scholarly journals Use of 16S-23S rRNA Intergenic Spacer Region PCR and Repetitive Extragenic Palindromic PCR Analyses of Escherichia coli Isolates To Identify Nonpoint Fecal Sources

2003 ◽  
Vol 69 (8) ◽  
pp. 4942-4950 ◽  
Author(s):  
Sylvie Seurinck ◽  
Willy Verstraete ◽  
Steven D. Siciliano
Keyword(s):  
Escherichia Coli ◽  
Natural Waters ◽  
Average Rate ◽  
Intergenic Spacer ◽  
23S Rrna ◽  
Pcr Analysis ◽  
Factors Affecting ◽  
Intergenic Spacer Region ◽  
E Coli ◽  
Repetitive Extragenic Palindromic

ABSTRACT Despite efforts to minimize fecal input into waterways, this kind of pollution continues to be a problem due to an inability to reliably identify nonpoint sources. Our objective was to find candidate source-specific Escherichia coli fingerprints as potential genotypic markers for raw sewage, horses, dogs, gulls, and cows. We evaluated 16S-23S rRNA intergenic spacer region (ISR)-PCR and repetitive extragenic palindromic (rep)-PCR analyses of E. coli isolates as tools to identify nonpoint fecal sources. The BOXA1R primer was used for rep-PCR analysis. A total of 267 E. coli isolates from different fecal sources were typed with both techniques. E. coli was found to be highly diverse. Only two candidate source-specific E. coli fingerprints, one for cow and one for raw sewage, were identified out of 87 ISR fingerprints. Similarly, there was only one candidate source-specific E. coli fingerprint for horse out of 59 BOX fingerprints. Jackknife analysis resulted in an average rate of correct classification (ARCC) of 83% for BOX-PCR analysis and 67% for ISR-PCR analysis for the five source categories of this study. When nonhuman sources were pooled so that each isolate was classified as animal or human derived (raw sewage), ARCCs of 82% for BOX-PCR analysis and 72% for ISR-PCR analysis were obtained. Critical factors affecting the utility of these methods, namely sample size and fingerprint stability, were also assessed. Chao1 estimation showed that generally 32 isolates per fecal source individual were sufficient to characterize the richness of the E. coli population of that source. The results of a fingerprint stability experiment indicated that BOX and ISR fingerprints were stable in natural waters at 4, 12, and 28°C for 150 days. In conclusion, 16S-23S rRNA ISR-PCR and rep-PCR analyses of E. coli isolates have the potential to identify nonpoint fecal sources. A fairly small number of isolates was needed to find candidate source-specific E. coli fingerprints that were stable under the simulated environmental conditions.

scholarly journals Study of The Diversity of 16S–23S rDNA Internal Transcribed Spacer (ITS) Typing of Escherichia Coli Strains Isolated From Various Biotopes in Tunisia

2021 ◽  
Author(s):  
Souhir Badi ◽  
Rim Ammeri ◽  
Mohamed Abbassi ◽  
Mejdi Snousssi ◽  
Paola Cremosini ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fragment Size ◽  
Genetic Relationships ◽  
Pcr Amplification ◽  
Intergenic Spacer ◽  
23S Rrna ◽  
Genotype I ◽  
Intergenic Spacer Region ◽  
E Coli ◽  
Rdna Its

Abstract We investigated the 16 S-23S rRNA intergenic spacer region (ISR)-PCR and the phylogenetic PCR analyzes of 150 Escherichia coli isolates as tools to explore their diversity, according to their sampling origins, and their relative dominance in these sampling sources. So, these genetic markers are used to explore phylogenetic and genetic relationships of these 150 E. coli isolates recovered from different environmental sources (water, food, animal, human and vegetables). These isolates are tested for their biochemical pattern and later genotyped through the 16S–23S rRNA intergenic spacer PCR amplification and their polymorphism investigation of PCR-amplified 16S-23S rDNA ITS. The main results of the pattern band profile revealed one to 4 DNA fragments. Distributing 150 E. coli isolates according to their ITS and by using RS-PCR, revealed 4 genotypes and 4 subtypes. The DNA fragment size ranged from 450 to 550 bp. DNA band patterns analysis revealed considerable genetic diversity in interspecies. Thus, the 450 and 550 bp size of the common bands in all E. coli isolates are highly diversified. Genotype I appeared as the most frequent with 77.3% (116 isolates), genotype II with 12% (18 isolates); genotype III with 9.7% (14 isolates), and the IV rarely occurred with 4% (2 isolates). Distributing the E. coli phylogroups showed 84 isolates (56%) of group A, 35 isolates (23.3%) of group B1, 28 isolates (18.7%) of group B2 and only 3 isolates (2%) of group D.

Source tracking ofEscherichia coliby 16S–23S intergenic spacer region denaturing gradient gel electrophoresis (DGGE) of therrnBribosomal operon

2007 ◽  
Vol 53 (10) ◽  
pp. 1174-1184 ◽  
Author(s):  
Thomas V. D’Elia ◽  
Chester R. Cooper ◽  
Carl G. Johnston
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Intergenic Spacer ◽  
Source Tracking ◽  
Combined Sewer Overflow ◽  
Intergenic Spacer Region ◽  
E Coli ◽  
Combined Sewer ◽  
Gradient Gel Electrophoresis

This research validates a novel approach for source tracking based on denaturing gradient gel electrophoresis (DGGE) analysis of DNA extracted from Escherichia coli isolates. Escherichia coli from different animal sources and from river samples upstream from, at, and downstream of a combined sewer overflow were subjected to DGGE to determine sequence variations within the 16S–23S intergenic spacer region (ISR) of the rrnB ribosomal operon. The ISR was analyzed to determine if E. coli isolates from various animal sources could be differentiated from each other. DNA isolated from the E. coli animal sources was PCR amplified to isolate the rrnB operon. To prevent amplification of all 7 E. coli ribosomal operons by PCR amplification using universal primers, sequence-specific primers were utilized for the rrnB operon. Another primer set was then used to prepare samples of the 16S–23S ISR for DGGE. Comparison of PCR–DGGE results between human and animal sources revealed differences in the distribution and frequency of the DGGE bands produced. Human and Canada Goose isolates had the most unique distribution patterns and the highest percent of unique isolates and were grouped separately from all other animal sources. Method validation suggests that there are enough host specificity and genetic differences for use in the field. Field results at and around a combined sewer overflow indicate that this method can be used for microbial source tracking.

scholarly journals Diversity of Salmonella Strains Isolated from the Aquatic Environment as Determined by Serotyping and Amplification of the Ribosomal DNA Spacer Regions

2000 ◽  
Vol 66 (4) ◽  
pp. 1544-1552 ◽  
Author(s):  
Julia Baudart ◽  
Karine Lemarchand ◽  
Anne Brisabois ◽  
Philippe Lebaron
Keyword(s):  
Pathogenic Bacteria ◽  
Natural Waters ◽  
Pcr Amplification ◽  
Intergenic Spacer ◽  
Rrna Genes ◽  
23S Rrna ◽  
Natural Environments ◽  
Storm Events ◽  
Intergenic Spacer Region ◽  
Coastal Watershed

ABSTRACT Salmonella species are pathogenic bacteria often detected in sewage, freshwater, marine coastal water, and groundwater.Salmonella spp. can survive for long periods in natural waters, and the persistence of specific and epidemic strains is of great concern in public health. However, the diversity of species found in the natural environment remains unknown. The aim of this study was to investigate the diversity of Salmonellastrains isolated from different natural aquatic systems within a Mediterranean coastal watershed (river, wastewater, and marine coastal areas). A total of 574 strains isolated from these natural environments were identified by both conventional serotyping and the ribosomal spacer-heteroduplex polymorphism (RS-HP) method (M. A. Jensen and N. Straus, PCR Methods Appl. 3:186–194, 1993). More than 40 different serotypes were found, and some serotypes probably mobilized from widespread animal-rearing activities were detected only during storm events. These serotypes may be good indicators of specific contamination sources. Furthermore, the RS-HP method based on the PCR amplification of the intergenic spacer region between the 16S and 23S rRNA genes can produce amplicon profiles allowing the discrimination of species at both serotype and intraserotype levels. This method represents a powerful tool that could be used for rapid typing of Salmonella isolates.

scholarly journals Detection and Characterization of an Elm Yellows (16SrV) Group Phytoplasma Infecting Virginia Creeper Plants in Southern Florida

Plant Disease ◽  
2001 ◽  
Vol 85 (10) ◽  
pp. 1055-1062 ◽  
Author(s):  
N. A. Harrison ◽  
H. M. Griffiths ◽  
M. L. Carpio ◽  
P. A.. Richardson
Keyword(s):  
Intergenic Spacer ◽  
23S Rrna ◽  
Pcr Analysis ◽  
Polymorphism Analysis ◽  
Intergenic Spacer Region ◽  
Flavescence Dorée ◽  
Specific Pcr ◽  
Rdna Sequences ◽  
Elm Yellows ◽  
Southern Florida

The polymerase chain reaction (PCR) employing phytoplasma-specific ribosomal RNA primer pair P1/P7 consistently amplified a product of expected size (1.8 kb) from 29 of 36 symptom-less Virginia creeper (Parthenocissus quinquefolia) plants growing in southern Florida. Restriction fragment length polymorphism analysis of P1/P7-primed PCR products indicated that most phytoplasmas detected in Virginia creeper were similar to phytoplasmas composing the elm yellows (16SrV) group. This relationship was verified by reamplification of P1/P7 products using an elm yellows (EY) group-specific rRNA primer pair fB1/rULWS1. rDNA products (1,571 bp) were generated by group-specific PCR from 28 phytoplasma-positive plants and 1 negatively testing plant identified by earlier P1/P7-primed PCR. Analysis of 16S rDNA sequences determined the Virginia creeper (VC) phytoplasma to be phylogenetically closest to the European alder yellows (ALY) agent, an established 16SrV-C subgroup strain. However, presence or absence of restriction sites for endonucleases AluI, BfaI, MspI, RsaI, and TaqI in the 16S rRNA and 16-23S rRNA intergenic spacer region of the VC phytoplasma collectively differentiated this strain from ALY and other 16SrV group phytoplasmas. Failure to detect the VC phytoplasma by PCR employing nonribosomal primer pair FD9f/FD9r suggests that this newly characterized agent varies from known European grapevine yellows (flavescence dorée) phyto-plasmas previously classified as 16SrV subgroup C or D strains.

scholarly journals Molecular surveillance of shiga toxigenic Escherichia coli in selected beef abattoirs in Osun State Nigeria

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Femi Ayoade ◽  
Judith Oguzie ◽  
Philomena Eromon ◽  
Omolola E. Omotosho ◽  
Tosin Ogunbiyi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sequence Analysis ◽  
Block Design ◽  
Latex Agglutination ◽  
Pcr Analysis ◽  
Accession Number ◽  
E Coli ◽  
Representative Isolate ◽  
Randomized Block Design ◽  
Toxigenic Strains

AbstractShiga toxigenic strains of E. coli (STEC) known to be etiological agents for diarrhea were screened for their incidence/occurrence in selected abattoirs sources in Osogbo metropolis of Osun State, Nigeria using a randomized block design. Samples were plated directly on selective and differential media and E. coli isolates. Multiplex PCR analysis was used to screen for the presence of specific virulence factors. These were confirmed serologically as non-O157 STEC using latex agglutination serotyping kit. Sequence analysis of PCR products was performed on a representative isolate showing the highest combination of virulence genes using the 16S gene for identification purposes only. Results showed that the average cfu/cm2 was significantly lower in the samples collected at Sekona-2 slaughter slab compared with those collected at Al-maleek batch abattoir and Sekona-1 slaughter slab in ascending order at P = 0.03. Moreover, the average cfu/cm2E. coli in samples collected from butchering knife was significantly lower when compared with that of the workers’ hand (P = 0.047) and slaughtering floor (P = 0.047) but not with the slaughter table (P = 0.98) and effluent water from the abattoir house (P = 0.39). These data suggest that the abattoir type may not be as important in the prevalence and spread of STEC as the hygiene practices of the workers. Sequence analysis of a representative isolate showed 100% coverage and 96.46% percentage identity with Escherichia coli O113:H21 (GenBank Accession number: CP031892.1) strain from Canada. This sequence was subsequently submitted to GenBank with accession number MW463885. From evolutionary analyses, the strain from Nigeria, sequenced in this study, is evolutionarily distant when compared with the publicly available sequences from Nigeria. Although no case of E. coli O157 was found within the study area, percent occurrence of non-O157 STEC as high as 46.3% at some of the sampled sites is worrisome and requires regulatory interventions in ensuring hygienic practices at the abattoirs within the study area.

scholarly journals Binding Site of the Bridged Macrolides in the Escherichia coli Ribosome

2005 ◽  
Vol 49 (1) ◽  
pp. 281-288 ◽  
Author(s):  
Liqun Xiong ◽  
Yakov Korkhin ◽  
Alexander S. Mankin
Keyword(s):  
Escherichia Coli ◽  
Tight Binding ◽  
Dimethyl Sulfate ◽  
23S Rrna ◽  
Side Chain ◽  
Macrolide Antibiotics ◽  
Side Chains ◽  
Alkyl Aryl ◽  
E Coli ◽  
Exit Tunnel

ABSTRACT Ketolides represent the latest group of macrolide antibiotics. Tight binding of ketolides to the ribosome appears to correlate with the presence of an extended alkyl-aryl side chain. Recently developed 6,11-bridged bicyclic ketolides extend the spectrum of platforms used to generate new potent macrolides with extended alkyl-aryl side chains. The purpose of the present study was to characterize the site of binding and the action of bridged macrolides in the ribosomes of Escherichia coli. All the bridged macrolides investigated efficiently protected A2058 and A2059 in domain V of 23S rRNA from modification by dimethyl sulfate and U2609 from modification by carbodiimide. In addition, bridged macrolides that carry extended alkyl-aryl side chains protruding from the 6,11 bridge protected A752 in helix 35 of domain II of 23S rRNA from modification by dimethyl sulfate. Bridged macrolides efficiently displaced erythromycin from the ribosome in a competition binding assay. The A2058G mutation in 23S rRNA conferred resistance to the bridged macrolides. The U2609C mutation, which renders E. coli resistant to the previously studied ketolides telithromycin and cethromycin, barely affected cell susceptibility to the bridged macrolides used in this study. The results of the biochemical and genetic studies indicate that in the E. coli ribosome, bridged macrolides bind in the nascent peptide exit tunnel at the site previously described for other macrolide antibiotics. The presence of the side chain promotes the formation of specific interactions with the helix 35 of 23S rRNA.

Genomic fingerprinting ofFrankiamicrosymbionts fromCeanothuscopopulations using repetitive sequences and polymerase chain reactions

1999 ◽  
Vol 77 (9) ◽  
pp. 1220-1230 ◽  
Author(s):  
Soon-Chun Jeong ◽  
David D Myrold
Keyword(s):  
Dna Sequencing ◽  
Dna Analysis ◽  
Repetitive Sequences ◽  
Intergenic Spacer ◽  
Rrna Genes ◽  
23S Rrna ◽  
Genomic Fingerprinting ◽  
Chain Reactions ◽  
Intergenic Spacer Region ◽  
Polymerase Chain

Specificity between Ceanothus species and their microsymbionts, Frankia, were investigated with nodules collected from three geographically separated copopulations of Ceanothus species. Nodules were analyzed using DNA sequencing and repetitive sequence polymerase chain reaction (rep-PCR) techniques. DNA sequencing of the intergenic spacer region between 16S and 23S rRNA genes suggested that Ceanothus-microsymbiotic Frankia are closely related at the intraspecific level. Diversity of the microsymbionts was further analyzed by genomic fingerprinting using repetitive sequences and PCR. A newly designed direct repeat (DR) sequence and a BOX sequence were used as PCR primers after justification that these primers can generate Frankia-specific fingerprints from nodule DNA. Analysis of the nodules using BOX- and DR-PCR showed that Ceanothus-microsymbiotic Frankia exhibited less diversity within each copopulation than among copopulations. These data suggested that geographic separation plays a more important role for divergence of Ceanothus-microsymbiotic Frankia than host plant.Key words: Frankia, Ceanothus, rep-PCR, diversity.

scholarly journals In vitro reconstitution of the GTPase-associated centre of the archaebacterial ribosome: the functional features observed in a hybrid form with Escherichia coli 50S subunits

2006 ◽  
Vol 396 (3) ◽  
pp. 565-571 ◽  
Author(s):  
Takaomi Nomura ◽  
Kohji Nakano ◽  
Yasushi Maki ◽  
Takao Naganuma ◽  
Takashi Nakashima ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Proteins ◽  
Synthetic Activity ◽  
23S Rrna ◽  
Elongation Factors ◽  
Hybrid Form ◽  
Pyrococcus Horikoshii ◽  
E Coli ◽  
Functional Features

We cloned the genes encoding the ribosomal proteins Ph (Pyrococcus horikoshii)-P0, Ph-L12 and Ph-L11, which constitute the GTPase-associated centre of the archaebacterium Pyrococcus horikoshii. These proteins are homologues of the eukaryotic P0, P1/P2 and eL12 proteins, and correspond to Escherichia coli L10, L7/L12 and L11 proteins respectively. The proteins and the truncation mutants of Ph-P0 were overexpressed in E. coli cells and used for in vitro assembly on to the conserved domain around position 1070 of 23S rRNA (E. coli numbering). Ph-L12 tightly associated as a homodimer and bound to the C-terminal half of Ph-P0. The Ph-P0·Ph-L12 complex and Ph-L11 bound to the 1070 rRNA fragments from the three biological kingdoms in the same manner as the equivalent proteins of eukaryotic and eubacterial ribosomes. The Ph-P0·Ph-L12 complex and Ph-L11 could replace L10·L7/L12 and L11 respectively, on the E. coli 50S subunit in vitro. The resultant hybrid ribosome was accessible for eukaryotic, as well as archaebacterial elongation factors, but not for prokaryotic elongation factors. The GTPase and polyphenylalanine-synthetic activity that is dependent on eukaryotic elongation factors was comparable with that of the hybrid ribosomes carrying the eukaryotic ribosomal proteins. The results suggest that the archaebacterial proteins, including the Ph-L12 homodimer, are functionally accessible to eukaryotic translation factors.

scholarly journals Identifying Host Sources of Fecal Pollution: Diversity of Escherichia coli in Confined Dairy and Swine Production Systems

2005 ◽  
Vol 71 (10) ◽  
pp. 5992-5998 ◽  
Author(s):  
Zexun Lu ◽  
David Lapen ◽  
Andrew Scott ◽  
Angela Dang ◽  
Edward Topp
Keyword(s):  
Escherichia Coli ◽  
Dairy Farm ◽  
Sampling Strategy ◽  
Farming Systems ◽  
Microbial Source Tracking ◽  
Fecal Pollution ◽  
Source Tracking ◽  
E Coli ◽  
Repetitive Extragenic Palindromic Pcr ◽  
Repetitive Extragenic Palindromic

ABSTRACT Repetitive extragenic palindromic PCR fingerprinting of Escherichia coli is one microbial source tracking approach for identifying the host source origin of fecal pollution in aquatic systems. The construction of robust known-source libraries is expensive and requires an informed sampling strategy. In many types of farming systems, waste is stored for several months before being released into the environment. In this study we analyzed, by means of repetitive extragenic palindromic PCR using the enterobacterial repetitive intergenic consensus primers and comparative analysis using the Bionumerics software, collections of E. coli obtained from a dairy farm and from a swine farm, both of which stored their waste as a slurry in holding tanks. In all fecal samples, obtained from either barns or holding tanks, the diversity of the E. coli populations was underrepresented by collections of 500 isolates. In both the dairy and the swine farms, the diversity of the E. coli community was greater in the manure holding tank than in the barn, when they were sampled on the same date. In both farms, a comparison of stored manure samples collected several months apart suggested that the community composition changed substantially in terms of the detected number, absolute identity, and relative abundance of genotypes. Comparison of E. coli populations obtained from 10 different locations in either holding tank suggested that spatial variability in the E. coli community should be accounted for when sampling. Overall, the diversity in E. coli populations in manure slurry storage facilities is significant and likely is problematic with respect to library construction for microbial source tracking applications.

Sign in / Sign up

Export Citation Format

Share Document