PCR-Independent Detection of Bacterial Species-Specific 16S rRNA at 10 fM by a Pore-Blockage Sensor

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.

Download Full-text

Species-Specific PCR for Identification of Common Contaminant Mollicutes in Cell Culture

Applied and Environmental Microbiology ◽

10.1128/aem.67.7.3195-3200.2001 ◽

2001 ◽

Vol 67 (7) ◽

pp. 3195-3200 ◽

Cited By ~ 46

Author(s):

Fanrong Kong ◽

Gregory James ◽

Susanna Gordon ◽

Anna Zelynski ◽

Gwendolyn L. Gilbert

Keyword(s):

16S Rrna ◽

Cell Cultures ◽

Bacterial Species ◽

Intergenic Spacer ◽

16S Rrna Genes ◽

Rrna Genes ◽

23S Rrna ◽

Rrna Gene ◽

Specific Pcr ◽

Species Specific

ABSTRACT Mycoplasma arginini, M. fermentans, M. hyorhinis, M. orale, and Acholeplasma laidlawii are the members of the class Mollicutes most commonly found in contaminated cell cultures. Previous studies have shown that the published PCR primer pairs designed to detect mollicutes in cell cultures are not entirely specific. The 16S rRNA gene, the 16S-23S rRNA intergenic spacer region, and the 5′ end of the 23S rRNA gene, as a whole, are promising targets for design of mollicute species-specific primer pairs. We analyzed the 16S rRNA genes, the 16S-23S rRNA intergenic spacer regions, and the 5′ end of the 23S rRNA genes of these mollicutes and developed PCR methods for species identification based on these regions. Using high melting temperatures, we developed a rapid-cycle PCR for detection and identification of contaminant mollicutes. Previously published, putative mollicute-specific primers amplified DNA from 73 contaminated cell lines, but the presence of mollicutes was confirmed by species-specific PCR in only 60. Sequences of the remaining 13 amplicons were identified as those of gram-positive bacterial species. Species-specific PCR primers are needed to confirm the presence of mollicutes in specimens and for identification, if required.

Download Full-text

Novel Approach to Quantitative Detection of Specific rRNA in a Microbial Community, Using Catalytic DNA

Applied and Environmental Microbiology ◽

10.1128/aem.71.8.4879-4884.2005 ◽

2005 ◽

Vol 71 (8) ◽

pp. 4879-4884 ◽

Cited By ~ 12

Author(s):

Hikaru Suenaga ◽

Rui Liu ◽

Yuko Shiramasa ◽

Takahiro Kanagawa

Keyword(s):

Microbial Community ◽

16S Rrna ◽

Bacterial Species ◽

Quantitative Detection ◽

Catalytic Function ◽

Novel Approach ◽

Specific Manner ◽

Novel Method ◽

Species Specific ◽

Wastewater Treatment Systems

ABSTRACT We developed a novel method for the quantitative detection of the 16S rRNA of a specific bacterial species in the microbial community by using deoxyribozyme (DNAzyme), which possesses the catalytic function to cleave RNA in a sequence-specific manner. A mixture of heterogeneous 16S rRNA containing the target 16S rRNA was incubated with a species-specific DNAzyme. The cleaved target 16S rRNA was separated from the intact 16S rRNA by electrophoresis, and then their amounts were compared for the quantitative detection of target 16S rRNA. This method was used to determine the abundance of the 16S rRNA of a filamentous bacterium, Sphaerotilus natans, in activated sludge, which is a microbial mixture used in wastewater treatment systems. The result indicated that this DNAzyme-based approach would be applicable to actual microbial communities.

Download Full-text

Polymerase chain reaction in the identification of periodontopathogens: A reliable and satisfactory method?

Archives of Biological Sciences ◽

10.2298/abs1204413j ◽

2012 ◽

Vol 64 (4) ◽

pp. 1413-1423

Author(s):

Natasa Nikolic-Jakoba ◽

Sandra Vojnovic ◽

A. Pavic ◽

S. Jankovic ◽

V. Lekovic ◽

...

Keyword(s):

16S Rrna ◽

Bacterial Species ◽

In Silico Analysis ◽

16S Rrna Gene Sequencing ◽

Rrna Gene ◽

Conventional Pcr ◽

Universal Primer ◽

Health And Disease ◽

Rrna Gene Sequencing ◽

Species Specific

Aggregatibacter actinomycetemcomitans is considered one of the bacterial species of etiological importance in periodontitis. The aim of this study was to evaluate the serotype of A. actinomycetemcomitans in the subgingival biofilm in subjects with periodontal health and disease. Pooled samples of subgingival plaque were taken for culture-based identification of microorganisms. Colonies suspected to be A. actinomycetemcomitans were selected for molecular identification using either multiplex or conventional PCR in serotype-specific genotyping and 16S rRNA gene sequencing. In silico analysis showed that most selected colonies belong to the genus Campylobacter, although positive signals for serotypes of A. actinomycetemcomitans were obtained with these samples. Identification of A. actinomycetemcomitans by conventional PCR for 16S rRNA with one species-specific and one universal primer was inconclusive because an almost identical signal with Campylobacter gracilis was obtained. Although PCR-based methods for the identification of A. actinomycetemcomitans are more rapid, sequencing should not be omitted.

Download Full-text

The structured diversity of specialized gut symbionts of the New World army ants

10.1101/084376 ◽

2016 ◽

Cited By ~ 1

Author(s):

Piotr Łukasik ◽

Justin A. Newton ◽

Jon G. Sanders ◽

Yi Hu ◽

Corrie S. Moreau ◽

...

Keyword(s):

16S Rrna ◽

Microbial Communities ◽

New World ◽

Bacterial Species ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Bacterial Strains ◽

Army Ants ◽

Army Ant ◽

Species Specific

Symbiotic bacteria play important roles in the biology of their arthropod hosts. Yet the microbiota of many diverse and influential groups remain understudied, resulting in a paucity of information on the fidelities and histories of these associations. Motivated by prior findings from a smaller scale, 16S rRNA-based study, we conducted a broad phylogenetic and geographical survey of microbial communities in the ecologically dominant New World army ants (Formicidae: Dorylinae). Amplicon sequencing of the 16S rRNA gene across 28 species spanning the five New World genera showed that the microbial communities of army ants consist of very few common and abundant bacterial species. The two most abundant microbes, referred to as Unclassified Firmicutes and Unclassified Entomoplasmatales, appear to be specialized army ant associates that dominate microbial communities in the gut lumen of three host genera, Eciton, Labidus and Nomamyrmex. Both are present in other army ant genera, including those from the Old World, suggesting that army ant symbioses date back to the Cretaceous. Extensive sequencing of bacterial protein-coding genes revealed multiple strains of these symbionts co-existing within colonies, but seldom within the same individual ant. Bacterial strains formed multiple host species-specific lineages on phylogenies, which often grouped strains from distant geographic locations. These patterns deviate from those seen in other social insects, and raise intriguing questions about the influence of army ant colony swarm-founding and within-colony genetic diversity on strain co-existence, and the effects of hosting a diverse suite of symbiont strains on colony ecology.

Download Full-text

Phenotypic and phylogenetic characterization of Lactobacillus species isolated from traditional Lighvan cheese

Food Production, Processing and Nutrition ◽

10.1186/s43014-021-00065-x ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

Haleh Forouhandeh ◽

Sepideh Zununi Vahed ◽

Hossein Ahangari ◽

Vahideh Tarhriz ◽

Mohammad Saeid Hejazi

Keyword(s):

16S Rrna ◽

Random Amplified Polymorphic Dna ◽

Bacterial Species ◽

16S Rrna Sequencing ◽

Lactobacillus Species ◽

Culture Methods ◽

Phylogenetic Characterization ◽

Rrna Sequencing ◽

Specific Culture ◽

Lighvan Cheese

Abstract Lighvan cheese (Lighvan panir) is among the most famous traditional cheese in Iran for its desired aroma and flavor. Undoubtedly, the lactic acid bacteria especially the genus Lactobacillus are the critical factors in developing the aroma, flavor, and texture in Lighvan cheese. In this study, the Lactobacillus population of the main Lighvan cheese was investigated. The Lactobacillus of the main Lighvan cheese was isolated using specific culture methods according to previously published Guidelines. Then, the phylogenetic features were investigated and the phenotypic characteristics were examined using specific culture methods. Twenty-eight Gram-positive bacterial species were identified belonged to the genus Lactobacillus. According to the same sequences as each other, three groups (A, B, and C) of isolates were categorized with a high degree of similarity to L. fermentum (100%) and L. casei group (L. casei, L. paracasei, and L. rhamnosus) (99.0 to 100%). Random amplified polymorphic DNA (RAPD) fingerprint analysis manifested the presence of three clusters that were dominant in traditional Lighvan cheese. Cluster І was divided into 4 sub-clusters. By the result of carbohydrate fermentation pattern and 16S rRNA sequencing, isolates were identified as L. rhamnosus. The isolates in clusters II and III represented L. paracasei and L. fermentum, respectively as they were identified by 16S rRNA sequencing and fermented carbohydrate patterns. Our result indicated that the specific aroma and flavor of traditional Lighvan cheese can be related to its Lactobacillus population including L. fermentum, L. casei, L. paracasei, and L. rhamnosus. Graphical abstract

Download Full-text

Rapid Detection, Identification, and Enumeration ofEscherichia coli Cells in Municipal Water by Chemiluminescent In Situ Hybridization

Applied and Environmental Microbiology ◽

10.1128/aem.67.1.142-147.2001 ◽

2001 ◽

Vol 67 (1) ◽

pp. 142-147 ◽

Cited By ~ 29

Author(s):

Henrik Stender ◽

Adam J. Broomer ◽

Kenneth Oliveira ◽

Heather Perry-O'Keefe ◽

Jens J. Hyldig-Nielsen ◽

...

Keyword(s):

In Situ Hybridization ◽

16S Rrna ◽

Sensitivity And Specificity ◽

Bacterial Species ◽

Rdna Sequence ◽

Sequence Information ◽

Sequence Alignments ◽

E Coli ◽

Municipal Water

ABSTRACT A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturableEscherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35°C, individual microcolonies of E. coliwere detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targetingP. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other thanE. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.

Download Full-text

The frequency of chimeric molecules as a consequence of PCR co-amplification of 16S rRNA genes from different bacterial species

Microbiology ◽

10.1099/13500872-142-5-1107 ◽

1996 ◽

Vol 142 (5) ◽

pp. 1107-1114 ◽

Cited By ~ 159

Author(s):

G. C. Y. Wang ◽

Y. Wang

Keyword(s):

16S Rrna ◽

Bacterial Species ◽

16S Rrna Genes ◽

Rrna Genes ◽

Chimeric Molecules

Download Full-text

16S rRNA Long-Read Sequencing of the Granulation Tissue from Nonsmokers and Smokers-Severe Chronic Periodontitis Patients

BioMed Research International ◽

10.1155/2018/4832912 ◽

2018 ◽

Vol 2018 ◽

pp. 1-10 ◽

Cited By ~ 3

Author(s):

Rebecca Chowdhry ◽

Neetu Singh ◽

Dinesh Kumar Sahu ◽

Ratnesh Kumar Tripathi ◽

Archana Mishra ◽

...

Keyword(s):

16S Rrna ◽

Granulation Tissue ◽

Ketone Bodies ◽

Bacterial Species ◽

Rrna Gene ◽

Differential Abundance ◽

Streptobacillus Moniliformis ◽

Increased Risk ◽

Long Read ◽

Significant Enrichment

Smoking has been associated with increased risk of periodontitis. The aim of the present study was to compare the periodontal disease severity among smokers and nonsmokers which may help in better understanding of predisposition to this chronic inflammation mediated diseases. We selected deep-seated infected granulation tissue removed during periodontal flap surgery procedures for identification and differential abundance of residential bacterial species among smokers and nonsmokers through long-read sequencing technology targeting full-length 16S rRNA gene. A total of 8 phyla were identified among which Firmicutes and Bacteroidetes were most dominating. Differential abundance analysis of OTUs through PICRUST showed significant (p>0.05) abundance of Phyla-Fusobacteria (Streptobacillus moniliformis); Phyla-Firmicutes (Streptococcus equi), and Phyla Proteobacteria (Enhydrobacter aerosaccus) in nonsmokers compared to smokers. The differential abundance of oral metagenomes in smokers showed significant enrichment of host genes modulating pathways involving primary immunodeficiency, citrate cycle, streptomycin biosynthesis, vitamin B6 metabolism, butanoate metabolism, glycine, serine, and threonine metabolism pathways. While thiamine metabolism, amino acid metabolism, homologous recombination, epithelial cell signaling, aminoacyl-tRNA biosynthesis, phosphonate/phosphinate metabolism, polycyclic aromatic hydrocarbon degradation, synthesis and degradation of ketone bodies, translation factors, Ascorbate and aldarate metabolism, and DNA replication pathways were significantly enriched in nonsmokers, modulation of these pathways in oral cavities due to differential enrichment of metagenomes in smokers may lead to an increased susceptibility to infections and/or higher formation of DNA adducts, which may increase the risk of carcinogenesis.

Download Full-text

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

Applied and Environmental Microbiology ◽

10.1128/aem.70.5.3171-3175.2004 ◽

2004 ◽

Vol 70 (5) ◽

pp. 3171-3175 ◽

Cited By ~ 56

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.

Download Full-text