Use of species-directed 16S rRNA gene PCR primers for detection of Atopobium vaginae in patients with bacterial vaginosis.
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ABSTRACT:
SUBMITTER: Ferris MJ
PROVIDER: S-EPMC535275 | biostudies-literature | 2004 Dec
REPOSITORIES: biostudies-literature
Publications
Use of species-directed 16S rRNA gene PCR primers for detection of Atopobium vaginae in patients with bacterial vaginosis.
Ferris Michael J MJ Masztal Alicia A Martin David H DH
Journal of clinical microbiology 20041201 12
Recent studies suggest that the association between a metronidazole-resistant anaerobe, Atopobium vaginae, and bacterial vaginosis (BV) warrants further investigation. In the present study, specific primers enhanced detection of A. vaginae and provided additional evidence that this bacterium is prevalent among patients with BV but absent among patients with normal vaginal flora. ...[more]
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Project description:BackgroundThe pathogenesis of bacterial vaginosis remains largely elusive, although some microorganisms, including Gardnerella vaginalis, are suspected of playing a role in the etiology of this disorder. Recently culture-independent analysis of microbial ecosystems has proven its efficacy in characterizing the diversity of bacterial populations. Here, we report on the results obtained by combining culture and PCR-based methods to characterize the normal and disturbed vaginal microflora.ResultsA total of 150 vaginal swab samples from healthy women (115 pregnant and 35 non-pregnant) were categorized on the basis of Gram stain of direct smear as grade I (n = 112), grade II (n = 26), grade III (n = 9) or grade IV (n = 3). The composition of the vaginal microbial community of eight of these vaginal swabs (three grade I, two grade II and three grade III), all from non-pregnant women, were studied by culture and by cloning of the 16S rRNA genes obtained after direct amplification. Forty-six cultured isolates were identified by tDNA-PCR, 854 cloned 16S rRNA gene fragments were analysed of which 156 by sequencing, yielding a total of 38 species, including 9 presumptively novel species with at least five species that have not been isolated previously from vaginal samples. Interestingly, cloning revealed that Atopobium vaginae was abundant in four out of the five non-grade I specimens. Finally, species specific PCR for A. vaginae and Gardnerella vaginalis pointed to a statistically significant co-occurrence of both species in the bacterial vaginosis samples.ConclusionsAlthough historically the literature regarding bacterial vaginosis has largely focused on G. vaginalis in particular, several findings of this study--like the abundance of A. vaginae in disturbed vaginal microflora and the presence of several novel species--indicate that much is to be learned about the composition of the vaginal microflora and its relation to the etiology of BV.
| S-EPMC419343 | biostudies-literature
Project description:Bacterial vaginosis (BV) is a polymicrobial syndrome characterized by a change in vaginal flora away from predominantly Lactobacillus species. The cause of BV is unknown, but the condition has been implicated in diverse medical outcomes. The bacterium Atopobium vaginae has been recognized only recently. It is not readily identified by commercial diagnostic kits. Its clinical significance is unknown but it has recently been isolated from a tuboovarian abcess.Nucleotide sequencing of PCR amplified 16S rRNA gene segments, that were separated into bands within lanes on polyacrylamide gels by denaturing gradient gel electrophoresis (DGGE), was used to examine bacterial vaginal flora in 46 patients clinically described as having normal (Lactobacillus spp. predominant; Nugent score < or = 3) and abnormal flora (Nugent score > or = 4). These women ranged in age from 14 to 48 and 82% were African American.The DGGE banding patterns of normal and BV-positive patients were recognizably distinct. Those of normal patients contained 1 to 4 bands that were focused in the centre region of the gel lane, while those of BV positive patients contained bands that were not all focused in the center region of the gel lane. More detailed analysis of patterns revealed that bands identified as Atopobium vaginae were present in a majority (12/22) of BV positive patients, while corresponding bands were rare (2/24) in normal patients. (P < 0.001) Two A. vaginae isolates were cultivated from two patients whose DGGE analyses indicated the presence of this organism. Two A. vaginae 16S rRNA gene sequences were identified among the clinical isolates. The same two sequences were obtained from DGGE bands of the corresponding vaginal flora. The sequences differed by one nucleotide over the short (approximately 300 bp) segment used for DGGE analysis and migrated to slightly different points in denaturing gradient gels. Both isolates were strict anaerobes and highly metronidazole resistant.The results suggest that A. vaginae may be an important component of the complex bacterial ecology that constitutes abnormal vaginal flora. This organism could play a role in treatment failure if further studies confirm it is consistently metronidozole resistant.
| S-EPMC362875 | biostudies-literature
Project description:ObjectivesBacterial vaginosis (BV) is characterised by a change in the microbial composition of the vagina. The BV-associated organisms outnumber the health-associated Lactobacillus species and form a polymicrobial biofilm on the vaginal epithelium, possibly explaining the difficulties with antibiotic treatment. A better understanding of vaginal biofilm with emphasis on Atopobium vaginae and Gardnerella vaginalis may contribute to a better diagnosis and treatment of BV.MethodsTo this purpose, we evaluated the association between the presence of both bacteria by fluorescence in situ hybridisation (FISH) and BV by Nugent scoring in 463 vaginal slides of 120 participants participating in a clinical trial in Rwanda.ResultsA bacterial biofilm was detected in half of the samples using a universal bacterial probe. The biofilm contained A. vaginae in 54.1% and G. vaginalis in 82.0% of the samples. A. vaginae was accompanied by G. vaginalis in 99.5% of samples. The odds of having a Nugent score above 4 were increased for samples with dispersed G. vaginalis and/or A. vaginae present (OR 4.5; CI 2 to 10.3). The probability of having a high Nugent score was even higher when a combination of adherent G. vaginalis and dispersed A. vaginae was visualised (OR 75.6; CI 13.3 to 429.5) and highest when both bacteria were part of the biofilm (OR 119; CI 39.9 to 360.8).ConclusionsOur study, although not comprehensive at studying the polymicrobial biofilm in BV, provided a strong indication towards the importance of A. vaginae and the symbiosis of A. vaginae and G. vaginalis in this biofilm.Trial registration numberNCT01796613.
| S-EPMC5136707 | biostudies-literature
Project description:BACKGROUND: Bacterial vaginosis (BV) is a condition with diverse etiology. This condition predisposes women to increased susceptibility to sexually transmitted diseases, including human immunodeficiency virus (HIV) infections and preterm birth. The diagnostic methods currently adopted in the evaluation of patient samples for BV are arguably Amsel criteria, and Nugent score that require microscopy and expert interpretation. These two methods are still subjective. OBJECTIVE: The objective of this study was to determine the organisms present in the vagina of 34 HIV negative Nigerian women diagnosed as having bacterial vaginosis by using molecular techniques. METHODS: The vaginal samples were subjected to DNA extraction, and amplified with eubacterial primers via PCR. The PCR products were separated using denaturing gradient gel electrophoresis (DGGE). Bands were excised, re-amplified, purified and sequenced. Sequence identification was performed using the BLAST algorithm and Genbank data base. RESULTS: Mycoplasma hominis (12/34; 35%) was the most common isolate and 9 (26%) contained one of two clones of an unusual Rainbow Trout intestinal bacterium, while unculturable Streptococcus sp, and other bacteria made up the remaining isolates. CONCLUSIONS: The findings indicate further diversity in the etiological agents associated with BV, and raise the question as to whether diagnosis and management of this condition needs to be re-evaluated in countries like Nigeria. There is some controversy over the clinical importance of BV, as it was once regarded as a disease caused by Gardnerella and presenting as an odourous discharge condition, but is now diagnosed without necessarily the presence of these organisms or signs. With the incidence of BV aligned to an increased risk of HIV in a country ravaged by this virus, the effective eradication of BV can only be achieved if appropriate therapies are delivered.
| S-EPMC1925267 | biostudies-literature
Project description:BACKGROUND: Previous studies have indicated that a recently described anaerobic bacterium, Atopobium vaginae is associated with bacterial vaginosis (BV). Thus far the four isolates of this fastidious micro-organism were found to be highly resistant to metronidazole and susceptible for clindamycin, two antibiotics preferred for the treatment of BV. METHODS: Nine strains of Atopobium vaginae, four strains of Gardnerella vaginalis, two strains of Lactobacillus iners and one strain each of Bifidobacterium breve, B. longum, L. crispatus, L. gasseri and L. jensenii were tested against 15 antimicrobial agents using the Etest. RESULTS: All nine strains of A. vaginae were highly resistant to nalidixic acid and colistin while being inhibited by low concentrations of clindamycin (range: < 0.016 microg/ml), rifampicin (< 0.002 microg/ml), azithromycin (< 0.016-0.32 microg/ml), penicillin (0.008-0.25 microg/ml), ampicillin (< 0.016-0.94 microg/ml), ciprofloxacin (0.023-0.25 microg/ml) and linezolid (0.016-0.125 microg/ml). We found a variable susceptibility for metronidazole, ranging from 2 to more than 256 microg/ml. The four G. vaginalis strains were also susceptible for clindamycin (< 0.016-0.047 microg/ml) and three strains were susceptible to less than 1 microg/ml of metronidazole. All lactobacilli were resistant to metronidazole (> 256 microg/ml) but susceptible to clindamycin (0.023-0.125 microg/ml). CONCLUSION: Clindamycin has higher activity against G. vaginalis and A. vaginae than metronidazole, but not all A. vaginae isolates are metronidazole resistant, as seemed to be a straightforward conclusion from previous studies on a more limited number of strains.
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Project description:BACKGROUND:Targeted amplicon sequencing of the 16S ribosomal RNA gene is one of the key tools for studying microbial diversity. The accuracy of this approach strongly depends on the choice of primer pairs and, in particular, on the balance between efficiency, specificity and sensitivity in the amplification of the different bacterial 16S sequences contained in a sample. There is thus the need for computational methods to design optimal bacterial 16S primers able to take into account the knowledge provided by the new sequencing technologies. RESULTS:We propose here a computational method for optimizing the choice of primer sets, based on multi-objective optimization, which simultaneously: 1) maximizes efficiency and specificity of target amplification; 2) maximizes the number of different bacterial 16S sequences matched by at least one primer; 3) minimizes the differences in the number of primers matching each bacterial 16S sequence. Our algorithm can be applied to any desired amplicon length without affecting computational performance. The source code of the developed algorithm is released as the mopo16S software tool (Multi-Objective Primer Optimization for 16S experiments) under the GNU General Public License and is available at http://sysbiobig.dei.unipd.it/?q=Software#mopo16S . CONCLUSIONS:Results show that our strategy is able to find better primer pairs than the ones available in the literature according to all three optimization criteria. We also experimentally validated three of the primer pairs identified by our method on multiple bacterial species, belonging to different genera and phyla. Results confirm the predicted efficiency and the ability to maximize the number of different bacterial 16S sequences matched by primers.
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Project description:Bacterial vaginosis (BV), a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA) probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR) as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1%) and a specificity of 89.4% (95% confidence interval: 76.1% - 96%) of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis.
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Project description:Bacterial endosymbionts of aquatic invertebrates remain poorly studied. This is at least partly due to a lack of suitable techniques and primers for their identification. We designed a pair of non-degenerate primers which enabled us to amplify a fragment of ca. 500 bp of the 16S rRNA gene from various known bacterial endosymbiont species. By using this approach, we identified four bacterial endosymbionts, two endoparasites and one uncultured bacterium in seven, taxonomically diverse, freshwater crustacean hosts from temporary waters across a wide geographical area. The overall efficiency of our new WOLBSL and WOLBSR primers for amplification of the bacterial 16S rRNA gene was 100%. However, if different bacterial species from one sample were amplified simultaneously, sequences were illegible, despite a good quality of PCR products. Therefore, we suggest using our primers at the first stage of bacterial endosymbiont identification. Subsequently, genus specific primers are recommended. Overall, in the era of next-generation sequencing our method can be used as a first simple and low-cost approach to identify potential microbial symbionts associated with freshwater crustaceans using simple Sanger sequencing. The potential to detected bacterial symbionts in various invertebrate hosts in such a way will facilitate studies on host-symbiont interactions and coevolution.
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Project description:Within the paradigm of clinical infectious disease research, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa represent the four most clinically relevant, and hence most extensively studied bacteria. Current culture-based methods for identifying these organisms are slow and cumbersome, and there is increasing need for more rapid and accurate molecular detection methods. Using bioinformatic tools, 962,279 bacterial 16S rRNA gene sequences were aligned, and regions of homology were selected to generate a set of real-time PCR primers that target 93.6% of all bacterial 16S rRNA sequences published to date. A set of four species-specific real-time PCR primer pairs were also designed, capable of detecting less than 100 genome copies of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa. All primers were tested for specificity in vitro against 50 species of Gram-positive and -negative bacteria. Additionally, the species-specific primers were tested against a panel of 200 clinical isolates of each species, randomly selected from a large repository of clinical isolates from diverse areas and sources. A comparison of culture and real-time PCR demonstrated 100% concordance. The primers were incorporated into a rapid assay capable of positive identification from plate or broth cultures in less than 90 minutes. Furthermore, our data demonstrate that current targets, such as the uidA gene in E.coli, are not suitable as species-specific genes due to sequence variation. The assay described herein is rapid, cost-effective and accurate, and can be easily incorporated into any research laboratory capable of real-time PCR.
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Project description:rRNA-based studies, which have become the most common method for assessing microbial communities, rely upon faithful amplification of the corresponding genes from the original DNA sample. We report here an analysis and reevaluation of commonly used primers for amplifying the DNA between positions 27 and 1492 of bacterial 16S rRNA genes (numbered according to the Escherichia coli rRNA). We propose a formulation for a forward primer (27f) that includes three sequences not usually present. We compare our proposed formulation to two common alternatives by using linear amplification-providing an assessment that is independent of a reverse primer-and in combination with the 1492 reverse primer (1492r) under the PCR conditions appropriate for making community rRNA gene clone libraries. For analyses of DNA from human vaginal samples, our formulation was better at maintaining the original rRNA gene ratio of Lactobacillus spp. to Gardnerella spp., particularly under stringent amplification conditions. Because our 27f formulation remains relatively simple, having seven distinct primer sequences, there is minimal loss of overall amplification efficiency and specificity.
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