scholarly journals Molecular evidence for the ubiquitous presence of Legionella species in Dutch tap water installations

2007 ◽  
Vol 5 (3) ◽  
pp. 375-383 ◽  
Author(s):  
Bram M. W. Diederen ◽  
Caroline M. A. de Jong ◽  
Ingrid Aarts ◽  
Marcel F. Peeters ◽  
Anneke van der Zee
Keyword(s):  
16S Rrna ◽  
Water Samples ◽  
Tap Water ◽  
Molecular Evidence ◽  
Rrna Gene ◽  
Specific Probe ◽  
Detection Rates ◽  
Specific Pcr ◽  
Legionella Species ◽  
Species Specific

Our aim was to investigate the occurrence and identity of Legionella spp. in Dutch tap water installations using culture, real-time PCR and sequence analysis. The PCR assays used were a 16S rRNA gene based PCR with both a Legionella species specific probe and a L. pneumophila specific probe and a L. pneumophila-specific PCR based on the sequence of the mip gene. A total of 357 water samples from 250 locations in The Netherlands was investigated. The detection rates of Legionella spp. were 2,2% (8 of 357) by culture, and 87,1% (311 of 357) by PCR. The majority of samples was found to contain Legionella species other than L. pneumophila. These comprised of Legionella Like Amoebal Pathogens (LLAPs), L. busanensis, L. worsliensis and others. Fourteen (3,9%) samples were positive for L. pneumophila by either culture, 16S rRNA based PCR and/or mip based PCR. It is apparent from this study that Legionella spp. DNA is ubiquitous in Dutch potable water samples. Our findings further suggest that LLAPs and viable but nonculturable (VBNC) Legionella represent a large proportion of the population in man-made environments.

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

2001 ◽  
Vol 67 (7) ◽  
pp. 3195-3200 ◽  
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.

scholarly journals Distribution of Bifidobacterial Species in Human Intestinal Microflora Examined with 16S rRNA-Gene-Targeted Species-Specific Primers

1999 ◽  
Vol 65 (10) ◽  
pp. 4506-4512 ◽  
Author(s):  
Takahiro Matsuki ◽  
Koichi Watanabe ◽  
Ryuichiro Tanaka ◽  
Masafumi Fukuda ◽  
Hiroshi Oyaizu
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Intestinal Tract ◽  
Bifidobacterium Longum ◽  
Rrna Gene ◽  
Human Feces ◽  
Specific Primers ◽  
Specific Pcr ◽  
Species Specific ◽  
Human Intestinal Tract

ABSTRACT In order to clarify the distribution of bifidobacterial species in the human intestinal tract, a 16S rRNA-gene-targeted species-specific PCR technique was developed and used with DNAs extracted from fecal samples obtained from 48 healthy adults and 27 breast-fed infants. To cover all of the bifidobacterial species that have been isolated from and identified in the human intestinal tract, species-specific primers for Bifidobacterium longum, B. infantis,B. dentium, and B. gallicum were developed and used with primers for B. adolescentis, B. angulatum, B. bifidum, B. breve, and the B. catenulatum group (B. catenulatum andB. pseudocatenulatum) that were developed in a previous study (T. Matsuki, K. Watanabe, R. Tanaka, and H. Oyaizu, FEMS Microbiol. Lett. 167:113–121, 1998). The specificity of the nine primers was confirmed by PCR, and the species-specific PCR method was found to be a useful means for identifying Bifidobacteriumstrains isolated from human feces. The results of an examination of bifidobacterial species distribution showed that the B. catenulatum group was the most commonly found taxon (detected in 44 of 48 samples [92%]), followed by B. longum andB. adolescentis, in the adult intestinal bifidobacterial flora and that B. breve, B. infantis, andB. longum were frequently found in the intestinal tracts of infants. The present study demonstrated that qualitative detection of the bifidobacterial species present in human feces can be accomplished rapidly and accurately.

scholarly journals Campylobacter canadensis sp. nov., from captive whooping cranes in Canada

2007 ◽  
Vol 57 (11) ◽  
pp. 2636-2644 ◽  
Author(s):  
G. Douglas Inglis ◽  
Bryanne M. Hoar ◽  
Douglas P. Whiteside ◽  
Douglas W. Morck
Keyword(s):  
16S Rrna ◽  
Novel Species ◽  
Sequence Similarity ◽  
23S Rrna ◽  
Rrna Gene ◽  
Polymorphism Analysis ◽  
Specific Pcr ◽  
Whooping Cranes ◽  
23S Rrna Gene ◽  
Species Specific

Ten isolates of an unknown Campylobacter species were isolated from cloacal swabs obtained from captive adult whooping cranes (Grus americana). All isolates were identified as Campylobacter based on generic PCR and grouped with other Campylobacter species based on 23S rRNA gene sequence. None of the isolates could be identified by species-specific PCR for known taxa, and all ten isolates formed a robust clade that was very distinct from known Campylobacter species based on 16S rRNA, rpoB and cpn60 gene sequences. The results of 16S rRNA gene nucleotide sequence (≤92 % sequence similarity to recognized Campylobacter species) and genomic DNA (no detectable relatedness) analyses were consistent with novel species status. Cells of the Campylobacter from whooping cranes were uniflagellar and typically sigmoid to allantoid in shape (0.48 μm wide and 2.61 μm long), but also spheroid to coccoid (0.59 μm wide and 0.73 μm long). The bacterium was oxidase-positive, able to reduce nitrite, able to grow at 3 ° and 42 °C, and grew anaerobically, as well as in an atmosphere devoid of H2, and on MacConkey agar. It was not α-haemolytic and was negative for hippurate and indoxyl acetate hydrolysis and alkaline phosphatase. It also was susceptible to cephalotin and was unable to grow on nutrient agar, on a medium containing 3.5 % NaCl or in ambient O2. The bacterium was unable to grow at 25 °C and growth was negative or very restricted at 30 °C. Fluorescent amplified fragment length polymorphism analysis indicated that nine of the recovered isolates were genetically distinct. A species-specific primer set targeting the cpn60 gene was developed. The name Campylobacter canadensis sp. nov. is proposed for the novel species, with the type strain L266T (=CCUG 54429T =LMG 24001T).

Identification of non-tuberculous mycobacteria: utility of the GenoType Mycobacterium CM/AS assay compared with HPLC and 16S rRNA gene sequencing

2009 ◽  
Vol 58 (7) ◽  
pp. 900-904 ◽  
Author(s):  
Andie S. Lee ◽  
Peter Jelfs ◽  
Vitali Sintchenko ◽  
Gwendolyn L. Gilbert
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rrna Gene Sequencing ◽  
Rapid Identification ◽  
Mycobacterium Abscessus ◽  
Clinical Isolates ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Clinically Significant ◽  
Species Specific

Non-tuberculous mycobacteria (NTM) causing clinical disease have become increasingly common and more diverse. A new reverse line probe assay, GenoType Mycobacterium CM/AS (Hain Lifescience), was evaluated for identification of a broad range of NTM. It was compared with phenotypic (HPLC) and molecular (DNA probes, in-house real-time multiplex species-specific PCR, 16S rRNA gene PCR and sequencing) identification techniques, which together provided the reference ‘gold standard’. A total of 131 clinical isolates belonging to 31 Mycobacterium species and 19 controls, including 5 non-Mycobacterium species, was used. Concordant results between the GenoType Mycobacterium assay and the reference identification were obtained in 119/131 clinical isolates (90.8 %). Identification of Mycobacterium abscessus and Mycobacterium lentiflavum by the assay was problematic. The GenoType Mycobacterium assay enables rapid identification of a broad range of potentially clinically significant Mycobacterium species, but some species require further testing to differentiate or confirm ambiguous results.

scholarly journals Detection of Legionellae in Hospital Water Samples by Quantitative Real-Time LightCycler PCR

2001 ◽  
Vol 67 (9) ◽  
pp. 3985-3993 ◽  
Author(s):  
Nele Wellinghausen ◽  
Cathrin Frost ◽  
Reinhard Marre
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Water Samples ◽  
Potable Water ◽  
Water Systems ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Pcr Assays ◽  
The 16S Rrna Gene

ABSTRACT Contamination of hospital water systems with legionellae is a well-known cause of nosocomial legionellosis. We describe a new real-time LightCycler PCR assay for quantitative determination of legionellae in potable water samples. Primers that amplify both a 386-bp fragment of the 16S rRNA gene from Legionellaspp. and a specifically cloned fragment of the phage lambda, added to each sample as an internal inhibitor control, were used. The amplified products were detected by use of a dual-color hybridization probe assay design and quantified with external standards composed ofLegionella pneumophila genomic DNA. The PCR assay had a sensitivity of 1 fg of Legionella DNA (i.e., less than one Legionella organism) per assay and detected 44Legionella species and serogroups. Seventy-seven water samples from three hospitals were investigated by PCR and culture. The rates of detection of legionellae were 98.7% (76 of 77) by the PCR assay and 70.1% (54 of 77) by culture; PCR inhibitors were detected in one sample. The amounts of legionellae calculated from the PCR results were associated with the CFU detected by culture (r= 0.57; P < 0.001), but PCR results were mostly higher than the culture results. Since L. pneumophila is the main cause of legionellosis, we further developed a quantitativeL. pneumophila-specific PCR assay targeting the macrophage infectivity potentiator (mip) gene, which codes for an immunophilin of the FK506 binding protein family. All but one of the 16S rRNA gene PCR-positive water samples were also positive in the mip gene PCR, and the results of the two PCR assays were correlated. In conclusion, the newly developedLegionella genus-specific and L. pneumophila species-specific PCR assays proved to be valuable tools for investigation of Legionella contamination in potable water systems.

scholarly journals The development of a 16S rRNA gene based PCR for the identification of Streptococcus pneumoniaeand comparison with four other species specific PCR assays

2010 ◽  
Vol 10 (1) ◽  
Author(s):  
Nabil Abdullah El Aila ◽  
Stefan Emler ◽  
Tarja Kaijalainen ◽  
Thierry De Baere ◽  
Bart Saerens ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Pcr Assays ◽  
Species Specific

scholarly journals Analysis of the 16S rRNA Gene Sequence of Anaplasma centrale and Its Phylogenetic Relatedness to Other Ehrlichiae

2001 ◽  
Vol 8 (2) ◽  
pp. 241-244 ◽  
Author(s):  
Hisashi Inokuma ◽  
Yutaka Terada ◽  
Tugihiko Kamio ◽  
Didier Raoult ◽  
Philippe Brouqui
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Distance Analysis ◽  
Specific Pcr ◽  
Species Specific ◽  
Anaplasma Centrale ◽  
The 16S Rrna Gene

ABSTRACT The nucleotide sequence of the Anaplasma centrale 16S rRNA gene was determined and compared with the sequences of ehrlichial bacteria. The sequence of A. centrale was closely related to Anaplasma marginale by both level-of-similarity (98.08% identical) and distance analysis. A species-specific PCR was developed based upon the alignment data. The PCR can detect A. centrale DNA extracted from 10 infected bovine red blood cells in a reaction mixture. A. centrale DNA was amplified in the reaction, but not other related ehrlichial species.

scholarly journals Temporal Assessment of the Impact of Exposure to Cow Feces in Two Watersheds by Multiple Host-Specific PCR Assays

2008 ◽  
Vol 74 (22) ◽  
pp. 6839-6847 ◽  
Author(s):  
Yong-Jin Lee ◽  
Marirosa Molina ◽  
Jorge W. Santo Domingo ◽  
Jonathan D. Willis ◽  
Michael Cyterski ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Water Samples ◽  
Fecal Pollution ◽  
Source Tracking ◽  
Rrna Gene ◽  
Gene Markers ◽  
Specific Pcr ◽  
Pcr Assays ◽  
The Impact

ABSTRACT Exposure to feces in two watersheds with different management histories was assessed by tracking cattle feces bacterial populations using multiple host-specific PCR assays. In addition, environmental factors affecting the occurrence of these markers were identified. Each assay was performed using DNA extracts from water and sediment samples collected from a watershed directly impacted by cattle fecal pollution (WS1) and from a watershed impacted only through runoff (WS2). In WS1, the ruminant-specific Bacteroidales 16S rRNA gene marker CF128F was detected in 65% of the water samples, while the non-16S rRNA gene markers Bac1, Bac2, and Bac5 were found in 32 to 37% of the water samples. In contrast, all source-specific markers were detected in less than 6% of the water samples from WS2. Binary logistic regressions (BLRs) revealed that the occurrence of Bac32F and CF128F was significantly correlated with season as a temporal factor and watershed as a site factor. BLRs also indicated that the dynamics of fecal-source-tracking markers correlated with the density of a traditional fecal indicator (P < 0.001). Overall, our results suggest that a combination of 16S rRNA gene and non-16S rRNA gene markers provides a higher level of confidence for tracking unknown sources of fecal pollution in environmental samples. This study also provided practical insights for implementation of microbial source-tracking practices to determine sources of fecal pollution and the influence of environmental variables on the occurrence of source-specific markers.

scholarly journals Phylogenetic analysis of Helicobacter species based on partial gyrB gene sequences

2007 ◽  
Vol 57 (3) ◽  
pp. 444-449 ◽  
Author(s):  
Minna Hannula ◽  
Marja-Liisa Hänninen
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Pcr Primers ◽  
Phylogenetic Study ◽  
Rrna Gene ◽  
Gene Sequences ◽  
Specific Pcr ◽  
Species Specific ◽  
The 16S Rrna Gene

Analysis of 16S rRNA gene sequences is one of the most common methods for investigating the phylogeny and taxonomy of bacteria. However, several studies have indicated that the 16S rRNA gene does not distinguish between certain Helicobacter species. We therefore selected for phylogenetic analysis an alternative marker, gyrB, encoding gyrase subunit B. The aim of this investigation was to examine the applicability of gyrB gene fragments (~1100 bp) for the phylogenetic study of 16 Helicobacter species and a total of 33 Helicobacter strains included in this study. Based on the sequenced fragments, a phylogenetic tree was obtained that contained two distinct clusters, with gastric species forming one cluster and enterohepatic species the other. The only exception was the gastric species Helicobacter mustelae, which clustered with the enterohepatic species. The calculated similarity matrix revealed the highest interspecies similarity between Helicobacter salomonis and Helicobacter felis (89 %) and the lowest similarity between Helicobacter pullorum and H. felis (60 %). The DNA G+C content of the sequenced fragments was ⩽40 mol% in enterohepatic species and >46 mol% in gastric species, excluding Helicobacter pylori and H. mustelae, with G+C contents of 34 and 42 mol%, respectively. In summary, the gyrB gene fragments provided superior resolution and reliability to the 16S rRNA gene for differentiating between closely related Helicobacter species. A further outcome of this study was achieved by designing gyrB gene-based species-specific PCR primers for the identification of Helicobacter bizzozeronii.

scholarly journals Atypical Helicobacter canadensis Strains Associated with Swine

2006 ◽  
Vol 72 (6) ◽  
pp. 4464-4471 ◽  
Author(s):  
G. Douglas Inglis ◽  
Malcolm McConville ◽  
Anno de Jong
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Fragment Length Polymorphism ◽  
23S Rrna ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Cardinal Characteristic ◽  
Swine Feces ◽  
Species Specific ◽  
Genomic Typing

ABSTRACT Forty-two Helicobacter isolates were isolated from swine feces in The Netherlands and Denmark. All 12 isolates sequenced (16S rRNA gene) formed a robust clade with Helicobacter canadensis (∼99% similarity). Species-specific PCR indicated that all of the isolates were H. canadensis isolates. Although the appearance of the porcine isolates was similar to the appearance of H. canadensis, only one of these isolates was able to hydrolyze indoxyl acetate, a cardinal characteristic of this taxon. Examination of the 23S rRNA and hsp60 genes revealed high levels of similarity between the porcine isolates and H. canadensis. However, amplified fragment length polymorphism genomic typing showed that isolates recovered from swine feces were genetically distinct from H. canadensis strains obtained from humans and geese.

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