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.     

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.     

Identification of waterborne bacteria by the analysis of 16S-23S rRNA intergenic spacer region

AIM: In this study, we evaluated, the use of universal primers, specific for the 16S-23S rRNA intergenic region, to detect and identify nine species that are of high interest for the microbiological control of water. METHODS AND RESULTS: The analysis of the fragments was carried out using a High Resolution acrylamide/bisacrylamide gels in a fluorescent automated DNA sequencer. The results showed specific profiles for each of the nine species but this technique failed to detect simultaneously micro-organisms in samples containing a mixed population. CONCLUSION: Nevertheless, the electrophoretic profiles obtained provided a very useful tool for the rapid and specific identification of water isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: A possible new methodology for a rapid identification of pathogens in water.     

Presence of a novel 16S-23S rRNA gene intergenic spacer insert in Bradyrhizobium canariense strains

Seven slow-growing bacterial strains isolated from root nodules of yellow serradella (Ornithopus compressus) that originated from Asinara Island on North Western Sardinia in Italy were characterized by partial 16S rRNA gene and intergenic spacer (ITS) sequencing as well as amplified fragment length polymorphism (AFLP) genomic fingerprinting. The results indicated that the O. compressus isolates belong to the Bradyrhizobium canariense species. The analysis of ITS sequences divided the branch of B. canariense strains into two statistically separated groups (ITS clusters I and II). All the strains in ITS cluster I showed the presence of unique oligonucleotide insert TTAGAGACTTAGGTTTCTK. This insert was neither found in other described species of the family Rhizobiaceae nor in any other bacterial families and can be used as a natural and high selective genetic marker for ITS cluster I of B. canariense strains. ITS grouping of O. compressus isolates was supported by the unweighted pair group method with arithmetic averages cluster analysis of their AFLP patterns, suggesting that the strains of ITS cluster II were genetically closer to each other than to isolates from the ITS cluster I. A taxonomic importance is supposed of the revealed 19 bp ITS insert for an intraspecific division within high heterogeneous B. canariense species.     

Strain-specific differentiation of environmental Escherichia coli isolates via denaturing gradient gel electrophoresis (DGGE) analysis of the 16S-23S intergenic spacer region

Denaturing gradient gel electrophoresis (DGGE) was applied to the 16S-23S rRNA intergenic spacer region (ISR) as a means to evaluate strain level differences in Escherichia coli. The ISRs of 81 environmental E. coli isolates obtained from bovine, poultry, and human sources yielded a total of 41 unique DGGE banding patterns, with identical patterns and common bands within each source and no overlapping patterns among sources. An additional 51 isolates from two nearby streams yielded 45 unique banding patterns with no overlap between sites. However, two of the isolates from the streams had identical banding patterns to those from two of the source isolates, resulting in a total of 84 unique DGGE banding patterns out of 132 isolates identified in this study. These results revealed high diversity among environmental E. coli isolates, which made it difficult to unambiguously ascribe strains found in water samples to specific host organisms.     

Analysis of DNA encoding 23S rRNA and 16S–23S rRNA intergenic spacer regions from Plesiomonas shigelloides

Amplification of the gene encoding 23S rRNA of Plesiomonas shigelloides by polymerase chain reaction (PCR), with primers complementary to conserved regions of 16S and the 3' end of 23S rRNA genes, resulted in a DNA fragment of approximately 3 kb. This fragment was cloned in Escherichia coli and its nucleotide sequence determined. The region encoding 23S rRNA shows high homology with the published sequences of 23S rRNA from other members of the gamma division of Proteobacteria. The sequence of the intergenic spacer region, between the 16S and 23S rRNA genes, was determined in a further two clones. In one the sequence of a single tRNA(Glu) was found which was absent from the other two. This variation in sequence suggests that the different clones may be derived from different ribosomal RNA operons.     

Use of 16S-23S rRNA intergenic spacer region PCR and repetitive extragenic palindromic PCR analyses of Escherichia coli isolates to identify nonpoint fecal sources

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 degrees 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.     

Source tracking of Escherichia coli by 16S-23S intergenic spacer region denaturing gradient gel electrophoresis (DGGE) of the rrnB ribosomal operon

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.     

Analysis of 16S-23S intergenic spacer regions of the rRNA operons in Edwardsiella ictaluri and Edwardsiella tarda isolates from fish

AIMS: To analyse interspecies and intraspecies differences based on the 16S-23S rRNA intergenic spacer region (ISR) sequences of the fish pathogens Edwardsiella ictaluri and Edwardsiella tarda. METHODS AND RESULTS: The 16S-23S rRNA spacer regions of 19 Edw. ictaluri and four Edw. tarda isolates from four geographical regions were amplified by PCR with primers complementary to conserved sequences within the flanking 16S-23S rRNA coding sequences. Two products were generated from all isolates, without interspecies or intraspecific size polymorphisms. Sequence analysis of the amplified fragments revealed a smaller ISR of 350 bp, which contained a gene for tRNA(Glu), and a larger ISR of 441 bp, which contained genes for tRNA(Ile) and tRNA(Ala). The sequences of the smaller ISR of different Edw. ictaluri isolates were essentially identical to each other. Partial sequences of larger ISR from several Edw. ictaluri isolates also revealed no differences from the one complete Edw. ictaluri large ISR sequence obtained. The sequences of the smaller ISR of Edw. tarda were 97% identical to the Edw. ictaluri smaller ISR and the larger ISR were 96-98% identical to the Edw. ictaluri larger ISR sequence. The Edw. tarda isolates displayed limited ISR sequence heterogeneity, with > or =97% sequence identity among isolates for both small and large ISR. CONCLUSIONS: There is a high degree of size and sequence similarity of 16S-23S ISR both among isolates within Edw. ictaluri and Edw. tarda species and between the two species. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results confirm a close genetic relationship between Edw. ictaluri and Edw. tarda and the relative homogeneity of Edw. ictaluri isolates compared with Edw. tarda isolates. Because no differences were found in ISR sequences among Edw. ictaluri isolates, sequence analysis of the ISR will not be useful to distinguish isolates of Edw. ictaluri. However, we identified restriction sites that differ between ISR sequences of Edw. ictaluri and Edw. tarda, which will be useful in distinguishing the two species.     

Differentiation of group VIII Spiroplasma strains with sequences of the 16S-23S rDNA intergenic spacer region

Spiroplasma species (Mollicutes: Spiroplasmataceae) are associated with a wide variety of insects, and serology has classified this genus into 34 groups, 3 with subgroups. The 16S rRNA gene has been used for phylogenetic analysis of spiroplasmas, but this approach is uninformative for group VIII because the serologically distinct subgroups generally have similarity coefficients >0.990. Therefore, we investigated the utility of the 16S-23S rRNA spacer region as a means to differentiate closely related subgroups or strains. We generated intergenic sequences and detailed serological profiles for 8 group VIII Spiroplasma strains. Sequence analyses using Maximum Parsimony, Neighbor Joining, and Maximum Likelihood placed the strains into 2 clades. One clade consisted of strains BARC 2649 and GSU5367. The other clade was divided into clusters containing representatives of the 3 designated group VIII subgroups (EA-1, DF-1, and TAAS-1) and 3 previously unclassified strains. The stability of the positions of the strains in various analytical models and the ability to provide robust support for groupings tentatively supported by serology indicates that the 16S-23S intergenic rDNA sequence will prove useful in intragroup analysis of group VIII spiroplasmas.     

Polymorphism in the dgt-dapD-tsf region of Escherichia coli K-12 strains.

Restriction analysis of the dapD region cloned from several strains of Escherichia coli, revealed a restriction-fragment length polymorphism (RFLP). This RFLP, which includes the BamHI, EcoRI and SalI sites, may be useful in classification of various E. coli strains.     

Characterization of the 23S and 5S rRNA genes and 23S-5S intergenic spacer region (ITS-2) of Photobacterium damselae

The 23S ribosomal RNA (rRNA) gene has been sequenced in strains of the fish pathogens Photobacterium damselae subsp. damselae (ATCC 33539) and subsp. piscicida (ATCC 29690), showing that 3 nucleotide positions are clearly different between subspecies. In addition, the 5S rRNA gene plus the intergenic spacer region between the 23S and 5S rRNA genes (ITS-2) were amplified, cloned and sequenced for the 2 reference strains as well as the field isolates RG91 (subsp. damselae) and DI21 (subsp. piscicida). A 100% similarity was found for the consensus 5S rRNA gene sequence in the 2 subspecies, although some microheterogeneity was detected as inter-cistronic variability within the same chromosome. Sequence analysis of the spacer region between the 23S and 5S rRNA genes revealed 2 conserved and 3 variable nucleotide sequence blocks, and 4 different modular organizations were found. The ITS-2 spacer region exhibited both inter-subspecies and intercistronic polymorphism, with a mosaic-like structure. The EMBL accession numbers for the 23S, 5S and ITS-2 sequences are: P. damselae subsp. piscicida 5S gene (AJ274379), P. damselae subsp. damselae 23S gene (Y18520), subsp. piscicida 23S gene (Y17901), P. damselae subsp. piscicida ITS-2 (AJ250695, AJ250696), P. damselae subsp. damselae ITS-2 (AJ250697, AJ250698).     

The mosaic nature of intergenic 16S-23S rRNA spacer regions suggests rRNA operon copy number variation in Clostridium difficile strains

Clostridium difficile is a major spore-forming environmental pathogen that causes serious health problems in patients undergoing antibiotic therapy. Consequently, reliable and sensitive methods for typing individual strains are required for epidemiological and environmental studies. Ribotyping is generally considered the best method, but it fails to account for sequence diversity which might exist in intergenic 16S-23S rRNA spacer regions (ISRs) within and among strains of this organism. Therefore, this study was undertaken to compare the sequence of each individual ISR in five strains of C. difficile to explore the extent of this diversity and see whether such information might provide the basis for more sensitive and discriminatory strain typing methods. After targeted PCR amplification, cloning, and sequencing, the diversity of the ISRs was used as a measure of rRNA operon copy number. In C. difficile strains 630, ATCC 43593, A, and B, 11, 11, 7, and 8 ISR length variants, respectively, were found (containing different combinations of sequence groups [i to xiii]), suggesting 11, 11, 7, and 8 rrn copies in the respective strains. Many ISRs of the same length differed markedly in their sequences, and some of these were restricted in occurrence to a single strain. Most of these ISRs did not contain any tRNA genes, and only single copies of the tRNA(Ala) gene were found in those that did. The presence of ISR sequence groups (i to xiii) varied between strains, with some found in one, two, three, four, or all five strains. We conclude that the intergenic 16S-23S rRNA spacer regions showed a high degree of diversity, not only among the rrn operons in different strains and different rrn copies in a single strain but also among ISRs of the same length. It appears that C. difficile ISRs vary more at the inter- and intragenic levels than those of other species as determined by empirical comparison of sequences. The precise characterization of these sequences has demonstrated a high level of mosaic sequence block rearrangements that are present or absent in multiple strain-variable rrn copies within and between five different strains of C. difficile.     

Variation in the 16S-23S rRNA intergenic spacer regions in Vibrio parahaemolyticus strains are due to indels nearby their tRNAGlu

Vibrio parahaemolyticus contains 11 rRNA operons each including one of six 16S-23S rRNA gene intergenic spacer classes differing in size and nucleotide sequence. Some of the spacer classes may differ between isolates. We observed that the differences in the spacers between isolates are generally in two spacer classes present in single copies in the genome, one class containing tRNA(Ala) and tRNA(Glu) and the other tRNA(Glu) exclusively. Moreover, these differences are due to indels located nearby their tRNA(Glu). Comparison of the nucleotide sequence between spacer classes suggests that intragenomic nonreciprocal recombination causes the size variations observed in the spacer regions of V. parahaemolyticus strains.     

DNA sequence of the 16S rRNA/23S rRNA intercistronic spacer of two rDNA operons of the archaebacterium Methanococcus vannielii

The DNA sequence of the spacer (plus flanking) regions separating the 16S rRNA and 23S rRNA genes of two presumptive rDNA operons of the archaebacterium Methanococcus vannielii was determined. The spacers are 156 and 242 base pairs in size and they share a sequence homology of 49 base pairs following the 3' terminus of the 16S rRNA gene and of about 60 base pairs preceding the 5' end of the 23S rRNA gene. The 242 base pair spacer, in addition contains a sequence which can be transcribed into tRNAAla, whereas no tRNA-like secondary structure can be delineated from the 156 base pair spacer region. Almost complete sequence homology was detected between the end of the 16S rRNA gene and the 3' termini of either Escherichia coli or Halobacterium halobium 16S rRNA, whereas the putative 5' terminal 23S rRNA sequence shared partial homology with E. coli 23S rRNA and eukaryotic 5.8S rRNA.     

Typing of Staphylococcus aureus by amplification of the 16S–23S rRNA intergenic spacer sequences

Abstract An acid phosphatase containing a 27-kDa polypeptide component has been identified in Escherichia coli by means of a zymogram technique. The enzyme is secreted in the periplasmic space and is able to hydrolyze several organic phosphate esters, but not diesters, showing preferential activity on p -nitrophenyl phosphate and other phenolic phosphate esters. Production of the enzyme apparently occurs only in cells growing on carbon sources other than glucose.     

Analysis of 16S-23S rRNA intergenic spacer regions of Vibrio cholerae and Vibrio mimicus

Vibrio cholerae identification based on molecular sequence data has been hampered by a lack of sequence variation from the closely related Vibrio mimicus. The two species share many genes coding for proteins, such as ctxAB, and show almost identical 16S DNA coding for rRNA (rDNA) sequences. Primers targeting conserved sequences flanking the 3' end of the 16S and the 5' end of the 23S rDNAs were used to amplify the 16S-23S rRNA intergenic spacer regions of V. cholerae and V. mimicus. Two major (ca. 580 and 500 bp) and one minor (ca. 750 bp) amplicons were consistently generated for both species, and their sequences were determined. The largest fragment contains three tRNA genes (tDNAs) coding for tRNAGlu, tRNALys, and tRNAVal, which has not previously been found in bacteria examined to date. The 580-bp amplicon contained tDNAIle and tDNAAla, whereas the 500-bp fragment had single tDNA coding either tRNAGlu or tRNAAla. Little variation, i.e., 0 to 0.4%, was found among V. cholerae O1 classical, O1 El Tor, and O139 epidemic strains. Slightly more variation was found against the non-O1/non-O139 serotypes (ca. 1% difference) and V. mimicus (2 to 3% difference). A pair of oligonucleotide primers were designed, based on the region differentiating all of V. cholerae strains from V. mimicus. The PCR system developed was subsequently evaluated by using representatives of V. cholerae from environmental and clinical sources, and of other taxa, including V. mimicus. This study provides the first molecular tool for identifying the species V. cholerae.     

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.     

Heteroduplex structures in 16S-23S rRNA intergenic transcribed spacer PCR products reveal ribosomal interoperonic polymorphisms within single Frankia strains

AIMS: Detection of polymorphisms in intergenic transcribed spacer (ITS) 16S-23S rRNA within single Frankia strains. METHODS AND RESULTS: Polymorphisms in the 16S-23S rRNA ITS were investigated in single-colony subcultures of seven Frankia isolates. Multiple ITS-polymerase chain reaction (PCR) bands were detected solely in isolates BMG5.5 and BMG5.11. The slow-migrating bands in the ITS-PCR agarose gel electrophoresis profiles of the isolates were revealed to be heteroduplexes on the basis of their migration shift in different electrophoretic matrices, southern hybridization and the single-strand DNA mung bean endonuclease digestion. Laser-scanned capillary electrophoresis detected two ITS-PCR fragments differing in length by three and six nucleotide insertions/deletions in strains BMG5.5 and BMG5.11, respectively. Sequence analysis of the cloned ITS showed that in strain BMG5.5 the two ITS differed by the presence of three to four copies of the 3-bp tandem repeat 5'-TGG-3'. In strain BMG5.11, the two ITS differed by the presence of two to three copies of the 6-bp tandem repeat 5'-CTTGGG-3'. CONCLUSIONS: We demonstrate the occurrence of ITS 16S-23S rRNa polymorphisms within single Frankia strains. SIGNIFICANCE AND IMPACT OF THE STUDY: We reported the occurrence of ITS 16S-23S rRNA polymorphisms within single Frankia strains from Elaeagnus host group recognized as the more flexible strains within Frankia genus. Furthermore, we underscored the applied interest of strains BMG5.11 and BMG5.5 in future ecological studies using ITS 16S-23S rRNA as molecular marker.