Molecular Approaches for the Measurement of Density, Diversity, and Phylogeny

Liu, Wen Tso; Stahl, David A.

doi:10.1128/9781555815882.ch12

Cited by 24 publications

(16 citation statements)

References 168 publications

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“…For example, the accuracy of the API 20E kit at identifying Enterobacteriaceae is approximately 80 to 90% for cultures obtained from clinical laboratories (41). Our findings and recent studies support the notion that 16S rRNA genes can serve as a more-robust approach than traditional biochemical tests for identifying TE to the genus or species level (42,43). By use of 16S rRNA gene-based phylogeny, TE could be successfully classified into at least 32 clusters and could be differentiated from NTE clusters (Fig.…”

Section: Figsupporting

confidence: 72%

Phenotypic and Phylogenetic Identification of Coliform Bacteria Obtained Using 12 Coliform Methods Approved by the U.S. Environmental Protection Agency

Zhang

Hong

LeChevallier

et al. 2015

Appl Environ Microbiol

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The current definition of coliform bacteria is method dependent, and when different culture-based methods are used, discrepancies in results can occur and affect the accuracy of identification of true coliforms. This study used an alternative approach to the identification of true coliforms by combining the phenotypic traits of the coliform isolates and the phylogenetic affiliation of 16S rRNA gene sequences with the use of lacZ and uidA genes. A collection of 1,404 isolates detected by 12 U.S. Environmental Protection Agency-approved coliform-testing methods were characterized based on their phylogenetic affiliations and responses to their original isolation media and lauryl tryptose broth, m-Endo, and MI agar media. Isolates were phylogenetically classified into 32 true-coliform, or targeted Enterobacteriaceae (TE), groups and 14 noncoliform, or nontargeted Enterobacteriaceae (NTE), groups. It was shown statistically that detecting true-positive (TP) events is more challenging than detecting true-negative (TN) events. Furthermore, most false-negative (FN) events were associated with four TE groups (i.e., Serratia group I and the Providencia, Proteus, and Morganella groups) and most false-positive (FP) events with two NTE groups, the Aeromonas and Plesiomonas groups. In Escherichia coli testing, 18 out of 145 E. coli isolates identified by enzymatic methods were validated as FN. The reasons behind the FP and FN reactions could be explained through analysis of the lacZ and uidA genes. Overall, combining the analyses of the 16S rRNA, lacZ, and uidA genes with the growth responses of TE and NTE on culture-based media is an effective way to evaluate the performance of coliform detection methods. Total coliform bacteria and Escherichia coli are considered costeffective bacterial indicators for the protection of public health. Detectable total coliforms indicate potential contamination associated with the water distribution system, while E. coli is a good indicator of fecal contamination, with a health goal (i.e., maximum contaminant level goal [MCLG]) of zero under the Revised Total Coliform Rule (RTCR) (1). When a positive result of total-coliform testing is observed, public water systems (PWSs) are required to collect and analyze three repeat samples (1). A system assessment is required when a PWS exceeds a specific frequency of total-coliform occurrence. When an E. coli maximum contaminant level (MCL) violation incurs, a PWS must have an assessment performed by the state or a state-approved entity and must correct any sanitary defects (1). False-positive (FP) results for either total-coliform or E. coli tests impose an unnecessary burden on water utilities. On the other hand, false-negative (FN) results expose consumers to potential health threats and delay response times for effective action. Therefore, accurate detection of total coliforms and E. coli in drinking water systems is crucial for both water utilities and consumers.To date, the U.S. EPA has approved 12 methods for the detection of coliform bacteria i...

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Section: Figsupporting

confidence: 72%

Phenotypic and Phylogenetic Identification of Coliform Bacteria Obtained Using 12 Coliform Methods Approved by the U.S. Environmental Protection Agency

Zhang

Hong

LeChevallier

et al. 2015

Appl Environ Microbiol

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“…The core of the molecular analyses was the 16S rRNA-based community fingerprints that allowed us to monitor the community, identify the active members due to "highlighted" fingerprint bands, and use the sequences of the bands for rough phylogenetic identification and for generation of highly specific primers. These primers were used to generate almost complete 16S rRNA gene sequences for comprehensive phylogenetic analysis (20,28) and to quantify the species of interest by real-time PCR (11,18). The material turnover in the stimulation experiments was used to calculate the stoichiometry of the process.…”

Section: Discussionmentioning

confidence: 99%

Identification of a Thiomicrospira denitrificans -Like Epsilonproteobacterium as a Catalyst for Autotrophic Denitrification in the Central Baltic Sea

Brettar

Labrenz

Flavier

et al. 2006

Appl Environ Microbiol

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Identification and functional analysis of key members of bacterial communities in marine and estuarine environments are major challenges for obtaining a mechanistic understanding of biogeochemical processes. In the Baltic Sea basins, as in many other marine environments with anoxic bodies of water, the oxic-anoxic interface is considered a layer of high bacterial turnover of sulfur, nitrogen, and carbon compounds that has a great impact on matter balances in the whole ecosystem. We focused on autotrophic denitrification by oxidation of reduced sulfur compounds as a biogeochemically important process mediating concomitant turnover of sulfur, nitrogen, and carbon. We used a newly developed approach consisting of molecular analyses in stimulation experiments and in situ abundance. The molecular approach was based on single-strand conformational polymorphism (SSCP) analysis of the bacterial community RNA, which allowed identification of potential denitrifiers based on the sequences of enhanced SSCP bands and monitoring of the overall bacterial community during the experiments. Sequences of the SSCP bands of interest were used to design highly specific primers that enabled (i) generation of almost complete 16S rRNA gene sequences using experimental and environmental DNA as templates and (ii) quantification of the bacteria of interest by real-time PCR. By using this approach we identified the bacteria responsible for autotrophic denitrification as a single taxon, an epsilonproteobacterium related to the autotrophic denitrifier Thiomicrospira denitrificans. This finding was confirmed by material balances in the experiments that were consistent with those obtained with continuous cultures of T. denitrificans. The presence and activity of a bacterium that is phylogenetically and physiologically closely related to T. denitrificans could be relevant for the carbon budget of the central Baltic Sea because T. denitrificans exhibits only one-half the efficiency for carbon dioxide fixation per mol of sulfide oxidized and mol of nitrate reduced of Thiobacillus denitrificans hypothesized previously for this function.

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“…Recently, new molecular tools have been applied for the routine identification of microbes, and had led to an increase in the number of identified bacteria (Gevers and Coenye, 2007;Liu and Stahl, 2007;Bittar and Rolain, 2010). Most of the advance molecular methods are based on 16S ribosomal DNA sequences, complete or partial genomes (Ben Amour et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Biochemical and molecular identification of lactic acid bacteria isolated from camel milk in Tunisia

Fguiri

Ziadi

Atigui

et al. 2015

Emir. J. Food Agric

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Molecular Approaches for the Measurement of Density, Diversity, and Phylogeny

Cited by 24 publications

References 168 publications

Phenotypic and Phylogenetic Identification of Coliform Bacteria Obtained Using 12 Coliform Methods Approved by the U.S. Environmental Protection Agency

Phenotypic and Phylogenetic Identification of Coliform Bacteria Obtained Using 12 Coliform Methods Approved by the U.S. Environmental Protection Agency

Identification of a Thiomicrospira denitrificans -Like Epsilonproteobacterium as a Catalyst for Autotrophic Denitrification in the Central Baltic Sea

Biochemical and molecular identification of lactic acid bacteria isolated from camel milk in Tunisia

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