiae hybrids and also highlight genome-wide differences in LOH patterns and rates of single nucleotide mutations between commonly used S. cerevisiae hybrid genetic backgrounds.

scholarly journals A METAGENOMIC ASSESSMENT OF BACTERIAL CONTAMINATION OF DUST EVENTS IN SENEGAL

2021 ◽  
Vol 9 (03) ◽  
pp. 509-526
Author(s):  
Alioune Marone ◽  
◽  
Malick Mbengue ◽  
Gregory Jenkins ◽  
Demba Ndao Niang ◽  
...  
Keyword(s):  
Respiratory Diseases ◽  
Rrna Gene ◽  
Source Population ◽  
Sequencing Analysis ◽  
Human Pathogens ◽  
Sequencing Data ◽  
Bacterial Populations ◽  
African Dust ◽  
Hypervariable Regions ◽  
Dust Events

Previous work in the Caribbean and West Africa have shown that air samples taken during dust events contain microorganisms (bacteria, fungi, viruses), including human pathogens that can cause many respiratory diseases. To better understand the potential downstream effect of bacteria dust on human health and public ecosystems, it is important to characterize the source population. In this study, we aimed to explore the bacterial populations of African dust samples collected between 2013-2017. The dust samples were collected using the spatula method, then the hypervariable regions (V3 and V4) of the 16S rRNA gene were amplified using PCR followed byMiSeq Illumina sequencing. Analysis of the sequencing data were performed using MG-RAST. At the phylum level, the proportions of Actinobacteria (22%), Firmicutes (20%), Proteobacteria (19%), and Bacteroidetes (13%) were respectively predominant in all dust samples. At the genus level, Bacillus(16%), Pseudomonas(10%), Nocardiodes and Exiguobacterium (5%) are the most dominated genera in African dust samples collected in this study.The study showed that molecular characterization of dust microbial population remains a very efficient method, also applicable to the search for viruses and fungi in this type of sample. It is important to note that the majority of microorganisms identified in this study can cause respiratory diseases.

scholarly journals Adaptation of Microbial Communities to Environmental Arsenic and Selection of Arsenite-Oxidizing Bacteria From Contaminated Groundwaters

2021 ◽  
Vol 12 ◽  
Author(s):  
Sarah Zecchin ◽  
Simona Crognale ◽  
Patrizia Zaccheo ◽  
Stefano Fazi ◽  
Stefano Amalfitano ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Inorganic Arsenic ◽  
16S Rrna Gene Sequencing ◽  
Northern Italy ◽  
Rrna Gene ◽  
Bacterial Populations ◽  
Main Driver ◽  
Rrna Gene Sequencing ◽  
Arsenite Oxidizing Bacteria ◽  
Card Fish

Arsenic mobilization in groundwater systems is driven by a variety of functionally diverse microorganisms and complex interconnections between different physicochemical factors. In order to unravel this great ecosystem complexity, groundwaters with varying background concentrations and speciation of arsenic were considered in the Po Plain (Northern Italy), one of the most populated areas in Europe affected by metalloid contamination. High-throughput Illumina 16S rRNA gene sequencing, CARD-FISH and enrichment of arsenic-transforming consortia showed that among the analyzed groundwaters, diverse microbial communities were present, both in terms of diversity and functionality. Oxidized inorganic arsenic [arsenite, As(III)] was the main driver that shaped each community. Several uncharacterized members of the genus Pseudomonas, putatively involved in metalloid transformation, were revealed in situ in the most contaminated samples. With a cultivation approach, arsenic metabolisms potentially active at the site were evidenced. In chemolithoautotrophic conditions, As(III) oxidation rate linearly correlated to As(III) concentration measured at the parental sites, suggesting that local As(III) concentration was a relevant factor that selected for As(III)-oxidizing bacterial populations. In view of the exploitation of these As(III)-oxidizing consortia in biotechnology-based arsenic bioremediation actions, these results suggest that contaminated aquifers in Northern Italy host unexplored microbial populations that provide essential ecosystem services.

scholarly journals Fitness Trade-Offs in blaTEM Evolution

2008 ◽  
Vol 52 (7) ◽  
pp. 2340-2345 ◽  
Author(s):  
Joanna E. Mroczkowska ◽  
Miriam Barlow
Keyword(s):  
Microbial Populations ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Bacterial Populations ◽  
Real Time Quantitative Pcr ◽  
Fitness Effects ◽  
Fitness Advantage ◽  
Extended Spectrum ◽  
Trade Offs ◽  
Pcr Method

ABSTRACT bla TEM-1 expression results in penicillin resistance, whereas expression of many bla TEM-1 descendants, called extended-spectrum β-lactamases (ESBLs), results simultaneously in resistance to penicillins and extended-spectrum cephalosporins. Despite the expanded resistance phenotypes conferred by many ESBLs, bla TEM-1 is still the most abundant bla TEM allele in many microbial populations. This study examines the fitness effects of the two amino acid substitutions, R164S and E240K, that have occurred repeatedly among ESBL bla TEM-1 descendants. Using a single-nucleotide polymorphism-specific real-time quantitative PCR method, we analyzed the fitness of strains expressing bla TEM-1, bla TEM-10, and bla TEM-12. Our results show that bacteria expressing the ancestral bla TEM-1 allele have a fitness advantage over those expressing either bla TEM-10 or bla TEM-12 when exposed to ampicillin. This observation, combined with the fact that penicillins are the most prevalent antimicrobials prescribed worldwide, may explain why bla TEM-1 has persisted as the most frequently encountered bla TEM allele in bacterial populations.

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