scholarly journals Development of Quantitative PCR Assays Targeting the 16S rRNA Genes of Enterococcus spp. and Their Application to the Identification of Enterococcus Species in Environmental Samples

2012 ◽  
Vol 79 (1) ◽  
pp. 196-204 ◽  
Author(s):  
Hodon Ryu ◽  
Michael Henson ◽  
Michael Elk ◽  
Carlos Toledo-Hernandez ◽  
John Griffith ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Quantitative Pcr ◽  
Water Samples ◽  
The Other ◽  
Rrna Gene ◽  
Fecal Samples ◽  
Content Type ◽  
Specific Assay ◽  
Enterococcal Species

ABSTRACTThe detection of environmental enterococci has been determined primarily by using culture-based techniques that might exclude some enterococcal species as well as those that are nonculturable. To address this, the relative abundances of enterococci were examined by challenging fecal and water samples against a currently available genus-specific assay (Entero1). To determine the diversity of enterococcal species, 16S rRNA gene-based group-specific quantitative PCR (qPCR) assays were developed and evaluated against eight of the most common environmental enterococcal species. Partial 16S rRNA gene sequences of 439 presumptive environmental enterococcal strains were analyzed to study further the diversity of enterococci and to confirm the specificities of group-specific assays. The group-specific qPCR assays showed relatively high amplification rates with targeted species (>98%), although some assays cross-amplified with nontargeted species (1.3 to 6.5%). The results with the group-specific assays also showed that different enterococcal species co-occurred in most fecal samples. The most abundant enterococci in water and fecal samples wereEnterococcus faecalisandEnterococcus faecium, although we identified more water isolates asEnterococcus casseliflavusthan as any of the other species. The prevalence of the Entero1 marker was in agreement with the combined number of positive signals determined by the group-specific assays in most fecal samples, except in gull feces. On the other hand, the number of group-specific assay signals was lower in all water samples tested, suggesting that other enterococcal species are present in these samples. While the results highlight the value of genus- and group-specific assays for detecting the major enterococcal groups in environmental water samples, additional studies are needed to determine further the diversity, distributions, and relative abundances of all enterococcal species found in water.

scholarly journals Highly Specific Sewage-DerivedBacteroidesQuantitative PCR Assays Target Sewage-Polluted Waters

2019 ◽  
Vol 85 (6) ◽  
Author(s):  
Shuchen Feng ◽  
Sandra L. McLellan
Keyword(s):  
Population Structure ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Water Samples ◽  
Rrna Gene ◽  
Content Type ◽  
Hypervariable Regions ◽  
Animal Hosts ◽  
Pcr Assays ◽  
The 16S Rrna Gene

ABSTRACTThe identification of sewage contamination in water has primarily relied on the detection of human-associatedBacteroidesusing markers within the V2 region of the 16S rRNA gene. Despite the establishment of multiple assays that target the HF183 cluster (i.e.,Bacteroides dorei) and otherBacteroidesorganisms (e.g.,Bacteroides thetaiotaomicron), the potential for more human-associated markers in this genus has not been explored in depth. We examined theBacteroidespopulation structure in sewage and animal hosts across the V4V5 and V6 hypervariable regions. Using near-full-length cloned sequences, we identified the sequences in the V4V5 and V6 hypervariable regions that are linked to the HF183 marker in the V2 region and found these sequences were present in multiple animals. In addition, the V4V5 and V6 regions contained human fecal marker sequences for organisms that were independent of the HF183 cluster. The most abundantBacteroidesin untreated sewage was not human associated but pipe derived. Two TaqMan quantitative PCR (qPCR) assays targeting the V4V5 and V6 regions of this organism were developed. Validation studies using fecal samples from seven animal hosts (n = 76) and uncontaminated water samples (n = 30) demonstrated the high specificity of the assays for sewage. FreshwaterBacteroideswere also identified in uncontaminated water samples, demonstrating that measures of totalBacteroidesdo not reflect fecal pollution. A comparison of two previously described humanBacteroidesassays (HB and HF183/BacR287) in municipal wastewater influent and sewage-contaminated urban water samples revealed identical results, illustrating the assays target the same organism. The detection of sewage-derivedBacteroidesprovided an independent measure of sewage-impacted waters.IMPORTANCEBacteroidesare major members of the gut microbiota, and host-specific organisms within this genus have been used extensively to gain information on pollution sources. This study provides a broad view of the population structure ofBacteroideswithin sewage to contextualize the well-studied HF183 marker for a human-associatedBacteroides. The study also delineates host-specific sequence patterns across multiple hypervariable regions of the 16S rRNA gene to improve our ability to use sequence data to assess water quality. Here, we demonstrate that regions downstream of the HF183 marker are nonspecific but other potential human-associated markers are present. Furthermore, we show the most abundantBacteroidesin sewage is free living, rather than host associated, and specifically found in sewage. Quantitative PCR assays that target organisms specific to sewer pipes offer measures that are independent of the human microbiome for identifying sewage pollution in water.

Mucilaginibacter sabulilitoris sp. nov., isolated from marine sand in a firth

2013 ◽  
Vol 63 (Pt_8) ◽  
pp. 2865-2871 ◽  
Author(s):  
Chul-Hyung Kang ◽  
Yong-Taek Jung ◽  
Jung-Hoon Yoon
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Strain ◽  
Type Species ◽  
Sequence Similarity ◽  
The Other ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
Marine Sand

A Gram-stain-negative, non-spore-forming, strictly aerobic, non-flagellated, non-gliding, rod-shaped bacterial strain, designated SMS-12T, was isolated from marine sand in a firth on the western coast of South Korea. Strain SMS-12T grew optimally at 25 °C, at pH 7.0–7.5 and in the absence of NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain SMS-12T fell within the clade comprising species of the genus Mucilaginibacter , forming a coherent cluster with the type strain of Mucilaginibacter lappiensis , with which it exhibited the highest 16S rRNA gene sequence similarity value of 97.5 %. Levels of sequence similarity to the type strains of the other species of the genus Mucilaginibacter and the other species used in the phylogenetic analysis were 93.3–96.4 % and <91.5 %, respectively. Strain SMS-12T contained MK-7 as the predominant menaquinone, and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH), iso-C15 : 0 and iso-C17 : 0 3-OH as the major fatty acids. The major polar lipids were phosphatidylethanolamine and one unidentified aminophospholipid; sphingolipids were present. The DNA G+C content was 41.8 mol% and the mean DNA–DNA relatedness with M. lappiensis KACC 14978T was 13 %. Differential phenotypic properties, together with phylogenetic and genetic distinctiveness, revealed that strain SMS-12T is separate from other species of the genus Mucilaginibacter . On the basis of the data presented, strain SMS-12T is considered to represent a novel species of the genus Mucilaginibacter , for which the name Mucilaginibacter sabulilitoris sp. nov. is proposed. The type strain is SMS-12T ( = KCTC 32111T = CCUG 62214T).

scholarly journals Analysis of the Gull Fecal Microbial Community Reveals the Dominance of Catellicoccus marimammalium in Relation to Culturable Enterococci

2013 ◽  
Vol 80 (2) ◽  
pp. 757-765 ◽  
Author(s):  
Amber M. Koskey ◽  
Jenny C. Fisher ◽  
Mary F. Traudt ◽  
Ryan J. Newton ◽  
Sandra L. McLellan
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rrna Gene Sequencing ◽  
Illumina Miseq ◽  
Rrna Gene ◽  
Sequence Comparisons ◽  
Fecal Indicator ◽  
Fecal Samples ◽  
Content Type

ABSTRACTGulls are prevalent in beach environments and can be a major source of fecal contamination. Gulls have been shown to harbor a high abundance of fecal indicator bacteria (FIB), such asEscherichia coliand enterococci, which can be readily detected as part of routine beach monitoring. Despite the ubiquitous presence of gull fecal material in beach environments, the associated microbial community is relatively poorly characterized. We generated comprehensive microbial community profiles of gull fecal samples using Roche 454 and Illumina MiSeq platforms to investigate the composition and variability of the gull fecal microbial community and to measure the proportion of FIB.EnterococcaceaeandEnterobacteriaceaewere the two most abundant families in our gull samples. Sequence comparisons between short-read data and nearly full-length 16S rRNA gene clones generated from the same samples revealedCatellicoccus marimammaliumas the most numerous taxon among all samples. The identification of bacteria from gull fecal pellets cultured on membrane-Enterococcusindoxyl-β-d-glucoside (mEI) plates showed that the dominant sequences recovered in our sequence libraries did not represent organisms culturable on mEI. Based on 16S rRNA gene sequencing of gull fecal isolates cultured on mEI plates, 98.8% were identified asEnterococcusspp., 1.2% were identified asStreptococcusspp., and none were identified asC. marimammalium. Illumina deep sequencing indicated that gull fecal samples harbor significantly higher proportions ofC. marimammalium16S rRNA gene sequences (>50-fold) relative to typical mEI culturableEnterococcusspp.C. marimammaliumtherefore can be confidently utilized as a genetic marker to identify gull fecal pollution in the beach environment.

Multilocus sequence analysis and 16S rRNA gene sequencing reveal that Yersinia frederiksenii genospecies 2 is Yersinia massiliensis

2013 ◽  
Vol 63 (Pt_8) ◽  
pp. 3124-3129 ◽  
Author(s):  
Roberto A. Souza ◽  
Priscilla F. M. Imori ◽  
Juliana P. Falcão
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Species ◽  
Phylogenetic Analyses ◽  
16S Rrna Gene Sequencing ◽  
The Other ◽  
Phylogenetic Position ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type

Since Yersinia frederiksenii was first described in 1980, it has been recognized genotypically as a heterogeneous species, comprising three phenotypically indistinguishable genospecies. In this study, the sequence of the 16S rRNA gene and the concatenated sequences of six housekeeping genes (glnA, gyrB, hsp60, recA, rpoB and sodA) of all the currently known species of the genus Yersinia were used to determine the phylogenetic position of Y. frederiksenii genospecies 2 in the genus Yersinia . The phylogenetic analyses grouped the Y. frederiksenii genospecies 2 strains in a monophyletic group together with representative strains of Yersinia massiliensis . Moreover, the Y. frederiksenii genospecies 2 strains were also grouped apart from the other species of the genus Yersinia and far from the other two genospecies of Y. frederiksenii . All of the observations made in this study support the conclusion that Y. frederiksenii genospecies 2 should be reclassified as Y. massiliensis .

scholarly journals Myroides albus sp. nov., isolated from the gut of plastic-eating larvae of the coleopteran insect Zophobas atratus

2020 ◽  
Vol 70 (10) ◽  
pp. 5460-5466 ◽  
Author(s):  
Mengli Xia ◽  
Lin Hu ◽  
Yi-Xin Huo ◽  
Yu Yang
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Phylogenetic Analyses ◽  
The Other ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
Coleopteran Insect ◽  
Zophobas Atratus ◽  
The 16S Rrna Gene

A bacterial strain, BIT-d1T, was isolated from the gut of plastic-eating larvae of the coleopteran insect Zophobas atratus. Its taxonomic position was analysed using a polyphasic approach. Cells were white-pigmented, Gram-stain-negative, non-motile, long rods without flagella. The 16S rRNA gene sequence (1401 bp) of strain BIT-d1T showed highest similarity (98.0%) to Myroides pelagicus SM1T and 96.6~92.6 % similarity to the other species of the genus Myroides . The results of phylogenetic analyses, based on the 16S rRNA gene, concatenated sequences of six housekeeping genes (gyrB, dnaK, tuf, murG, atpA and glyA) and genome sequences, placed strain BIT-d1T in a separate lineage among the genus Myroides , family Flavobacteriaceae . The major isoprenoid quinone was menaquinone-6 (MK-6) and the major fatty acids were C15 : 0 iso, C17 : 0 iso 3-OH and summed feature 9 (comprising iso-C17 : 1  ω9c and/or C16 : 0 10-methyl), which were similar to other members in the genus Myroides. In silico DNA–DNA hybridization and average nucleotide identity calculations plus physiological and biochemical tests exhibited the genotypic and phenotypic differentiation of strain BIT-d1T from the other members of the genus Myroides . Therefore, strain BIT-d1T is considered to represent a novel species within the genus Myroides , for which the name Myroides albus sp. nov is proposed. The type strain is BIT-d1T (=CGMCC 1.17043T=KCTC 72447T).

scholarly journals Comparison of Gull Feces-Specific Assays Targeting the 16S rRNA Genes of Catellicoccus marimammalium and Streptococcus spp.

2012 ◽  
Vol 78 (6) ◽  
pp. 1909-1916 ◽  
Author(s):  
Hodon Ryu ◽  
John F. Griffith ◽  
Izhar U. H. Khan ◽  
Stephen Hill ◽  
Thomas A. Edge ◽  
...  
Keyword(s):  
16S Rrna ◽  
Water Samples ◽  
Environmental Water ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Taqman Assay ◽  
Rrna Gene ◽  
Recreational Waters ◽  
Fecal Samples ◽  
Content Type

ABSTRACTTwo novel gull-specific quantitative PCR (qPCR) assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR green assay targetingStreptococcusspp. (gull3) and a hydrolysis TaqMan assay targetingCatellicoccus marimammalium(gull4). The objectives of this study were to compare the host specificity of a previousC. marimammaliumqPCR assay (gull2) with that of the new markers and to examine the presence of the three gull markers in environmental water samples from different geographic locations. Most of the gull fecal samples tested (n= 255) generated positive signals with the gull2 and gull4 assays (i.e., >86%), whereas only 28% were positive with gull3. Low prevalence and abundance of tested gull markers (0.6 to 15%) were observed in fecal samples from six nonavian species (n= 180 fecal samples), whereas the assays cross-reacted to some extent (13 to 31%) with other (nongull) avian fecal samples. The gull3 assay was positive against fecal samples from 11 of 15 avian species, including gull. Of the presumed gull-impacted water samples (n= 349), 86%, 59%, and 91% were positive with the gull2, the gull3, and the gull4 assays, respectively. Approximately 5% of 239 non-gull-impacted water samples were positive with the gull2 and the gull4 assays, whereas 21% were positive witg the gull3 assay. While the relatively high occurrence of gull2 and gull4 markers in waters impacted by gull feces suggests that these assays could be used in environmental monitoring studies, the data also suggest that multiple avian-specific assays will be needed to accurately assess the contribution of different avian sources in recreational waters.

scholarly journals Identification of New Single Nucleotide Polymorphism-Based Markers for Inter- and Intraspecies Discrimination of Obligate Bacterial Parasites (Pasteuria spp.) of Invertebrates

2011 ◽  
Vol 77 (18) ◽  
pp. 6388-6394 ◽  
Author(s):  
Tim H. Mauchline ◽  
Rachel Knox ◽  
Sharad Mohan ◽  
Stephen J. Powers ◽  
Brian R. Kerry ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
The Other ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Single Nucleotide ◽  
Content Type ◽  
Protein Encoding ◽  
Encoding Genes ◽  
The 16S Rrna Gene

ABSTRACTProtein-encoding and 16S rRNA genes ofPasteuria penetranspopulations from a wide range of geographic locations were examined. Most interpopulation single nucleotide polymorphisms (SNPs) were detected in the 16S rRNA gene. However, in order to fully resolve all populations, these were supplemented with SNPs from protein-encoding genes in a multilocus SNP typing approach. Examination of individual 16S rRNA gene sequences revealed the occurrence of “cryptic” SNPs which were not present in the consensus sequences of anyP. penetranspopulation. Additionally, hierarchical cluster analysis separatedP. penetrans16S rRNA gene clones into four groups, and one of which contained sequences from the most highly passaged population, demonstrating that it is possible to manipulate the population structure of this fastidious bacterium. The other groups were made from representatives of the other populations in various proportions. Comparison of sequences among threePasteuriaspecies, namely,P. penetrans,P. hartismeri, andP. ramosa, showed that the protein-encoding genes provided greater discrimination than the 16S rRNA gene. From these findings, we have developed a toolbox for the discrimination ofPasteuriaat both the inter- and intraspecies levels. We also provide a model to monitor genetic variation in other obligate hyperparasites and difficult-to-culture microorganisms.

scholarly journals Phyllobacterium loti sp. nov. isolated from nodules of Lotus corniculatus

2014 ◽  
Vol 64 (Pt_3) ◽  
pp. 781-786 ◽  
Author(s):  
Maximo Sánchez ◽  
Martha-Helena Ramírez-Bahena ◽  
Alvaro Peix ◽  
María J. Lorite ◽  
Juan Sanjuán ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Sequence Similarity ◽  
Carbon Sources ◽  
Lotus Corniculatus ◽  
Culture Conditions ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
The 16S Rrna Gene

Strain S658T was isolated from a Lotus corniculatus nodule in a soil sample obtained in Uruguay. Phylogenetic analysis of the 16S rRNA gene and atpD gene showed that this strain clustered within the genus Phyllobacterium . The closest related species was, in both cases, Phyllobacterium trifolii PETP02T with 99.8 % sequence similarity in the 16S rRNA gene and 96.1 % in the atpD gene. The 16S rRNA gene contains an insert at the beginning of the sequence that has no similarities with other inserts present in the same gene in described rhizobial species. Ubiquinone Q-10 was the only quinone detected. Strain S658T differed from its closest relatives through its growth in diverse culture conditions and in the assimilation of several carbon sources. It was not able to reproduce nodules in Lotus corniculatus. The results of DNA–DNA hybridization, phenotypic tests and fatty acid analyses confirmed that this strain should be classified as a representative of a novel species of the genus Phyllobacterium , for which the name Phyllobacterium loti sp. nov. is proposed. The type strain is S658T( = LMG 27289T = CECT 8230T).

scholarly journals Faecalicoccus acidiformans gen. nov., sp. nov., isolated from the chicken caecum, and reclassification of Streptococcus pleomorphus (Barnes et al. 1977), Eubacterium biforme (Eggerth 1935) and Eubacterium cylindroides (Cato et al. 1974) as Faecalicoccus pleomorphus comb. nov., Holdemanella biformis gen. nov., comb. nov. and Faecalitalea cylindroides gen. nov., comb. nov., respectively, within the family Erysipelotrichaceae

2014 ◽  
Vol 64 (Pt_11) ◽  
pp. 3877-3884 ◽  
Author(s):  
Celine De Maesschalck ◽  
Filip Van Immerseel ◽  
Venessa Eeckhaut ◽  
Siegrid De Baere ◽  
Margo Cnockaert ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Species ◽  
Gene Sequence ◽  
Sequence Similarity ◽  
16S Rrna Gene Sequence ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Content Type ◽  
Link Type

Strains LMG 27428T and LMG 27427 were isolated from the caecal content of a chicken and produced butyric, lactic and formic acids as major metabolic end products. The genomic DNA G+C contents of strains LMG 27428T and LMG 27427 were 40.4 and 38.8 mol%. On the basis of 16S rRNA gene sequence similarity, both strains were most closely related to the generically misclassified Streptococcus pleomorphus ATCC 29734T. Strain LMG 27428T could be distinguished from S. pleomorphus ATCC 29734T based on production of more lactic acid and less formic acid in M2GSC medium, a higher DNA G+C content and the absence of activities of acid phosphatase and leucine, arginine, leucyl glycine, pyroglutamic acid, glycine and histidine arylamidases, while strain LMG 27428 was biochemically indistinguishable from S. pleomorphus ATCC 29734T. The novel genus Faecalicoccus gen. nov. within the family Erysipelotrichaceae is proposed to accommodate strains LMG 27428T and LMG 27427. Strain LMG 27428T ( = DSM 26963T) is the type strain of Faecalicoccus acidiformans sp. nov., and strain LMG 27427 ( = DSM 26962) is a strain of Faecalicoccus pleomorphus comb. nov. (type strain LMG 17756T = ATCC 29734T = DSM 20574T). Furthermore, the nearest phylogenetic neighbours of the genus Faecalicoccus are the generically misclassified Eubacterium cylindroides DSM 3983T (94.4 % 16S rRNA gene sequence similarity to strain LMG 27428T) and Eubacterium biforme DSM 3989T (92.7 % 16S rRNA gene sequence similarity to strain LMG 27428T). We present genotypic and phenotypic data that allow the differentiation of each of these taxa and propose to reclassify these generically misnamed species of the genus Eubacterium formally as Faecalitalea cylindroides gen. nov., comb. nov. and Holdemanella biformis gen. nov., comb. nov., respectively. The type strain of Faecalitalea cylindroides is DSM 3983T = ATCC 27803T = JCM 10261T and that of Holdemanella biformis is DSM 3989T = ATCC 27806T = CCUG 28091T.

scholarly journals Pseudoseohaeicola caenipelagi gen. nov., sp. nov., isolated from a tidal flat

2015 ◽  
Vol 65 (Pt_6) ◽  
pp. 1819-1824 ◽  
Author(s):  
Sooyeon Park ◽  
Ji-Min Park ◽  
Chul-Hyung Kang ◽  
Song-Gun Kim ◽  
Jung-Hoon Yoon
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Tidal Flat ◽  
Type Strain ◽  
Type Species ◽  
Phylogenetic Trees ◽  
Sequence Similarity ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type

A Gram-stain-negative, non-motile, aerobic and pleomorphic bacterium, designated BS-W13T, was isolated from a tidal flat on the South Sea, South Korea, and its taxonomic position was investigated using a polyphasic approach. Strain BS-W13T grew optimally at 25 °C, at pH 7.0–8.0 and in the presence of 1.0–2.0 % (w/v) NaCl. Neighbour-joining and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences showed that strain BS-W13T clustered with the type strain of Seohaeicola saemankumensis , showing the highest sequence similarity (95.96 %) to this strain. Strain BS-W13T exhibited 16S rRNA gene sequence similarity values of 95.95, 95.91, 95.72 and 95.68 % to the type strains of Sulfitobacter donghicola , Sulfitobacter porphyrae , Sulfitobacter mediterraneus and Roseobacter litoralis , respectively. Strain BS-W13T contained Q-10 as the predominant ubiquinone and C18 : 1ω7c as the major fatty acid. The polar lipid profile of strain BS-W13T, containing phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid and one unidentified lipid as major components, was distinguishable from those of some phylogenetically related taxa. The DNA G+C content of strain BS-W13T was 58.1 mol%. The phylogenetic data and differential chemotaxonomic and other phenotypic properties revealed that strain BS-W13T constitutes a novel genus and species within family Rhodobacteraceae of the class Alphaproteobacteria , for which the name Pseudoseohaeicola caenipelagi gen. nov., sp. nov. is proposed. The type strain is BS-W13T ( = KCTC 42349T = CECT 8724T).

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