Identification of the genus Geobacillus using genus-specific primers, based on the 16S-23S rRNA gene internal transcribed spacer

The aim of this study was to develop an easy and accurate technique for the identification of the genus Geobacillus. For this purpose, Geobacillus genus-specific primers GEOBAC (GEOBAC-F and GEOBAC-R) based on the 16S-23S rRNA gene internal transcribed spacer (ITS) region sequences have been designed. In total, 52 sequences from three species of the genus Geobacillus (Geobacillus stearothermophilus, Geobacillus kaustophilus and Geobacillus lituanicus) were examined for the design of these primers. Analysis of the sequences revealed three highly conservative regions common to these species: 5' and 3' end regions of 16S-23S rRNA gene ITSs and box A. Some sequences possessed two additional conservative regions - genes of tRNA(Ile) and tRNA(Ala). These particular sequences were chosen for the construction of the primers. The designed primers targeted the gene of tRNA(Ile) and the 3' end region of ITSs. This technique was validated with both the reference strains of the genus Geobacillus and the thermophilic aerobic endospore-forming environmental isolates. Different Geobacillus species could be grouped according to the number and size of GEOBAC-PCR products and identified on the basis of the AluI and TaqI restriction analysis of these products.     

Unusual intragenomic and interspecific variability of <Emphasis Type="Italic">Geobacillus</Emphasis> 16S-23S rRNA internal transcribed spacers

The aim of this study was to evaluate the inter-and intraspecific as well as intragenomic variability of Geobacillus 16S–23S rRNA internal transcribed spacers without tRNA genes and to compare these sequences with sequences bearing tRNA genes. In this study the structural analysis was performed in a unique way because the length and the sequence of the structural blocks were adjusted to fit the structure of 16S–23S rRNA internal transcribed spacers of five different Geobacillus species. Our study demonstrated the mosaic-like structure of 16S–23S rRNA internal transcribed spacers in Geobacillus. Some characteristics of these spacers of geobacilli were not previously reported for other bacteria: unusually short conserved sequence in the 5′ end region, some identical conserved blocks in both 5′ and 3′ regions of 16S–23S rRNA internal transcribed spacers, the same sequence blocks in both 16S–23S and 23S–5S rRNA intergenic spacers. Our study demonstrated quite uniform arrangement of the sequence blocks in Geobacillus thermodenitrificans. This species diverged early in the phylogenetic tree of the genus Geobacillus. For the phylogenetically recent species Geobacillus kaustophilus and Geobacillus lituanicus the low inter-and intraspecific, but high intragenomic variability, as a consequence of recent phylogenetic events, was established.     

Variability of the 16S-23S rRNA gene internal transcribed spacer in Pseudomonas avellanae strains

The 16S-23S rRNA gene internal transcribed spacer region (ITS1) from 34 strains of Pseudomonas avellanae and some strains of Pseudomonas syringae pathovars was amplified and assessed by restriction fragment length polymorphism (RFLP) using 10 restriction enzymes. In addition, the ITS1 region of four representative P. avellanae strains was sequenced and compared by the neighbour-joining algorithm with that of P. syringae pathovars. Two main groups of P. avellanae strains were observed that did not correlate with their origin. The ITS1 region sequencing revealed a high similarity with the P. syringae complex. One group of P. avellanae strains showed high similarity to P. s. pv. actinidiae and P. s. pv. tomato; another group showed similarity with P. s. pv. tabaci and P. s. pv. glycinea. Two strains clustered with P. s. pv. pisi. The difficulties to unambiguously classify the strains associated with hazelnut decline in Greece and Italy are discussed.     

Development of a novel triplex PCR assay for the detection and differentiation of thermophilic species of Campylobacter using 16S-23S rDNA internal transcribed spacer (ITS) region

AIM: Campylobacter species are significantly implicated in human gastrointestinal infections. Of 20 species of Campylobacter, C. jejuni, C. coli and C. lari have been considered as the most important causative agents of human infections. In order to better understand the occurrence and epidemiology of these thermophilic Campylobacter species, an improved and rapid detection method is warranted. A novel triplex polymerase chain reaction (PCR) assay was developed based on the variable 16S-23S rDNA internal transcribed spacer (ITS) region to identify and discriminate between these species in water samples. METHODS AND RESULTS: Campylobacter species-specific primers for C. jejuni, C. coli and C. lari derived from highly variable sequences in the ITS region were used. Specificity of the newly designed primers and PCR conditions were verified using other species of Campylobacter as well as 31 different negative control species. The assay was further validated with 97 Campylobacter cultures from water samples. CONCLUSIONS: The assay was found to be simple, easy to perform, and had a high sensitivity, specificity and reproducibility. It enabled simultaneous detection and differentiation of multiple Campylobacter species in water samples. SIGNIFICANCE AND IMPACT OF STUDY: Use of the newly developed PCR assay, coupled with a previously developed rapid DNA template preparation step, will enable improved detection capabilities for Campylobacter species in environmental matrices.     

Differentiation of bacterial strains by thermal gradient gel electrophoresis using non-GC-clamped PCR primers for the 16S-23S rDNA intergenic spacer region

The method for DNA fingerprinting of the 16S-23S rDNA intergenic spacer region was modified to increase resolution of bacterial strains by thermal gradient gel electrophoresis (TGGE) analysis. By utilizing the high melting temperature region of the tRNA gene located in the middle of the 16S-23S rDNA intergenic spacer region as an internal clamp for TGGE, multiple melting domain problems were solved. PCR primers lacking a stretch of GC-rich sequences (GC-clamp) amplified the intergenic spacer region more efficiently than GC-clamped primers. Therefore, PCR artifacts were avoided by using low, 17-cycle, PCR. The method was successfully applied to diverse bacterial species for strain differentiation by TGGE without requiring a special PCR primer set.     

PCR amplification of species specific sequences of 16S rDNA and 16S-23S rDNA intergenic spacer region for identification of Streptococcus phocae

Streptococcus phocae, a bacterial pathogen of seals, could reliably be identified by PCR amplification using oligonucleotide primers designed according to species specific segments of the previously sequenced 16S rRNA gene and the 16S-23S rDNA intergenic spacer region of this species. The PCR mediated assay allowed an identification of S. phocae isolated from harbor and gray seals and from Atlantic salmons. No cross-reaction could be observed with 13 different other streptococcal species and subspecies and with Lactococcus garvieae strains investigated for control purposes.     

Identification of Lactobacillus alimentarius and Lactobacillus farciminis with 16S-23S rDNA intergenic spacer region polymorphism and PCR amplification using species-specific oligonucleotide

AIMS: The restriction fragment length polymorphism (RFLP) method was used to differentiate Lactobacillus species having closely related identities in the 16S-23S rDNA intergenic spacer region (ISR). Species-specific primers for Lact. farciminis and Lact. alimentarius were designed and allowed rapid identification of these species. METHODS AND RESULTS: The 16S-23S rDNA spacer region was amplified by primers tAla and 23S/p10, then digested by HinfI and TaqI enzymes and analysed by electrophoresis. Digestion by HinfI was not sufficient to differentiate Lact. sakei, Lact. curvatus, Lact. farciminis, Lact. alimentarius, Lact. plantarum and Lact. paraplantarum. In contrast, digestion carried out by TaqI revealed five different patterns allowing these species to be distinguished, except for Lact. plantarum from Lact. paraplantarum. The 16S-23S rDNA spacer region of Lact. farciminis and Lact. alimentarius were amplified and then cloned into vector pCR(R)2.1 and sequenced. The DNA sequences obtained were analysed and species-specific primers were designed from these sequences. The specificity of these primers was positively demonstrated as no response was obtained for 14 other species tested. RESULTS AND CONCLUSIONS: The species-specific primers for Lact. farciminis and Lact. alimentarius were shown to be useful for identifying these species among other lactobacilli. The RFLP profile obtained upon digestion with HinfI and TaqI enzymes can be used to discriminate Lact. farciminis, Lact. alimentarius, Lact. sakei, Lact. curvatus and Lact. plantarum. SIGNIFICANCE AND IMPACT OF THE STUDY: In this paper, we have established the first species-specific primer for PCR identification of Lact. farciminis and Lact. alimentarius. Both species-specific primer and RFLP, could be used as tools for rapid identification of lactobacilli up to species level.     

Identification of Escherichia coli through analysis of 16S rRNA and 16S-23S rRNA internal transcribed spacer region sequences

A bacterial strain, designated BzDS03 was isolated from water sample, collected from Dal Lake Srinagar. The strain was characterized by using 16S ribosomal RNA gene and 16S-23S rRNA internal transcribed spacer region sequences. Phylogenetic analysis showed that 16S rRNA sequence of the isolate formed a monophyletic clade with genera Escherichia. The closest phylogenetic relative was Escherichia coli with 99% 16S rRNA gene sequence similarity. The result of Ribosomal database project's classifier tool revealed that the strain BzDS03 belongs to genera Escherichia.16S rRNA sequence of isolate was deposited in GenBank with accession number FJ961336. Further analysis of 16S-23S rRNA sequence of isolate confirms that the identified strain BzDS03 be assigned as the type strain of Escherichia coli with 98% 16S-23S rRNA sequence similarity. The GenBank accession number allotted for 16S-23S rRNA intergenic spacer sequence of isolate is FJ961337.     

tRNA genes were found in Piscirickettsia salmonis 16S-23S rDNA spacer region (ITS)

Piscirickettsia salmonis is the etiological agent of Salmonid Rickettsial Septicemia, a disease affecting salmon aquaculture industry. We analyzed the 16S-23S rDNA spacer region (internal transcribed spacer, ITS) of Chilean P. salmonis isolates LF-89 and EM-90. Two main ITS amplification products were obtained by PCR using L1 and G1 primers, differing from that described where only one ITS region was found. By Southern blot, it was established that these two amplification products contained sequences related to P. salmonis ITS. Sequence analysis confirmed that P. salmonis had two ITS regions: ITS A and ITS B. In both isolates, the smaller (ITS B) corresponded to ITS sequences previously described for each one, and the larger (ITS A) were almost the same as their respective ITS B sequences interrupted by an insert which contained two tRNAs genes: tRNA-Ile and tRNA-Ala.     

Detection of Laribacter hongkongensis using species-specific duplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region (ISR)

Aims: For the rapid detection of Laribacter hongkongensis, which is associated with human community‐acquired gastroenteritis and traveller’s diarrhoea, we developed a duplex species‐specific PCR assay. Methods and Results: Full‐length of the 16S–23S rRNA intergenic spacer region (ISR) sequences of 52 L. hongkongensis isolates were obtained by PCR‐based sequencing. Two species‐specific primer pairs targeting 16S rRNA gene and ISR were designed for duplex PCR detection of L. hongkongensis. The L. hongkongensis species‐specific duplex PCR assay showed 100% specificity, and the minimum detectable level was 2·1 × 10^?2 ng μl^?1 genomic DNA which corresponds to 5000 CFU ml^?1. Conclusions: The high specificity and sensitivity of the assay make it suitable for rapid detection of L. hongkongensis. Significance and Impact of the Study: This species‐specific duplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify L. hongkongensis and may have applications in both clinical and environmental microbiology.     

Genetic relationships among mycoplasmas based on the 16S-23S rRNA spacer sequence

The nucleotide sequences of the spacer regions between the 16S and 23S rRNA genes of 20 Mycoplasma species were determined following amplification by PCR. Although the spacer regions lacked spacer tRNA genes, they contained the box B and box A sequences in this order from the 5' terminus. The sequence alignment indicated that the 20 species were divided into four clusters, the M. pneumoniae, M. hominis, M. hyorhinis and M. fermentans clusters, and a single floating species, M. hyopneumoniae.     

Structural analysis and genetic variation of the 16S-23S rDNA internal spacer region from Micrococcus luteus strains

AIMS: To clone and sequence the 16S-23S ribosomal DNA (rDNA) internal spacer region (ISR) from Micrococcus luteus. METHODS AND RESULTS: The primer pair for 16S-23S rDNA ISR amplified a fragment of about 850 bp in length for two strains, JCM3347 and JCM3348 and a fragment of about 790 bp for a strain, ATCC9341. After sequencing the ISRs were identified by the comparison of the ISRs and the flanking regions of ISR. CONCLUSIONS: Although the sequence difference of the ISR occurred at only one position between the two JCM strains, the highly variable length (440 and 370 bp) and sequence similarity (about 40%) were demonstrated between the ISRs of the two JCM strains and a ATCC strain. SIGNIFICANCE AND IMPACT OF THE STUDY: A CCTCCT sequence was first detected at the 3'-end of the 16S rDNA of the three strains. Moreover, highly similar sequence to the 21-bp region containing a putative rRNA processing site was observed in the ISR of the three strains. Interestingly, no intercistronic tRNAs were demonstrated in the ISRs from the three strains.     

Comprehensive detection of bacterial populations by PCR amplification of the 16S-23S rRNA spacer region

PCR amplification of the spacer region between the 16S and 23S rRNA genes is commonly employed for the analysis of bacterial communities. In this analysis, the intergenic spacers are amplified by PCR using primers complementary to conserved regions in the 3' 16S rDNA and 5' 23S rDNA. By this method, the observation of every bacterial population may be limited by several causes. To explore the extent of bacterial populations overlooked by this method, we have used an empirical approach. In a sample containing about 50 colonies, we tested the capability to amplify by PCR the spacers from each colony. We also examined the ability to observe the spacers from each colony in the product obtained after amplification of the DNA extracted from the whole sample, as it is usually performed by this method. Contrarily to our expectations that a significant fraction of colonies would not yield amplification products, spacers were successfully amplified from every colony of two different samples examined. Overall, our results suggest that in spite of well-based theoretical limitations, the analysis of bacterial communities by amplification of the spacer regions can render a comprehensive representation of the more abundant bacterial clades in the sample.     

The phylogenetic diversity of thermophilic members of the genus Bacillus as revealed by 16S rDNA analysis

Abstract Sixteen thermophilic strains of the genus Bacillus , representing eight validly described and six invalidly described species, as well as one unassigned strain, were investigated by comparative 16S rDNA analyses and the sequences compared to the existing database for the genera Bacillus and Alicyclobacillus . The majority of strains were found to cluster in two groups represented by B. stearothermophilus and B. pallidus. Bacillus smithii, B. thermocloacae , and B. thermoruber are phylogenetically well separated and cluster within the radiation of mesophilic bacilli. The as yet undescribed taxon 'B. flavothermus' warrants species status. B. schlegelii and B. tusciae group peripherally with members of Alicyclobacillus and may be reclassified when more phenotypic data support their phylogenetic position.     

Cloning and sequencing of 16S rDNA and 16S-23S rDNA internal spacer region (ISR) from urease-positive thermophilic Campylobacter (UPTC)

AIMS: To clone and sequence the 16S rDNA and 16S-23S rDNA internal spacer region (ISR) from urease-positive thermophilic Campylobacter (UPTC). METHODS AND RESULTS: The primer sets for 16S rDNA and 16S-23S rDNA ISR amplified almost the full length of 16S rDNA and 16S-23S rDNA ISR. About 1500 bp for 16S rDNA and about 720 bp for 16S-23S rDNA ISR of the rrn operon of four strains of UPTC were identified after molecular cloning and sequencing. CONCLUSIONS: The four strains and CCUG18267 of UPTC showed approximately 99% sequence homology of 16S rDNA to each other, 96-97% to Camp. coli, 97-98% to Camp. jejuni and 97-98% to Camp. lari. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, the nucleotide sequence of 16S-23S rDNA ISR of UPTC has been analysed. The sequence of ISR was almost identical among the four strains of UPTC. It is interesting that the UPTC intercistronic tRNAs demonstrated an order of tRNA of 5'-16S-tRNAAla-tRNAIle-23S-3' in the organisms.     

Ribotyping and rapid identification of Staphylococcus xylosus by 16-23S spacer amplification

Ninety-five strains of Staphylococcus xylosus isolated from goat milk, French sausage or mice were analyzed together with 35 Staphylococcus type strains by 16-23S spacer amplification and ribotyping. The results obtained by PCR amplification of the 16-23S spacer region permitted the distinction of each type strain and additionally generated a DNA banding pattern characteristic for 93 of the 95 Staphylococcus xylosus strains. Ribotyping proved to be an efficient epidemiological tool for Staphylococcus xylosus species as it clustered the 95 strains into 23 distinct types.     

Combining denaturing gradient gel electrophoresis of 16S rDNA V3 region and 16S-23S rDNA spacer region polymorphism analyses for the identification of staphylococci from Italian fermented sausages

Separation of amplified V3 region from 16S rDNA by denaturing gradient gel electrophoresis (PCR-DGGE) and 16S-23S rDNA intergenic spacer region polymorphism (ISR-PCR) analyses were tested as tool for differentiation of staphylococcal strains commonly isolated from fermented sausages. Variable V3 regions of 25 staphylococcal reference strains and 96 wild strains of species belonging to the genera Staphylococcus, Micrococcus and Kocuria were analyzed. PCR-DGGE profiles obtained were species-specific for S. sciuri, S. haemolyticus, S. hominis, S. auricularis, S. condimenti, S. kloosi, S. vitulus, S. succinus, S. pasteuri, S. capitis and S. (Macrococcus) caseolyticus. Moreover, 7 groups could be distinguished gathering the remaining species as result of the separation of the V3 rDNA amplicons in DGGE. Furthermore, the combination of the results obtained by PCR-DGGE and ISR-PCR analyses allowed a clear differentiation of all the staphylococcal species analysed, with exception of the pairs S. equorum-S. cohnii and S. carnosus-S. schleiferi. The suitability of both molecular techniques and of the combination their results for the identification of staphylococci was validated analysing partial nucleotide sequence of the 16S rDNA of a representative number of wild strains.