Community DNA Extraction Research Articles

Community DNA Extraction from Bacterial Colonies

Community DNA Extraction from Bacterial Colonies

Two-Dose Ceftiofur Treatment Increases Cephamycinase Gene Quantities and Fecal Microbiome Diversity in Dairy Cows Diagnosed with Metritis.

Antimicrobial resistance is a significant concern worldwide; meanwhile, the impact of 3rd generation cephalosporin (3GC) antibiotics on the microbial communities of cattle and resistance within these communities is largely unknown. The objectives of this study were to determine the effects of two-dose ceftiofur crystalline-free acid (2-CCFA) treatment on the fecal microbiota and on the quantities of second-and third-generation cephalosporin, fluoroquinolone, and macrolide resistance genes in Holstein-Friesian dairy cows in the southwestern United States. Across three dairy farms, 124 matched pairs of cows were enrolled in a longitudinal study. Following the product label regimen, CCFA was administered on days 0 and 3 to cows diagnosed with postpartum metritis. Healthy cows were pair-matched based on lactation number and calving date. Fecal samples were collected on days 0, 6, and 16 and pooled in groups of 4 (n = 192) by farm, day, and treatment group for community DNA extraction. The characterization of community DNA included real-time PCR (qPCR) to quantify the following antibiotic resistance genes: blaCMY-2, blaCTX-M, mphA, qnrB19, and the highly conserved 16S rRNA back-calculated to gene copies per gram of feces. Additionally, 16S rRNA amplicon sequencing and metagenomics analyses were used to determine differences in bacterial community composition by treatment, day, and farm. Overall, blaCMY-2 gene copies per gram of feces increased significantly (p ≤ 0.05) in the treated group compared to the untreated group on day 6 and remained elevated on day 16. However, blaCTX-M, mphA, and qnrB19 gene quantities did not differ significantly (p ≥ 0.05) between treatment groups, days, or farms, suggesting a cephamycinase-specific enhancement in cows on these farms. Perhaps unexpectedly, 16S rRNA amplicon metagenomic analyses showed that the fecal bacterial communities from treated animals on day 6 had significantly greater (p ≤ 0.05) alpha and beta diversity than the untreated group. Two-dose ceftiofur treatment in dairy cows with metritis elevates cephamycinase gene quantities among all fecal bacteria while paradoxically increasing microbial diversity.

Microbial structure and function diversity of open dumpsite compost used as fertilizer by peasant farmers

A study was carried out to assess the microbial communities and functions in addition to several key environmental variables (pH, temperature, carbon, nitrogen, carbon to nitrogen ratio, and moisture) of open dumpsite compost. Sampling was done on days 45 and 90 following composting of the organic fraction of the municipal solid waste (MSW). Following community DNA extraction, sequencing was done on Illumina Miseq platform. Pair-ends of the sequences reads were assembled using the make-contigs tool while open reading frame (ORFs) was done using FragGeneScan and these were functionally annotated to several bioinformatics pipelines. Structural composition revealed 5 kingdoms namely bacteria, protozoa, unknown, archaea, and fungi but with varying degree of abundance. After unknown phyla, proteobacteria was the second dominant phyla with abundance of 42.45 and 40.30% , respectively on days 45 and 90 and they were well represented in the class, order and family taxas in both samples. Majority of the genus in both samples were uncultured genus. However, potential pathogenic species belonging to the genuses Clostridium, Escherichia, Pseudomonas, Enterococcus, Micrococcus, Shigella, Klebsiella and Bacillus amongst other were detected in our samples on 90days. Physiological diversity assessment revealed that carbohydrate (CHO) metabolisms were the most dominant and was almost similar in both samples. At day 45, the CHO metabolisms were dominated by phylotypes belonging to the alphaproteobacteria and betaproteobacteria classes of bacteria but it was more diverse on day 90 as Chlorobi, Gammaproteobacteria, Firmicutes, Actinobacteria, Tenericutes, and Planctomycetes were also involved. Other metabolisms such as amino acid, nucleotides, vitamins and co-factors, xenobiotics, genetic information and well as environmental information processing were also captured. This practice should be discouraged to avoid incidence of food-borne diseases.

Adaptions of Lichen Microbiota Functioning Under Persistent Exposure to Arsenic Contamination.

Host-associated microbiota play an important role in the health and persistence of more complex organisms. In this study, metagenomic analyses were used to reveal microbial community adaptations in three lichen samples as a response to different arsenic concentrations at the sampling sites. Elevated arsenic concentrations at a former mining site expanded the spectrum and number of relevant functions in the lichen-associated microorganisms. Apparent changes affected the abundance of numerous detoxification-related genes, they were substantially enhanced in arsenic-polluted samples. Complementary quantifications of the arsenite S-adenosylmethionine methyltransferase (arsM) gene showed that its abundance is not strictly responding to the environmental arsenic concentrations. The analyzed samples contained rather low numbers of the arsM gene with a maximum of 202 gene copies μl-1 in total community DNA extracts. In addition, bacterial isolates were screened for the presence of arsM. Positive isolates were exposed to different As(III) and As(V) concentrations and tolerated up to 30 mM inorganic arsenic in fluid media, while no substantial biotransformations were observed. Obtained data deepens our understanding related to adaptions of whole microbial communities to adverse environmental conditions. Moreover, this study provides the first evidence that the integrity of bacteria in the lichen holobiont is maintained by acquisition of specific resistances.

Modifications and optimization of manual methods for polymerase chain reaction and 16S rRNA gene sequencing quality community DNA extraction from goat rumen digesta.

Background and Aim:A critical prerequisite for studying rumen microbial community by high throughput molecular biology methods is good quality community DNA. Current methods of extraction use kits designed for samples from the different origin for rumen. This puts stress on the development of a relevant manual method for DNA extraction. The objective of this study was to modify the existing methods of community DNA extraction and thereby systematic comparison of their efficiency based on DNA yield, purity, 16S rRNA gene sequencing, and identification to determine the optimal DNA extraction methods whose DNA products reflect targeted bacterial communities special to rumen.Materials and Methods:Enzymatic method, Chemical method, Enzymatic + Chemical method, and Enzymatic + Chemical + Physical method were modified toward evaluation of community DNA extraction from solid, squeezed, and liquid fractions of goat rumen digesta. Each method was assessed critically for nucleic acid yield and its quality. The methods resulting in high nucleic acid yield, optimal purity ratios with intact band on agarose gel electrophoresis were optimized further. Optimized methods were studied using standard polymerase chain reaction (PCR) with universal bacterial primers and 16S rRNA primers of targeted rumen bacteria. Methods denoting the presence of targeted rumen bacteria were assessed further with 16S rRNA gene sequencing and identification studies. It led toward methods efficacy estimation for molecular biology applications. Effect of rumen sample preservation on community DNA extraction was also studied. Their mean standard deviation values were calculated to understand sampling criticality.Results:Modified Chemical method (Cetrimonium bromide) and Enzymatic+Chemical+Physical (ECP) method (Lysozyme-Cetrimonium bromide-Sodium Dodecyl Sulfate-freeze-thaw) could extract 835 ng/µl and 161 ng/µl community DNA from 1.5 g solid and 2 ml squeezed rumen digesta with purity ratios of 1.8 (A260 nm/A280 nm) and 2.3 (A260 nm/A230 nm) respectively. Comparative analysis showed the better efficiency of ECP method and chemical method toward freshly squeezed rumen digesta and solid rumen digesta. However, sample preservation at −80°C for 1.5 months drastically affected the yield and purity ratios of community DNA. New protocol revealed targeted microbial community having Gram-positive as well as Gram-negative bacteria such as Prevotella ruminicola, Streptococcus lutetiensis, Ruminococcus flavefaciens, Fibrobacter succinogenes, and Selenomonas ruminantium.Conclusion:To date, this is the first report of modified methods wherein least chemicals and steps lead toward PCR and 16S rRNA gene sequencing quality community DNA extraction from goat rumen digesta. Detection of targeted rumen bacteria in solid and squeezed rumen digesta proves their strongest association with rumen fiber mat. It also marks the presence of distinct microbial communities in solid and squeezed rumen fractions that in turn differs the performance of each different method employed and yield of nucleic acid obtained. It also leaves a possibility of the presence of complex microbial consortia in squeezed rumen digesta whose DNA extraction methods need more attention. Finally, manual protocols of community DNA extraction may vary in different ruminant which suggests undertaking rigorous research in their establishment.

CTAB influenced differential elution of metagenomic DNA from saltpan and marine sediments.

A simple, reliable method for genomic DNA extraction from sediments with minimum contaminants was developed to address the risk of poor quality DNA in metagenomic studies. Nine DNA extraction methods using 20% cetyl-trimethyl-ammonium bromide (CTAB) were performed and compared to develop an extraction protocol that can offer humic acid-free metagenomic DNA from marine and saltpan sediments. Community DNA extraction was executed via., Zhou et al. modified protocol using 20% CTAB treatment at different steps to compare the efficacy of humic acid removal. Out of nine DNA extraction methods, method 6 significantly improved the quality of DNA with efficient removal of humic substances. 16S rRNA gene amplification and spectrophotometric analysis confirmed the efficiency of method 6 to remove DNA inhibitors from marine sediments as well as saltpan samples. Inhibitors extracted along with metagenomic DNA outcome increased DNA yield and PCR inhibition in method 1 and 3. However, repeated 20% CTAB wash in method 6 ensured 16S amplification and least yield and concentration. Current study explains a detailed protocol based on 20% CTAB wash for the extraction of humic acid-free DNA from diverse sediment samples.

Sampling, Extraction, and High-Throughput Sequencing Methods for Environmental Microbial and Viral Communities.

The emergence of high-throughput sequencing technologies has deepened our understanding of complex microbial communities and greatly facilitated the study of as-yet uncultured microbes and viruses. Studies of complex microbial communities require high-quality data to generate valid results. Here, we detail current methods of microbial and viral community sample acquisition, DNA extraction, sample preparation, and sequencing on Illumina high-throughput platforms. While using appropriate analytical tools is important, it must not overshadow the need for establishing a proper experimental design and obtaining sufficient numbers of samples for statistical purposes. Researchers must also take care to sample biologically relevant sites and control for potential confounding factors (e.g., contamination).

Endophytic Microbial Community DNA Extraction from the Plant Phyllosphere.

The plant phyllosphere, which represents all plant parts that are above the ground, is considered one of the most extensive ecosystems to be colonized by microorganisms, both at the surface as epiphytes or as endophytes within the plant. These plant-associated microbial communities are reservoirs of microbial diversity and they can be important for plant health. The characterization of microbial communities in diverse plants, such as Espeletia plants that are endemic to the Paramo ecosystem in the Andes Mountains, can shed light regarding possible interactions among microorganisms and microbial functional properties. Obtaining DNA from plant endophytic microbial communities involves various steps to ensure that samples are free of contamination from microorganisms present on the plant surface (epiphytes). Plant leaves are first surface sterilized, cut into pieces, homogenized using glass beads, and then used for DNA extraction using a commercially available kit. DNA samples are then quantified and analyzed using Qubit® 2.0 for use in PCR amplification of 16S rRNA genes.

Modification of extraction method for community DNA isolation from salt affected compact wasteland soil samples

To overcome the issue of interferences by salt and compactness in release of bacterial cell required for lysis, method described by Yeates et al. (1998), was optimized for isolation of genomic material (Deoxyribo Nucleic Acid, DNA) from soil microbial community by addition of Al(NH4)SO4. Very low total viable count was observed in the samples tested and hence use of higher amount of soil is required primarily for DNA isolation from wasteland soils. The method proves itself efficient where commercially available bead beating and enzymatic lysis methods could not give isolation of any amount of community genomic DNA due to compact nature and salt concentrations present in soil.•The protocol was found efficient for soil samples with high clay content for microbial community DNA extraction.•Variation in lysis incubation and amount of soil may help with soil samples containing low microbial population.•Addition of Al(NH4)SO4 is crucial step in humic acid removal from extracted DNA samples for soil samples containing high salinity and clay particles.

An Improved Method for High Quality Metagenomics DNA Extraction from Human and Environmental Samples.

To explore the natural microbial community of any ecosystems by high-resolution molecular approaches including next generation sequencing, it is extremely important to develop a sensitive and reproducible DNA extraction method that facilitate isolation of microbial DNA of sufficient purity and quantity from culturable and uncultured microbial species living in that environment. Proper lysis of heterogeneous community microbial cells without damaging their genomes is a major challenge. In this study, we have developed an improved method for extraction of community DNA from different environmental and human origin samples. We introduced a combination of physical, chemical and mechanical lysis methods for proper lysis of microbial inhabitants. The community microbial DNA was precipitated by using salt and organic solvent. Both the quality and quantity of isolated DNA was compared with the existing methodologies and the supremacy of our method was confirmed. Maximum recovery of genomic DNA in the absence of substantial amount of impurities made the method convenient for nucleic acid extraction. The nucleic acids obtained using this method are suitable for different downstream applications. This improved method has been named as the THSTI method to depict the Institute where the method was developed.

Back to the Future of Soil Metagenomics.

Direct extraction and characterization of microbial community DNA through PCR amplicon surveys and metagenomics has revolutionized the study of environmental microbiology and microbial ecology. In particular, metagenomic analysis of nucleic acids provides direct access to the genomes of the “uncultivated majority.” Accelerated by advances in sequencing technology, microbiologists have discovered more novel phyla, classes, genera, and genes from microorganisms in the first decade and a half of the twenty-first century than since these “many very little living animalcules” were first discovered by van Leeuwenhoek (Table 1). The unsurpassed diversity of soils promises continued exploration of a range of industrial, agricultural, and environmental functions. The ability to explore soil microbial communities with increasing capacity offers the highest promise for answering many outstanding who, what, where, when, why, and with whom questions such as: Which microorganisms are linked to which soil habitats? How do microbial abundances change with changing edaphic conditions? How do microbial assemblages interact and influence one another synergistically or antagonistically? What is the full extent of soil microbial diversity, both functionally and phylogenetically? What are the dynamics of microbial communities in space and time? How sensitive are microbial communities to a changing climate? What is the role of horizontal gene transfer in the stability of microbial communities? Do highly diverse microbial communities confer resistance and resilience in soils?

Enhanced method for microbial community DNA extraction and purification from agricultural yellow loess soil.

In this study, novel DNA extraction and purification methods were developed to obtain high-quantity and reliable quality DNA from the microbial community of agricultural yellow loess soil samples. The efficiencies of five different soil DNAextraction protocols were evaluated on the basis of DNA yield, quality and DNA shearing. Our suggested extraction method, which used CTAB, EDTA and cell membrane lytic enzymes in the extraction followed by DNA precipitation using isopropanol, yielded a maximum DNA content of 42.28 ± 5.59 µg/g soil. In addition, among the five different purification protocols, the acid-treated polyvinyl polypyrrolidone (PVPP) spin column purification method yielded high-quality DNA and recovered 91% of DNA from the crude DNA. Spectrophotometry revealed that the ultraviolet A 260/A 230 and A 260/A 280 absorbance ratios of the purified DNA were 1.82 ± 0.03 and 1.94 ± 0.05, respectively. PCR-based 16S rRNA amplification showed clear bands at ~1.5 kb with acid-treated PVPP-purified DNA templates. In conclusion, our suggested extraction and purification protocols can be used to recover high concentration, high purity, and high-molecular-weight DNA from clay and silica-rich agricultural soil samples.

Microbial Diversity of a Remote Aviation Fuel Contaminated Sediment of a Lentic Ecosystem in Ibeno, Nigeria

Environmental pollution from Oil & Gas Exploration & Production (O&G E&P) activities remains one of the major problems in the oil-producing communities of Nigeria. This results from improper oily wastes disposal as well as incessant oil spills in the region. The operator’s lack of responsible business practices in wastes management and the over-dependence of the economy on oil and gas earnings, in the most part, exacerbate the problems of environmental pollution. Government's lack of political will power which treats issues on oil pollution with levity and long period of neglect of these polluted sites leave the environment ecologically destabilized. Studies to ascertain the ecological status of remote aviation fuel-contaminated sediment of a lentic ecosystem in Inua Eyet Ikot village, Ibeno, Nigeria, have been carried out using conventional microbiological culture-dependent methods. This methodology is known to reveal only <1% of the microbial diversity present. These results were therefore considered inaccurate and grossly misleading. In this present study, sediment samples from this lentic ecosystem in Ibeno, Nigeria previously polluted by Aviation fuel in 2001 (about 14 years ago) were collected and analyzed to assess its prokaryotic diversity using both the conventional culture-dependent as well as culture-independent (16S metagenomic) techniques involving community DNA extraction, DNA sequencing and bioinformatics analyses. The culture-dependent techniques revealed the presence of only six genera of bacteria and no archaea was detected. The 16S metagenomic methods revealed that the sediment of the polluted lentic ecosystem harbors a much more diverse population of bacteria and archaea. These results corroborate the ‘great plate count anomaly’ principle and demonstrate that the use of 16S metagenomic tools will redefine the actual ecological status of the environment.

Spatiotemporal variability in archaeal communities of tropical coastal waters

Spatiotemporal variations in the archaeal community structure from four locations along the central west coast of India were analyzed by denaturing gradient gel electrophoresis (DGGE) profiling and sequencing of prominent bands on the DGGE gels. A total of 36 water samples were collected during pre-monsoon, post-monsoon and monsoon seasons from three depths (surface, mid-depth , and close to bottom ∼20 m). The community DNA extracts from these samples were subjected to DGGE analysis. The results of this study clearly indicate a significant difference in the community structure between sampling locations and seasons. Location-wise variation was more pronounced during pre-monsoon and post-monsoon seasons. During monsoon, however, the depth-wise variation was pronounced, suggesting monsoon influence on archaeal community structure. Sequencing of DGGE bands suggested the presence of marine group I and II archaea. Canonical correspondence analysis indicated that nitrate, nitrite and chlorophyll a were the significant explanatory variables responsible for > 60 % variation in archaeal community in these coastal waters.

Comparison of methods for total community DNA extraction and purification from soilless substrate

Four protocols of soilless substrate DNA extraction were tested in three soilless substrates with different characterization, in order to find an efficient unbiased DNA extraction procedure which could analyze the microbial community structure in soilless substrate samples. Total DNA was extracted from each soilless substrate by the four protocols, and then was purified by Sephadex G-200 spin column after being precipitated by PEG8000. The results showed that more than 97.5% of the humic acids were also removed by Sephadex G-200 spin column. The agarose gel electrophoresis showed that the fragments of crude and purified DNA had a length of about 23 kb. In addition, the restriction map digested by restriction endonucleases showed that each of the protocols was adapted to extract microbial genome DNA from soilless substrate samples. Moreover, the protocol iii showed more bands by denaturing gradient gel electrophoresis analysis and was adapted to extract microbial genome DNA from soilless substrates with higher organic matter and humic.

Natural zinc enrichment in peatlands: Biogeochemistry of ZnS formation

Peatlands effectively retain heavy metals and prevent stream and watershed contamination. Sulfate reduction is considered the most significant process of metal immobilization in natural wetlands and microbial sulfate reduction is the presumed mechanism that results in the precipitation of metal sulfides. In this study, we examined the biogeochemical mechanisms involved in zinc retention and accumulation in a metalliferous peatland of western New York. In the reducing conditions of these peatlands zinc sulfides occurred as framboidal aggregates of sphalerite and polytypic wurtzite (2nH, n⩾2) nanocrystallites associated with bacterial cells and organic matter. Bacterial cells were co-located with ZnS inside peat particles where the microenvironment remained anoxic. The peat zinc sulfide was depleted in 34S isotopes relative to the sulfate supplied to the peatland by 18–34 per mill, implicating its biological formation. Extraction of microbial community DNA from peat samples yielded diverse PCR amplicons from dissimilatory sulfite reductase (dsrAB) genes, indicating varied bacterial taxa capable of reducing forms of oxidized sulfur. Nanocrystals with distinct structural features were observed in samples containing contrasting dsrAB sequences. The results of this investigation provide clear evidence that microorganisms can influence the chemical forms of heavy metals in peatland environments. Our findings also provide insight into the conditions necessary to promote the immobilization of chalcophile elements in engineered systems for the treatment of acid mine drainage and wastewater effluents.

Diversity of the Predominant Spoilage Bacteria in Water‐Boiled Salted Duck during Storage

The spoilage microbiota in water-boiled salted duck during storage at 4 degrees C was determined using culture-dependent and independent methods. Analysis of the denaturing gradient gel electrophoresis (DGGE) patterns of PCR amplicons targeting the V3 region of the 16S rDNA and sequencing of the bands allowed profiling of the microbiota present in the duck. Community DNA extracts were prepared directly from water-boiled salted duck and from culturable bacterial fractions harvested from both MRS and PCA media. The spoilage bacteria mainly consisted of Staphylococcus saprophyticus, Macrococcus caseolyticus, Weissella, Halomonas sp. or Cobetia sp., and Exiguobacterium sp. based on sequencing and homology search of the DGGE bands. It appeared that both the bacterial counts and diversity increased during storage time. By plating method, bacterial counts in MRS agar increased from 10(4) to 10(8) CFU/g from day 1 to 10, while total bacterial counts in PCA agar reached 10(9) CFU/g after 10 d. Total of 14 strains isolated from PCA and MRS agar were identified as M. caseolyticus (2), S. saprophyticus (7), S. sciuri (1), W. paramesenteroides (2), and W. confusa (2) by 16S rDNA sequencing. The identification of the spoilage-related microbiota is helpful to better understand the bacteria ecology in water-boiled salted duck and may lead to the discovery of appropriate preservation strategies.

Microbiological pattern of arterial catheters in the intensive care unit.

BackgroundIntravascular catheter related infection (CRI) is one of the most serious nosocomial infections. Diagnostic criteria include a positive culture from the catheter tip along with blood, yet in many patients with signs of infection, current culture techniques fail to identify pathogens on catheter segments. We hypothesised that a molecular examination of the bacterial community on short term arterial catheters (ACs) would improve our understanding of the variety of organisms that are present in this niche environment and would help develop new methods for the diagnosis of CRI.ResultsThe whole bacterial community presenting on all ACs was evaluated by molecular methods, i.e., a strategy of whole community DNA extraction, PCR amplification followed by cloning and 16S rDNA sequence analysis. Ten ACs were removed from patients suspected of CRI and 430 clones from 5 "colonised" and 5 "uncolonised" (semi-quantitative method) AC libraries were selected for sequencing and subsequent analysis. A total of 79 operational taxonomic units (OTUs) were identified at the level of 97% similarity belonging to six bacterial divisions. An average of 20 OTUs were present in each AC, irrespective of colonisation status. Conventional culture failed to reveal the majority of these bacteria.ConclusionsThere was no significant difference in the bacterial diversity between the 'uncolonised' and 'colonised' ACs. This suggests that vascular devices cultured conventionally and reported as non infective may at times potentially be a significant source of sepsis in critically ill patients. Alternative methods may be required for the accurate diagnosis of CRI in critically ill patients.

Modern analogues and the early history of microbial life

Revealing the geological history of microbial life is very challenging. Microbes rarely are preserved with morphological fidelity, and even when they are, morphology is a poor guide to phylogeny and metabolism. Biological studies of environments considered analogous to those of paleobiological interest on the ancient Earth can inform interpretations and suggest new approaches. This paper reviews recent advances in our understanding of the biological diversity of two environments relevant to Archean paleobiology: those of extreme acidity and temperature (the Mt. Hood and White Island volcanoes), and high salinity (living stromatolites in Shark Bay). The combination of traditional microbial isolation with the use of modern molecular techniques has revealed that the microbial communities in these environments are much more diverse than originally thought. Through the extraction of whole microbial community DNA, enzymatic amplification of evolutionarily conserved genes, and cloning and sequencing of these genes, more specific and informed inferences concerning functional complexity in these extreme environments have now been made. Studies of the modern stromatolites have demonstrated that they have a very diverse range of micoorganisms, and contrary to previous interpretations, cyanobacteria are not the most abundant microbes present. In addition, many of the microorganisms are unique with no known close relatives, and these microorganisms may also possess novel physiologies vital to the integrity and persistence of stromatolites through space and time. Microbes in the volcanoes studied are present ubiquitously and include geochemically significant sulfur- and iron-cycling taxa. The findings from the studies reviewed here suggest that the Archean biota may have been functionally diverse and much more complex than has yet been revealed. The importance of studying modern analogues is stressed in that the biogeochemical processes occurring in these communities leave morphological, mineralogical, lipid and isotopic signals that could be sought in the rock record.

Diversity of lactic acid bacteria from modified atmosphere packaged sliced cooked meat products at sell-by date assessed by PCR-denaturing gradient gel electrophoresis

The predominant lactic acid bacteria (LAB) microbiota associated with three types of modified atmosphere packaged (MAP) sliced cooked meat products (i.e. ham, turkey and chicken) was analyzed at sell-by date using a combination of culturing and molecular population fingerprinting. Likewise routine analyses during industrial MAP production, meat samples were plated on the general heterotrophic Plate Count Agar (PCA) and on the LAB-specific de Man, Rogosa, Sharpe (MRS) agar under different temperature and atmosphere conditions. Subsequently, community DNA extracts were prepared from culturable bacterial fractions harvested from both media and used for PCR targeting the V3 hyper-variable region of the 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) of PCR amplicons (PCR-DGGE). Irrespective of aerobic or anaerobic incubation conditions, V3-16S rDNA DGGE fingerprints of culturable fractions from PCA and MRS medium displayed a high level of similarity indicating that LAB constituted the most dominant group in the culturable bacterial community. Comparison of DGGE profiles of fractions grown at 20, 28 or 37 °C indicated that part of the culturable community consisted of psychrotrophs. Four DGGE bands were common among cooked ham, turkey and chicken products, suggesting that these represent the microbiota circulating in the plant where all three MAP product types were sliced and packaged. Based on band sequencing and band position analysis using LAB reference strains, these four bands could be assigned to Lactobacillus sakei and/or the closely related Lactobacillus fuchuensis, Lactobacillus curvatus, Carnobacterium divergens and Leuconostoc carnosum. In conclusion, the PCR-DGGE approach described in this study allows to discriminate, identify and monitor core and occasional LAB microbiota of MAP sliced cooked meat products and provides valuable complementary information to the current plating procedures routinely used in industrial plants.