16S-23S rRNA Intergenic Spacer Region Research Articles

Linking Community Profiles, Gene Expression and N-Removal in Anammox Bioreactors Treating Municipal Anaerobic Digestion Reject Water

Anaerobic ammonium oxidation (anammox) requires 60% less oxygen and no external organic carbon compared to conventional biological nitrogen removal (BNR). Nevertheless, full-scale installations of anammox are uncommon, primarily owing to the lack of well-established process monitoring and control strategies that result in stable anammox reactor performance. The overarching goal of this study was to develop and apply molecular biomarkers that link microbial community structure and activity to anammox process performance in a bioreactor fed with actual anaerobic digestion centrate from a full-scale operational wastewater treatment facility. Over long-term operation, Candidatus "Brocadia sp. 40" emerged as the dominant anammox population present in the reactor. There was good correspondence between reactor nitrogen removal performance and anammox bacterial concentrations. During the period of reactor operation, there was also a marked shift in biomass morphology from discrete cells to granular aggregates, which was paralleled by a shift also to more stable nitrogen removal and the succession and establishment of bacteria related to the Chlorobi/Bacteroidetes superfamily. Based on batch assays, hydrazine oxidoreductase (hzo) expression and concentrations of the 16S-23S rRNA intergenic spacer region (ISR) were good quantitative biomarkers of oxygen- and nitrite-mediated inhibition. When applied to a continuous anammox reactor, both molecular biomarkers show promise as monitoring tools for "predicting" reactor performance.

A panel of real-time PCR assays for specific detection of three phytoplasmas from the apple proliferation group

We report here on the development of combination of assays for fast, reliable, specific and sensitive detection and discrimination of ‘ Candidatus Phytoplasma mali’, ‘ Ca. P. prunorum’ and ‘ Ca. P. pyri’ from the 16Sr-X (apple proliferation – AP) group. These phytoplasmas are causal agents of diseases of fruit trees within the family Rosaceae, namely apple proliferation (AP), European stone fruit yellows (ESFY) and pear decline (PD). The designed panel of assays uses TaqMan ® minor groove binder probes (MGB). It comprises the same set of primers and specific probes for species-specific amplification within the 16S–23S rRNA intergenic spacer region, a set of primers and probes for amplification of the 16S ribosomal DNA region for the universal phytoplasma detection, and an additional set of primers and probe for 18S rRNA as an endogenous quality control of DNA extraction. The performance characteristics of the panel were evaluated. The advantages of new assays were shown in a comparative study with the conventional PCR, which proved their higher sensitivity combined with three-fold shorter time of testing process; and in comparison with two reported multiplex real-time PCR assays for detection of ‘ Ca. P. mali’ or ‘ Ca. P. pyri’. New panel of assays were tested on the DNA samples of ‘ Ca. P. mali’, ‘Ca. P. prunorum’, ‘ Ca. P. pyri’, other phytoplasmas and other bacteria isolated from plant material. Additionally, 198 symptomatic and asymptomatic fruit tree field samples collecting during several growing seasons were tested with new assays as well. The results of this study indicate that the combination of three specific assays may be applied in routine phytoplasma surveys and in the certification programs.

Population dynamics of Vibrio spp. associated with marine sponge microcosms

Vibrio is a diverse genus of marine-associated bacteria with at least 74 species and more expected as additional marine ecospheres are interrogated. This report describes a phylogenetic reconstruction of Vibrio isolates derived from one such unique ecosystem, marine sponges (Phylum Porifera) collected from depths of 150 to 1242 feet. 16S rRNA gene sequencing along with molecular typing of 16S-23S rRNA intergenic spacer regions clustered many sponge-associated Vibrio (spp) with current known species. That is, several benthic Vibrio species commensal with Porifera sponges seemed genetically linked to vibrios associated with coastal or shallow-water communities, signalling a panmictic population structure among seemingly ecologically disparate strains. Conversely, phylogenetic analysis provided evidence for at least two novel Vibrio speciation events within this specific sponge microcosm. Collectively, these findings earmark this still relatively unknown environment as a bastion of taxonomic and phylogenetic variability for the genus and probably other bacterial taxa.

Mycoplasma mucosicanis sp. nov., isolated from the mucosa of dogs

Fourteen Mycoplasma strains were isolated from the oral cavity and genital tract of asymptomatic dogs. Isolates had been preliminarily identified by conventional serological testing as Mycoplasma bovigenitalium, but in 16S-23S rRNA intergenic spacer PCR-RFLP assays the isolates exhibited an RFLP pattern distinct from M. bovigenitalium PG11(T). Analysis of the 16S rRNA gene placed a representative of the isolates (strain 1642(T)) in the M. bovigenitalium subcluster of the Mycoplasma bovis cluster of mycoplasmas, with the highest sequence similarities to Mycoplasma californicum ST-6(T) (96.4 %), M. bovigenitalium PG11(T) (96.3 %) and Mycoplasma phocirhinis 852(T) (96.2 %). 16S rRNA gene sequence similarities almost equidistant from three recognized species and results obtained by sequence analysis of the 16S-23S rRNA intergenic spacer region, polar lipid profiles and serological reactions indicated that this organism represents a novel species of the genus Mycoplasma for which the name Mycoplasma mucosicanis sp. nov. is proposed, with strain 1642(T) ( = ATCC BAA-1895(T) = DSM 22457(T)) as the type strain.

16S–23S rRNA Intergenic Spacer Region Variability in the Genus Frankia

16S-23S rRNA internally transcribed spacer (ITS) sequences from 53 Frankia strains were sequenced and sized from polymerase chain reaction amplification products and compiled with 14 selected 16S-23S ITS sequences from public database. Frankia genomes included two to three ITS copies lacking length polymorphism except for nine strains. No tRNA gene was encountered in this region. Frankia strains exhibited various lengths (369 to 452nt) and a wide range of sequence similarity (35-100%) in the ITS region. The average pairwise distance varied from 0.368 (clusters 1 and 2) to 0.964 (clusters 3 and 4) and was 0.397, 0.138, 0.129, and 0.016, respectively, for cluster 4 (saprophytic non-infective/non-effective), clusters 1 and 3 (facultative symbiotic), and cluster 2 (obligate symbiotic). This suggests a gradual erosion of Frankia diversity concomitantly with a shift from saprophytic non-infective/non-effective to facultative and symbiotic lifestyle. Comparative sequence analyses of the 16S-23S rRNA intergenic spacer region of Frankia strains are not useful to assign them to their respective cluster or host infection group. Accurate assignment required the inclusion of the adjacent 16S and 23S rRNA gene fragments.

The use of bacteria for the identification of vaginal secretions

We have used the 16S–23S rRNA intergenic spacer region for identifying vaginal specific bacteria. Lactobacillus crispatus and Lactobacillus gasseri were detected in vaginal secretions but not in semen, blood or saliva. Our data indicated that both L. crispatus and L. gasseri were detected in vaginal secretions from women with different levels of expression of hormonal genes including pregnant, pre- and post-menopausal women, and a woman who has had a hysterectomy. Therefore, we have demonstrated that these Lactobacilli are promising new markers for the forensic identification of vaginal secretions. We have incorporated the Lactobacilli markers into a mRNA multiplex system to produce an 11-plex assay that can identify circulatory blood, menstrual blood, saliva, semen (in the presence and absence of spermatozoa) and vaginal secretions.

Molecular Detection of Bartonella alsatica in European Wild Rabbits (Oryctolagus cuniculus) in Andalusia (Spain)

A sample of 279 European wild rabbits, Oryctolagus cuniculus (141 males, 138 females), captured alive in Andalusia (Spain) and belonging to the two haplotype classes previously described for this species (230 and 49 corresponding with haplotypes A and B, respectively), were tested for the presence of Bartonella alsatica DNA. Two species-specific nested polymerase chain reaction assays targeting for 16S-23S rRNA intergenic spacer region and RNA polymerase β subunit genes have been developed. Forty-eight (17.20%) rabbits were infected with B. alsatica. Two-way contingency table analyses and the calculation of Cramer's V statistic showed no differences in infection rate, considering haplotype lineage or sex. The risk of infection of human population, especially for hunters in close contact with this demonstrated human pathogen, should be considered.

PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species

BackgroundThe genus Vibrio is a diverse group of Gram-negative bacteria comprised of 74 species. Furthermore, the genus has and is expected to continue expanding with the addition of several new species annually. Consequently, it is of paramount importance to have a method which is able to reliably and efficiently differentiate the numerous Vibrio species.ResultsIn this study, a novel and rapid polymerase chain reaction (PCR)-based intergenic spacer (IGS)-typing system for vibrios was developed that is based on the well-known IGS regions located between the 16S and 23S rRNA genes on the bacterial chromosome. The system was optimized to resolve heteroduplex formation as well as to take advantage of capillary gel electrophoresis technology such that reproducible analyses could be achieved in a rapid manner. System validation was achieved through testing of 69 archetypal Vibrio strains, representing 48 Vibrio species, from which an 'IGS-type' profile database was generated. These data, presented here in several cluster analyses, demonstrated successful differentiation of the 69 type strains showing that this PCR-based fingerprinting method easily discriminates bacterial strains at the species level among Vibrio. Furthermore, testing 36 strains each of V. parahaemolyticus and V. vulnificus, important food borne pathogens, isolated from a variety of geographical locations with the IGS-typing method demonstrated distinct IGS-typing patterns indicative of subspecies divergence in both populations making this technique equally useful for intraspecies differentiation, as well.ConclusionThis rapid, reliable and efficient IGS-typing system, especially in combination with 16S rRNA gene sequencing, has the capacity to not only discern and identify vibrios at the species level but, in some cases, at the sub-species level, as well. This procedure is particularly well-suited for preliminary species identification and, lends itself nicely to epidemiological investigations providing information more quickly than other time-honoured methods traditionally used in these types of analyses.

Detection of Bartonella spp. in neotropical felids and evaluation of risk factors and hematological abnormalities associated with infection

Although antibodies to Bartonella henselae have been described in all neotropical felid species, DNA has been detected in only one species, Leopardus wiedii. The aim of this study was to determine whether DNA of Bartonella spp. could be detected in blood of other captive neotropical felids and evaluate risk factors and hematological findings associated with infection. Blood samples were collected from 57 small felids, including 1 Leopardus geoffroyi, 17 L. wiedii, 22 Leopardus tigrinus, 14 Leopardus pardalis, and 3 Puma yagouaroundi; 10 blood samples from Panthera onca were retrieved from blood banks. Complete blood counts were performed on blood samples from small felids, while all samples were evaluated by PCR. DNA extraction was confirmed by amplification of the cat GAPDH gene. Bartonella spp. were assessed by amplifying a fragment of their 16S-23S rRNA intergenic spacer region; PCR products were purified and sequenced. For the small neotropical felids, risk factors [origin (wild-caught or zoo-born), gender, felid species, and flea exposure] were evaluated using exact multiple logistic regression. Hematological findings (anemia, polycythemia/hyperproteinemia, leukocytosis and leukopenia) were tested for association with infection using Fisher's exact test. The 635bp product amplified from 10 samples (10/67=14.92%) was identified as B. henselae by sequencing. Small neotropical felid males were more likely to be positive than females (95% CI=0.00–0.451, p=0.0028), however other analyzed variables were not considered risk factors (p>0.05). Hematological abnormalities were not associated with infection (p>0.05). This is the first report documenting B. henselae detection by PCR in several species of neotropical felids.

First Report of 'Candidatus Phytoplasma australiense' in Potato.

In January of 2009, potato plants (Solanum tuberosum) from a commercial crop in the Waikato Region, New Zealand were observed to have symptoms of upward rolling and purpling of the leaves. The symptoms appeared similar to those of "zebra chip", a disorder of potato recently found to be associated with 'Candidatus Liberibacter solanacearum' in New Zealand and the United States (4). Total DNA from the leaf midveins and tubers from one of the symptomatic plants was separately extracted with an InviMag Plant DNA Mini Kit (Invitek GmbH, Berlin, Germany) and a KingFisher mL workstation (Thermo Scientific, Waltham, MA). DNA extracted from leaf midveins and tubers tested negative for 'Ca. L. solanacearum' by nested-PCR using primer pair OA2/OI2c (4) followed by Lib16S01F/Lib16S01R (5'-TTCTACGGGATAACGCACGG-3' and 5'-CGTCAGTATCAGGCCAGTGAG-3'), which amplifies a 580-bp region of the 16S rRNA gene. However, DNA extracted from the tuber tissue tested positive for phytoplasma by TaqMan real-time PCR (3). No phytoplasma was detected in the DNA extracted from leaf tissue. The 16S rRNA gene, 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene of the phytoplasma were amplified with primers P1/P7 (1). The PCR product was cloned into the pCR 4-TOPO vector (Invitrogen, Carlsbad, CA) and sequenced (GenBank Accession No. FJ943262). BLAST analysis showed 100% identity to 'Ca. Phytoplasma australiense' (16SrXII, Stolbur group). A fragment of approximately 850-bp of the Tuf gene was also amplified (2) and sequenced directly (GenBank Accession No. FJ943263). BLAST analysis showed 100% identity to Tuf gene variant IX of 'Ca. P. australiense' (2). An additional 14 plants showing similar leaf symptoms and also production of aerial tubers were collected from seven different potato fields from the Auckland and Waikato regions. Total DNA from the leaf midveins, stem, and tubers were separately extracted from each of the plants. The samples were tested for phytoplasma by nested-PCR using primer pair R16F2/R16R2, followed by NGF/NGR (1), and tested for 'Ca. L. solanacearum' by nested-PCR as described above. Seven plants tested positive only for phytoplasma, three tested positive for only 'Ca. L. solanacearum', and four plants tested positive for both pathogens. The pathogens were most commonly detected in samples extracted from the stem with 9 and 5 of the 14 samples testing positive for phytoplasma and liberibacter, respectively. Six of each of the leaf and tuber samples tested positive for phytoplasma. Liberibacter was detected in one of the leaf samples and in four of the tuber samples. 'Ca. P. australiense' has only been reported from New Zealand and Australia. The only other known hosts of 'Ca. P. australiense' in New Zealand are strawberry and native plants belonging to the genera Cordyline, Coprosma, and Phormium (2). In Australia, 'Ca. P. australiense' is associated with Australian grapevine yellows and Papaya dieback (2). To our knowledge, this is the first report of 'Ca. P. australiense' infecting potato as well as the first report of phytoplasma and 'Ca. L. solanacearum' mixed infections in potato.

Fourteen Years of Severe Arthralgia in a Man without Gastrointestinal Symptoms: Atypical Whipple's Disease

We report here the case of Whipple's disease in a 60-year-old man with severe arthralgia and systemic disorders but without gastrointestinal manifestations. The patient had different clinical diagnoses over a period of 14 years. We identified Tropheryma whipplei by real-time PCR. Molecular typing was also performed by sequencing the 16S-23S rRNA intergenic spacer region and domain III of the 23S rRNA gene.

Isolation of Bartonella species from rodents in Taiwan including a strain closely related to ‘Bartonella rochalimae’ from Rattus norvegicus

An increasing number of Bartonella species originally isolated from small mammals have been identified as emerging human pathogens. During an investigation of Bartonella infection in rodent populations carried out in Taiwan in 2006, a total of 58 rodents were tested. It was determined that 10.3 % (6/58) of the animals were Bartonella bacteraemic. After PCR/RFLP analysis, four isolates were identified as Bartonella elizabethae and one isolate as Bartonella tribocorum. However, there was one specific isolate with an unrecognized PCR/RFLP pattern. After further sequence and phylogenetic analyses of the gltA, ftsZ and rpoB genes, and the 16S-23S rRNA intergenic spacer region, the results indicated that this specific isolate from Rattus norvegicus was closely related to human pathogenic 'Bartonella rochalimae'. Further studies need to be conducted to evaluate whether this rodent species could be a reservoir for 'B. rochalimae'.

Phenotypic and Genotypic Characterization of Arcanobacterium haemolyticum Isolates from Infections of Horses

The present study was designed to characterize phenotypically and genotypically seven Arcanobacterium haemolyticum strains obtained from infections of six horses. All seven strains showed the cultural and biochemical properties typical of A. haemolyticum and were susceptible to most of the antibiotics tested. The species identification could be confirmed by amplification and sequencing of the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region and by PCR amplification of species-specific parts of the gene encoding phospholipase D in A. haemolyticum. Use of the latter could possibly improve future identification of this generally human pathogenic bacterial species which, according to the present results, seems to occur also in infections of horses.

Molecular approaches for identification and characterization of lactic acid bacteria

The last few years have produced a revolution in the development of very sensitive, rapid, automated, molecular detection methods for a variety of various species of lactic acid bacteria (LAB) associated with food and dairy products. Nowadays many such strains of LAB are considered probiotics. The genome-based methods are useful in identifying bacteria as a complementary or alternative tool to phenotypical methods. Over the years, identification methodologies using primers that target different sequences, such as the 16S ribosomal RNA (rRNA)-encoding gene, the 16S-23S rRNA intergenic spacer region, the 23S rRNA-encoding, recA and ldhD genes; randomly amplified polymorphic DNA, restriction fragment length polymorphism, denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, amplification rDNA restriction analysis, restriction enzyme analysis, rRNA, pulse field gel electrophoresis and amplification fragment length polymorphism have played a significant role in probiotic bacteriology. Hence, the aim of this review is to provide an overview of some rapid and reliable polymerase chain reaction-based molecular methods used for identifying and differentiating closely related species and strains of LAB associated with food and industry.

Genetic diversity of root-nodulating bacteria isolated from pea (Pisum sativum) in subtropical regions of China.

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S-23S rRNA intergenic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S-23S IGS RFLP patterns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. leguminosarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.

Phytoplasma phylogenetics based on analysis of secA and 23S rRNA gene sequences for improved resolution of candidate species of 'Candidatus Phytoplasma'

Phytoplasma phylogenetics has focused primarily on sequences of the non-coding 16S rRNA gene and the 16S-23S rRNA intergenic spacer region (16-23S ISR), and primers that enable amplification of these regions from all phytoplasmas by PCR are well established. In this study, primers based on the secA gene have been developed into a semi-nested PCR assay that results in a sequence of the expected size (about 480 bp) from all 34 phytoplasmas examined, including strains representative of 12 16Sr groups. Phylogenetic analysis of secA gene sequences showed similar clustering of phytoplasmas when compared with clusters resolved by similar sequence analyses of a 16-23S ISR-23S rRNA gene contig or of the 16S rRNA gene alone. The main differences between trees were in the branch lengths, which were elongated in the 16-23S ISR-23S rRNA gene tree when compared with the 16S rRNA gene tree and elongated still further in the secA gene tree, despite this being a shorter sequence. The improved resolution in the secA gene-derived phylogenetic tree resulted in the 16SrII group splitting into two distinct clusters, while phytoplasmas associated with coconut lethal yellowing-type diseases split into three distinct groups, thereby supporting past proposals that they represent different candidate species within 'Candidatus Phytoplasma'. The ability to differentiate 16Sr groups and subgroups by virtual RFLP analysis of secA gene sequences suggests that this gene may provide an informative alternative molecular marker for pathogen identification and diagnosis of phytoplasma diseases.

Sensitive and rapid detection of freshwater crustacean Spiroplasmas by ISRs-sequence-targeted species-specific primers

For the rapid and sensitive identification of the Spiroplasmas in the Chinese mitten crab Eriocheir sinensis, a species-specific PCR primer-pair was designed according to Spiroplasma sp. strain CRAB-specific segments of the previously sequenced 16S–23S rRNA intergenic spacer region (ISR). The PCR-mediated assay allowed identification of Spiroplasmas from different aquatic crustacean hosts, E. sinensis and red swamp crayfish Procambarus clarkia, in different geographical areas in China. No cross-reaction could be observed with other three available terrestrial plants/insects Spiroplasma strains (Spiroplasma sp. CH-1, Spiroplasma sp. CR-1 and Spiroplasma sp. M10). The sensitivity of detection of the Spiroplasma sp. strain CRAB could get as little as 100 fg of DNA template. The presented PCR method is useful for sensitive and specific detection of Spiroplasma from freshwater cultured crustaceans in the trade of aquatic products.

Molecular identification of Arcanobacterium bialowiezense and Arcanobacterium bonasi based on 16S–23S rRNA intergenic spacer region sequences

In the present study, the 16S–23S rDNA intergenic spacer region (ISR) of Arcanobacterium ( A.) bialowiezense DSM 17162, A. bonasi DSM 17163, A. bernardiae DSM 9152, A. haemolyticum DSM 20595, A. hippocoleae DSM 15539, A. phocae DSM 10002, A. pluranimalium DSM 13483 and A. pyogenes DSM 20630 was amplified, sequenced and compared with the corresponding 16S rRNA gene sequences yielding comparable phylogenetic relationships. The ISR sequence of A. bialowiezense and A. bonasi allowed the design of species-specific oligonucleotide primers which could successfully be used for PCR-mediated identification of previously characterized A. bialowiezense and A. bonasi isolated from infections of the European bison. The presented molecular identification might help to improve a future diagnosis of both newly described bacterial pathogens.

Impact of Environmental Stress-Tolerant Transgenic Potato on Genotypic Diversity of Microbial Communities and Soil Enzyme Activities under Stress Conditions

Transgenic crops able to tolerate environmental stress are being developed throughout the world. However, little data is available on the impact of environmental stress-tolerant transgenic crops on soil microorganisms and biochemistry. Recently developed transgenic potato plants carrying an environmental stress-related gene, DREB1A, with a stress-inducible promoter, are being evaluated for growth performance in greenhouses. In this study, we investigated microbial diversity and soil function to assess potential environmental risks of these transgenic potato lines. Genotypic diversity of the 16S-23S rRNA intergenic spacer region and activity levels of four enzymes were used as indicators of microbial genetic diversity and soil function, respectively. Salinity had a major effect on both bacterial (88-93%) and fungal (54-55%) diversity, while the transgene had a relatively small effect on genotypic structure (0-5%) based on the analysis of variance. However, a few genotypes appeared only in soils planted with the transgenic lines. Some enzyme activities were found to differ significantly between the transgenic and non-transgenic lines, although the results were not repeatable in the second trial. These results suggest that abiotic growth environments had a stronger impact on soil microorganisms and biochemistry than did plant genotypes.

Molecular evidence ofBartonella DNA in ixodid ticks in Czechia

We report the development of a novel method for detection of Bartonella DNA in ixodid ticks. The assay is based on a specific amplification of a part of 16S rRNA gene and 16S-23S rRNA intergenic spacer region of Bartonella sp. by nested PCR and Southern blot hybridization with specific DNA probe; the method is highly sensitive and specific. The screening of 327 unfed ticks collected in different urban and suburban areas of Czechia in 2003-2005 revealed the presence of Bartonella DNA in four Ixodes ricinus individuals (1.2%), two males, one female and one nymph.