Specific PCR primers from the 16S-23S rRNA spacer region for the rapid detection and identification of Actinobacillus seminis

Actinobacillus seminis is a common cause of ovine epididymitis and ram infertility. The ability to detect and identify this organism promptly is important commercially for the quality control of ram semen samples. Actinobacillus seminis is a fastidious and slow-growing bacterium and primary isolation and presumptive identification can be difficult and time-consuming. In this study, two ribosomal operons, termed rrnA and rrnB, have been characterized in the A. seminis genome, and these contain one and two tRNAs, respectively, in the spacer region between the 16S and 23S rRNA genes. Species-specific primers for A. seminis were developed from the sequence of the spacer region of rrnB for the identification and detection of A. seminis by PCR. The PCR assay was specific for A. seminis and gave no amplification products with phenotypically similar organisms such as Histophilus ovis. Storage solution used to preserve semen for long-term storage was found to inhibit the PCR. Therefore, for diagnostic purposes, the assay would best be performed after primary isolation or perhaps on fresh semen prior to storage if obvious contamination is indicated.     

Evaluation of a new method for identification of bacteria based on sequence homology of 16S rRNA gene

A new identification method for bacteria based on partial sequences of divergent regions of the 16S rRNA gene was evaluated. The method involves PCR-based amplification of 16S rRNA gene fragments, followed by sequencing and comparison of sequences of about 300 nucleotides with those in the database of NCBI (National Center for Biotechnology Information) via the Internet. Most of the bacteria tested could be identified at the species level even if some unread nucleotides were present in the sequence. Although this method still requires improvement, it has the potential to be a highly reliable and practical identification method for bacteria.     

A rapid dot-blot method for species identification of bloodstains

A very simple and rapid test for species identification is reported. Extracts of bloodstains were applied to a synthetic porous membrane and dried. The membrane was then quenched with glycine buffered saline containing BSA and Tween 20. A suspension of colloidal gold particles (GP) coated with rabbit antiserum to human IgG was poured onto, gently whirled and aspirated through the membrane. Spots from the human and monkey bloodstains became red, whereas those from other species of animals remained unstained. This test was completed within 3 to 4 min, and the antibody-coated GP reagent was prepared within 20 min using a very small quantity of antiserum. Cellulose acetate membranes of 0.45 microns or more in pore size were appropriate to this test.     

An evaluation of intergenic rRNA gene sequence length polymorphism analysis for the identification of Legionella species

There are currently more than 40 species of Legionella and the identification of most of these by standard methods is technically difficult. The aim of this study was to assess the suitability of a previously published PCR-based method of identifying Legionella spp. Intergenic 16S-23S rDNA spacer regions were amplified with primers complementary to conserved regions of the rRNA genes. Following electrophoretic separation of the products, data analyses were performed with the Taxotron software package. Computer-assisted analysis (with an empirically derived error tolerance of 3%) could differentiate only 26 of the 43 strains (representing 43 species), with the remaining 17 species clustering into four groups (group I, comprising 10 species; group II, three species; group III, two species and group IV, two species). Analysis of well-characterised 'non-type' strains of some Legionella spp. (e.g., from type culture collections) resulted in patterns distinct from the corresponding type strain in most cases. Furthermore, recent isolates (identified by conventional methods) were identified by this PCR method to the presumed correct species (or species group) in only a minority of cases. Well characterised strains and recent isolates of Legionella showed heterogeneity within many species. This intra-species variation severely limits the usefulness of the method for the identification of isolates. However, this property may be useful for epidemiological typing within such species.     

Multiplex PCR for the identification of Arcobacter and differentiation of Arcobacter butzleri from other arcobacters

A multiplex polymerase chain reaction (PCR) assay to identify Arcobacter isolates and to distinguish A. butzleri from other arcobacters is described. The test uses two primer sets. Set I targets a section of the 16S rRNA genes of Arcobacter spp. Set II amplifies a portion of the 23S rRNA genes unique to A. butzleri. Specificity of the primer sets was evaluated using ATCC reference strains of A. butzleri, A. cryaerophilus, A. skirrowii, Bacteroides spp., Campylobacter spp., Helicobacter spp. and Wolinella succinogenes. Upon PCR amplification, all of the Arcobacter isolates yielded a 1223 bp product, whereas A. butzleri ATCC 49616 exhibited both a 1223 bp and a 686 bp product. No PCR product was observed for other closely related ATCC strains (n = 37). We next analyzed by multiplex PCR field strains of Arcobacter spp. (n = 108) which had been previously characterized to the species level by either DNA-DNA hybridization, dot blot hybridization, ribotyping or by serology. The 1223 bp multiplex PCR product identified all of the isolates as Arcobacter. The presence of both the 1223 and 686 bp amplicons identified 66 strains as A. butzleri. Speciation by multiplex PCR agreed with results obtained by the other methods. The multiplex PCR assay is specific, rapid and easy to interpret and, thus, will aid in elucidating the prevalence, epidemiology and zoonotic potential of Arcobacter.     

PCR assay based on DNA coding for 16S rRNA for detection and identification of mycobacteria in clinical samples

A PCR and a reverse cross blot hybridization assay were developed for the detection and identification of mycobacteria in clinical samples. The PCR amplifies a part of the DNA coding for 16S rRNA with a set of primers that is specific for the genus Mycobacterium and that flanks species-specific sequences within the genes coding for 16S rRNA. The PCR product is analyzed in a reverse cross blot hybridization assay with probes specific for M. tuberculosis complex (pTub1), M. avium (pAvi3), M. intracellulare (pInt5 and pInt7), M. kansasii complex-M. scrofulaceum complex (pKan1), M. xenopi (pXen1), M. fortuitum (pFor1), M. smegmatis (pSme1), and Mycobacterium spp. (pMyc5a). The PCR assay can detect 10 fg of DNA, the equivalent of two mycobacteria. The specificities of the probes were tested with 108 mycobacterial strains (33 species) and 31 nonmycobacterial strains (of 17 genera). The probes pAvi3, pInt5, pInt7, pKan1, pXen1, and pMyc5a were specific. With probes pTub1, pFor1, and pSme1, slight cross hybridization occurred. However, the mycobacterial strains from which the cross-hybridizing PCR products were derived belonged to nonpathogenic or nonopportunistic species which do not occur in clinical samples. The test was used on 31 different clinical specimens obtained from patients suspected of having mycobacterial disease, including a patient with a double mycobacterial infection. The samples included sputum, bronchoalveolar lavage, tissue biopsy samples, cerebrospinal fluid, pus, peritoneal fluid, pleural fluid, and blood. The results of the PCR assay agreed with those of conventional identification methods or with clinical data, showing that the test can be used for the direct and rapid detection and identification of mycobacteria in clinical samples.     

Phylogenetic positions of Clostridium chauvoei and Clostridium septicum based on 16S rRNA gene sequences

The sequences of the 16S rRNA genes (rrs genes) of Clostridium chauvoei, the causative agent of blackleg in cattle, and the phenotypically related organism Clostridium septicum were determined. After amplification of 1,507-bp PCR fragments from the corresponding rrs genes, the sequences were determined in a single round of sequencing by using conserved region primers. A sequence similarity analysis of the sequences revealed the close phylogenetic relationship of C. chauvoei and C. septicum in Clostridium cluster I (M. D. Collins, P. A. Lawson, A. Willems, J. J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994), which includes Clostridium carnis, Clostridium perfringens, Clostridium botulinum, and Clostridium tetani. We found that 99.3% of the nucleotides in the genes of C. chauvoei and C. septicum are identical.     

Phylogenetic analyses of Chlamydia psittaci strains from birds based on 16S rRNA gene sequence

The nucleotide sequences of 16S ribosomal DNA (rDNA) were determined for 39 strains of Chlamydia psittaci (34 from birds and 5 from mammals) and for 4 Chlamydia pecorum strains. The sequences were compared phylogenetically with the gene sequences of nine Chlamydia strains (covering four species of the genus) retrieved from nucleotide databases. In the neighbor-joining tree, C. psittaci strains were more closely related to each other than to the other Chlamydia species, although a feline pneumonitis strain was distinct (983 to 98.6% similarity to other strains) and appeared to form the deepest subline within the species of C. psittaci (bootstrap value, 99%). The other strains of C. psittaci exhibiting similarity values of more than 99% were branched into several subgroups. Two pigeon strains and one turkey strain formed a distinct clade recovered in 97% of the bootstrapped trees. The other pigeon strains seemed to be distinct from the strains from psittacine birds, with 88% of bootstrap value. In the cluster of psittacine strains, three parakeet strains and an ovine abortion strain exhibited a specific association (level of sequence similarity, 99.9% or more; bootstrap value, 95%). These suggest that at least four groups of strains exist within the species C. psittaci. The 16S rDNA sequence is a valuable phylogenetic marker for the taxonomy of chlamydiae, and its analysis is a reliable tool for identification of the organisms.     

Molecular evolution of Mycoplasma capricolum subsp. capripneumoniae strains, based on polymorphisms in the 16S rRNA genes

Mycoplasma capricolum subsp. capripneumoniae belongs to the so-called Mycoplasma mycoides cluster and is the causal agent of contagious caprine pleuropneumonia (CCPP). All members of the M. mycoides cluster have two rRNA operons. The sequences of the 16S rRNA genes of both rRNA operons from 20 strains of M. capricolum subsp. capripneumoniae of different geographical origins in Africa and Asia were determined. Nucleotide differences which were present in only one of the two operons (polymorphisms) were detected in 24 positions. The polymorphisms were not randomly distributed in the 16S rRNA genes, and some of them were found in regions of low evolutionary variability. Interestingly, 11 polymorphisms were found in all the M. capricolum subsp. capripneumoniae strains, thus defining a putative ancestor. A sequence length difference between the 16S rRNA genes in a poly(A) region and 12 additional polymorphisms were found in only one or some of the strains. A phylogenetic tree was constructed by comparative analysis of the polymorphisms, and this tree revealed two distinct lines of descent. The nucleotide substitution rate of strains within line II was up to 50% higher than within line I. A tree was also constructed from individual operonal 16S rRNA sequences, and the sequences of the two operons were found to form two distinct clades. The topologies of both clades were strikingly similar, which supports the use of 16S rRNA sequence data from homologous operons for phylogenetic studies. The strain-specific polymorphism patterns of the 16S rRNA genes of M. capricolum subsp. capripneumoniae may be used as epidemiological markers for CCPP.     

Specific identification of the enteropathogens Campylobacter jejuni and Campylobacter coli by using a PCR test based on the ceuE gene encoding a putative virulence determinant

A PCR method for the rapid identification and discrimination of thermophilic Campylobacter jejuni and Campylobacter coli was developed by using a gene encoding a protein involved in siderophore transport (ceuE). A nucleotide sequence divergence of approximately 13% in the ceuE genes of C. jejuni and C. coli facilitated the design of two species-specific PCR primer sets. The specificity of the PCR amplification reactions was confirmed by using two nonradioactively labelled species-specific internal oligonucleotide hybridization probes for each of these species.     

Overexpression of the thiostrepton-resistance gene from Streptomyces azureus in Escherichia coli and characterization of recognition sites of the 23S rRNA A1067 2'-methyltransferase in the guanosine triphosphatase center of 23S ribosomal RNA

The thiostrepton-resistance gene encoding the 23S rRNA A1067 methyltransferase from Streptomyces azureus has been overexpressed in Escherichia coli using a T7-RNA-polymerase-dependent expression vector. The protein was efficiently expressed at levels up to 20% of total soluble protein and purified to near homogeneity. Kinetic parameters for S-adenosyl-L-methionine (Km = 0.1 mM) and an RNA fragment containing nucleotides 1029-1122 of the 23S ribosomal RNA from E. coli (Km = 0.001 mM) were determined. S-Adenosyl-L-homocysteine showed competitive product inhibition (Ki = 0.013 mM). Binding of either thiostrepton or protein L11 inhibited methylation. RNA sequence variants of the RNA fragment with mutations in nucleotides 1051-1108 were tested as substrates for the methylase. The experimental data indicate that methylation is dependent on the secondary structure of the hairpin including nucleotide A1067 and the exact sequence U(1066)-A(1067)-G(1068)-A(1069)-A(1070) of the single strand.     

Nuclear counterparts of the cytoplasmic mitochondrial 12S rRNA gene: a problem of ancient DNA and molecular phylogenies

Monkey mummy bones and teeth originating from the North Saqqara Baboon Galleries (Egypt), soft tissue from a mummified baboon in a museum collection, and nineteenth/twentieth-century skin fragments from mangabeys were used for DNA extraction and PCR amplification of part of the mitochondrial 12S rRNA gene. Sequences aligning with the 12S rRNA gene were recovered but were only distantly related to contemporary monkey mitochondrial 12S rRNA sequences. However, many of these sequences were identical or closely related to human nuclear DNA sequences resembling mitochondrial 12S rRNA (isolated from a cell line depleted in mitochondria) and therefore have to be considered contamination. Subsequently in a separate study we were able to recover genuine mitochondrial 12S rRNA sequences from many extant species of nonhuman Old World primates and sequences closely resembling the human nuclear integrations. Analysis of all sequences by the neighbor-joining (NJ) method indicated that mitochondrial DNA sequences and their nuclear counterparts can be divided into two distinct clusters. One cluster contained all temporary cytoplasmic mitochondrial DNA sequences and approximately half of the monkey nuclear mitochondriallike sequences. A second cluster contained most human nuclear sequences and the other half of monkey nuclear sequences with a separate branch leading to human and gorilla mitochondrial and nuclear sequences. Sequences recovered from ancient materials were equally divided between the two clusters. These results constitute a warning for when working with ancient DNA or performing phylogenetic analysis using mitochondrial DNA as a target sequence: Nuclear counterparts of mitochondrial genes may lead to faulty interpretation of results.     

PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli direct from diarrheic samples

Three sets of primers were designed for PCR detection and differentiation of Campylobacter jejuni and Campylobacter coli. The first PCR assay was designed to coidentify C. jejuni and C. coli based on their 16S rRNA gene sequences. The second PCR assay, based on the hippuricase gene sequence, identified all tested reference strains of C. jejuni and also strains of that species which lack detectable hippuricase activity. The third PCR assay, based on the sequence of a cloned (putative) aspartokinase gene and the downstream open reading frame, identified all tested reference strains of C. coli. The assays will find immediate application in the rapid identification to species level of isolates. The assays combine with a protocol for purification of total DNA from fecal samples to allow reproducible PCR identification of campylobacters directly from stools. Of 20 clinical samples from which campylobacters had been cultured, we detected C. jejuni in 17, C. coli in 2, and coinfection of C. jejuni and Campylobacter hyointestinalis in 1. These results were concordant with culture and phenotypic identification to species level. Strain typing by PCR-restriction fragment length polymorphism of the flagellin (flaA) gene detected identical flaA types in fecal DNA and the corresponding campylobacter isolate. Twenty-five Campylobacter-negative stool samples gave no reaction with the PCR assays. These PCR assays can rapidly define the occurrence, species incidence, and flaA genotypes of enteropathogenic campylobacters.     

Identification of the enteropathogens Campylobacter jejuni and Campylobacter coli based on the cadF virulence gene and its product

Campylobacter jejuni and Campylobacter coli are common causes of gastroenteritis in humans. Infection with C. jejuni or C. coli is commonly acquired by eating undercooked chicken. The goal of this study was to develop specific detection assays for C. jejuni and C. coli isolates based on the cadF virulence gene and its product. The cadF gene from C. jejuni and C. coli encodes a 37-kDa outer membrane protein that promotes the binding of these pathogens to intestinal epithelial cells. A fragment of approximately 400 bp was amplified from 38 of 40 (95%) C. jejuni isolates and 5 of 6 (83.3%) C. coli isolates with primers designed to amplify an internal fragment of the cadF gene. PCR was then used to amplify Campylobacter DNA from store-bought chickens. A 400-bp band was amplified from 26 of the 27 chicken carcasses tested by the PCR-based assay. The CadF protein was detected in every C. jejuni and C. coli isolate tested, as judged by immunoblot analysis with a rabbit anti-C. jejuni 37-kDa serum. In addition, methanol-fixed samples of whole-cell C. jejuni and C. coli were detected with the rabbit anti-37-kDa serum by using an indirect-immunofluorescence microscopy assay. These findings indicate that the cadF gene and its product are conserved among C. jejuni and C. coli isolates and that a PCR assay based on the cadF gene may be useful for the detection of Campylobacter organisms in food products.     

Strategy for the detection of Helicobacter species by amplification of 16S rRNA genes and identification of H. felis in a human gastric biopsy

The aim of the present work was to develop polymerase chain reactions (PCRs) based on the conserved nucleotide sequence of the 16S rRNA gene for detection of bacteria of the Helicobacter genus in human antral biopsy samples. The assay for Helicobacter spp was developed by amplifying a 399-bp 16S rRNA gene sequence specific to the genus Helicobacter. The identity of the amplicon was confirmed by hybridization with an internal probe and by restriction by endonuclease VspI showing two expected fragments of 295 and 104 base pairs. A total of 65 dyspeptic patients from France and New Caledonia were screened for Helicobacter spp infection through the use of the following diagnostic assays on biopsy specimens collected through endoscopy: direct detection of bacteria in histological sections by Giemsa and Warthin Starry staining, urease test and bacterial isolation, PCR for Helicobacter pylori ureC/glmM gene, and PCR targeted to 16S rRNA genes. The 16S rRNA gene PCR assay was able to detect down to 680 bacterial cells, as assessed by agarose gel electrophoresis, and down to 4 bacterial cells by hybridization of amplicon with the internal probe. The 16S rRNA PCR test was 100% specific and sensitive; results obtained with this test were in agreement with the visualization of bacteria by histology. Urease test and culture were 86.4% and 22.7% sensitive, and 96.5 and 100% specific, respectively. The H. pylori ureC/glmM gene-based PCR was 100% specific and only 95.4% sensitive, since one biopsy from a Melanesian patient contained a Helicobacter strain other than H. pylori. For this Melanesian patient, a branch-specific PCR targeting the epsilon branch of Proteobacteria was used to amplify a 967-bp amplicon. This amplicon was sequenced and matched with the H. felis sequence. This was confirmed using an H. felis-specific urease PCR test.     

The 16S-23S rRNA intergenic spacer region of Bartonella (Rochalimaea) species is longer than usually described in other bacteria

The isozymes of prostaglandin G/H synthase (PGHS) are shown to be differentially inhibited in vitro by currently marketed nonsteroidal anti-inflammatory drugs (NSAIDs) using microsomal rhPGHS-1 and rhPGHS-2. Comparison of selectivity ratios (IC50 rhPGHS-1/IC50 rhPGHS-2) demonstrated a 10-fold selectivity of etodolac (Lodine) for rhPGHS-2, whereas the other NSAIDs evaluated demonstrated no preference or a slight preference for inhibition of rhPGHS-1. In vitro enzyme results were supported by a human whole blood assay where etodolac also demonstrated a 10-fold selectivity for inhibition of PGHS-2 mediated TxB2 production. Taken together, these data may be key to explaining the clinically observed gastrointestinal safety of etodolac versus other marketed NSAIDs.     

A qualitative evaluation of the published oligonucleotides specific for the 16S rRNA gene sequences of the ammonia-oxidizing bacteria

Over the past few years, there has been an increasing interest in making oligonucleotides specific for ammonia-oxidizing bacteria (AOB), in order to detect and monitor these slow growing bacteria in environmental samples, in enrichment cultures and in wastewater treatment plants. Based on 16S rDNA sequences, a broad selection of oligonucleotides have been designed, either encompassing all known AOB in the beta-subgroup of the Proteobacteria (beta AOB), or subclasses within beta AOB. Thirty different oligonucleotides have so far been published, with varying specificity. The first AOB-specific oligonucleotides published were obtained as a result of an alignment of only eleven 16S rDNA sequences from AOB. Including the present study, there are now forty nearly full length 16S rDNA sequences available from these bacteria, in addition to a number of partial sequences, so that an improved evaluation of the published oligonucleotides can be done. Two new 16S rRNA gene sequences from Nitrosospira are presented here, in a phylogenetic analysis containing every 16S rRNA gene sequences (> 1 kb) available from AOB. On the basis of an alignment of all these sequences, combined with searches in the nucleotide sequence databases, an evaluation of the thirty published oligonucleotides is presented. The analysis expose the strength and weakness of each oligonucleotide and discuss the use of oligonucleotides specific for 16S rRNA genes in future studies of AOB. The present work also identifies one new, broad range primer, specific for the AOB in the beta-subgroup of the Proteobacteria.