Identification of 16S rRNA mutations in Mycoplasma genitalium potentially associated with tetracycline resistance in vivo but not selected in vitro in M. genitalium and Chlamydia trachomatis

2021 ◽  
Author(s):  
Chloé Le Roy ◽  
Arabella Touati ◽  
Carla Balcon ◽  
Justine Garraud ◽  
Jean-Michel Molina ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Tetracycline Resistance ◽  
Mycoplasma Genitalium ◽  
Rrna Gene ◽  
Resistant Mutants ◽  
The 16S Rrna Gene

Abstract Objectives Tetracyclines are widely used for the treatment of bacterial sexually transmitted infections (STIs) and recently have been used successfully for post-exposure prophylaxis of STIs in MSM. We investigated the in vitro and in vivo development of tetracycline resistance in Chlamydia trachomatis and Mycoplasma genitalium and evaluated 16S rRNA mutations associated with acquired resistance in other bacteria. Methods In vitro selection of resistant mutants of reference strains of C. trachomatis and M. genitalium was undertaken by serial passage in medium containing subinhibitory concentrations of tetracycline or doxycycline, respectively. The 16S rRNA gene of the two microorganisms was amplified and sequenced at different passages, as were those of 43 C. trachomatis- and 106 M. genitalium-positive specimens collected in France from 2013 to 2019. Results No tetracycline- or doxycycline-resistant strains of C. trachomatis and M. genitalium, respectively, were obtained after 30 serial passages. The tetracycline and doxycycline MICs were unchanged and analysis of the 16S rRNA gene, the molecular target of tetracyclines, of C. trachomatis and M. genitalium revealed no mutation. No mutation in the 16S rRNA gene was detected in C. trachomatis-positive specimens. However, six M. genitalium-positive specimens harboured a mutation potentially associated with tetracycline resistance without known prior tetracycline treatment for patients. Conclusions Tetracyclines did not select in vitro-resistant mutants of C. trachomatis or M. genitalium. However, 16S rRNA mutations either responsible for or associated with tetracycline resistance in other bacteria, including mycoplasma species, were identified in several M. genitalium-positive specimens.

scholarly journals Mutations in the 16S rRNA Genes of Helicobacter pylori Mediate Resistance to Tetracycline

2002 ◽  
Vol 184 (8) ◽  
pp. 2131-2140 ◽  
Author(s):  
Catharine A. Trieber ◽  
Diane E. Taylor
Keyword(s):  
Helicobacter Pylori ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Tetracycline Resistance ◽  
Clinical Isolates ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Nucleotide Substitutions ◽  
The 16S Rrna Gene

ABSTRACT Low-cost and rescue treatments for Helicobacter pylori infections involve combinations of several drugs including tetracycline. Resistance to tetracycline has recently emerged in H. pylori. The 16S rRNA gene sequences of two tetracycline-resistant clinical isolates (MIC = 64 μg/ml) were determined and compared to the consensus H. pylori 16S rRNA sequence. One isolate had four nucleotide substitutions, and the other had four substitutions and two deletions. Natural transformation with the 16S rRNA genes from the resistant organisms conferred tetracycline resistance on susceptible strains. 16S rRNA genes containing the individual mutations were constructed and tested for the ability to confer resistance. Only the 16S rRNA gene containing the triple mutation, AGA965-967TTC, was able to confer tetracycline resistance on H. pylori 26695. The MICs of tetracycline for the transformed strains were equivalent to those for the original clinical isolates. The two original isolates were also metronidazole resistant, but this trait was not linked to the tetracycline resistance phenotype. Serial passage of several H. pylori strains on increasing concentrations of tetracycline yielded mutants with only a very modest increase in tetracycline resistance to a MIC of 4 to 8 μg/ml. These mutants all had a deletion of G942 in the 16S rRNA genes. The mutations in the 16S rRNA are clearly responsible for tetracycline resistance in H. pylori.

scholarly journals Handling of Spurious Sequences Affects the Outcome of High-Throughput 16S rRNA Gene Amplicon Profiling

2020 ◽  
Author(s):  
Thomas Clavel ◽  
Sandra Reitmeier ◽  
Thomas CA Hitch ◽  
Nicole Treichel ◽  
Nikolaos Fikas ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Data Processing ◽  
Relative Abundance ◽  
Large Scale ◽  
Peat Soil ◽  
Rrna Gene ◽  
Dnase Treatment

Abstract Background: 16S rRNA gene amplicon sequencing is a very popular approach for studying microbiomes. However, varying standards exist for sample and data processing and some basic concepts, such as the occurrence of spurious sequences, have not been investigated in a comprehensive manner. Methods: Using defined communities of bacteria in vitro and in vivo, we searched for sequences not matching the expected species (i.e., spurious taxa) and determined a minimum threshold of occurrence suitable for robust data analysis. The presence and origin of spurious taxa were investigated via large-scale amplicon queries and gut samples from germfree mice spiked with target mock DNA. We also assessed the effect of varying sequence-filtering stringency on diversity readouts in human fecal and peat soil communities. Our findings are based on data generated in three sequencing facilities and analyzed via both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) approaches.Results: 16S rRNA gene amplicon data-processing based on OTUs clustering and singleton removal, a commonly used approach that discards any taxa represented by only one sequence across all samples, delivered an average approximately 50% (mock communities) to 80% (gnotobiotic mice) spurious taxa. The fraction of spurious taxa was generally lower based on ASV analysis, but varied depending on the gene region targeted and the barcoding system used. A relative abundance of 0.25% was found as an effective threshold below which the analysis of spurious taxa can be prevented to a large extent in both OTU- and ASV-based analysis approaches. Most spurious taxa (approx. 70%) detected in simplified communities occurred in samples multiplexed in the same sequencing run and were present in only one of ten runs. DNase treatment of gut content from germfree mice partly helped to exclude spurious taxa from the analysis of spiked mock DNA, but was not necessary when applying the 0.25% relative abundance threshold. Using this cut-off improved the reproducibility of analysis, i.e., specifically by reducing variation in richness estimates by 38% compared with singleton filtering in a benchmarking experiment using six human fecal samples across seven sequencing runs. Beta-diversity analyses of human fecal communities was markedly affected by both the filtering strategy and the type of phylogenetic distances used for comparing samples, highlighting the importance of carefully analyzing data before drawing conclusions. Conclusions: Handling of artifact sequences during bioinformatic processing of 16S rRNA gene amplicon data requires careful attention to avoid the generation of misleading findings. Applying a minimum relative abundance threshold between 0.10 and 0.30% is superior to the singleton removal approach, although study-specific analysis strategies may be needed depending on, for instance, the type of samples analyzed and the sequencing depth achieved. Additionally, we propose the concept of effective richness to facilitate the comparison of results across studies.

scholarly journals In vitro analysis of the pea chloroplast 16S rRNA gene promoter.

1989 ◽  
Vol 9 (12) ◽  
pp. 5650-5659 ◽  
Author(s):  
E Sun ◽  
B W Wu ◽  
K K Tewari
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Topoisomerase I ◽  
Transcriptional Start Site ◽  
Gene Promoter ◽  
In Vitro Transcription ◽  
Rrna Gene ◽  
Start Site

A cloned pea chloroplast 16S rRNA gene promoter has been characterized in detail by use of a homologous in vitro transcription system that contains a highly purified chloroplast RNA polymerase. The in vivo and in vitro 16S rRNA transcriptional start site has been identified to be a T on the plus strand, 158 bases upstream of the mature 5' end of the gene. BAL 31 deletions of the 16S rRNA leader region demonstrated that the bases between -66 to +30 relative to the transcriptional start site (+1) are necessary for specific 16S transcription. Disruption of canonical TTGACA or TATAAT elements within this region caused complete transcriptional inactivation and prevented protein binding. The topological requirement for 16S transcription was examined by using a construct that synthesized a transcript from the 16S promoter and released it from a pea plastid putative terminator sequence. This minigene was relaxed in vitro with a topoisomerase I from pea chloroplast. It was shown that the 16S promoter was most active when the minigene plasmid was supercoiled.

scholarly journals Quantification of Expression of Staphylococcus epidermidis Housekeeping Genes with Taqman Quantitative PCR during In Vitro Growth and under Different Conditions

2001 ◽  
Vol 183 (24) ◽  
pp. 7094-7101 ◽  
Author(s):  
S. J. Vandecasteele ◽  
W. E. Peetermans ◽  
R. Merckx ◽  
J. Van Eldere
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Quantitative Pcr ◽  
Housekeeping Genes ◽  
Rrna Gene ◽  
In Vitro Growth ◽  
The 16S Rrna Gene

ABSTRACT The aims of the present study were (i) to develop and test a sensitive and reproducible method for the study of gene expression in staphylococci and (ii) to study the expression of five housekeeping genes which are involved in nucleic acid metabolism (gmk, guanylate kinase; the dihydrofolate reductase [DHFR] gene), glucose metabolism (tpi, triosephosphate isomerase), and protein metabolism (the 16S rRNA gene;hsp-60, heat-shock protein 60) during in vitro exponential and stationary growth. A modified method for instant mRNA isolation was combined with gene quantification via Taqman real-time quantitative PCR. The detection limit of our method was 10 copies of RNA. The average intersample variability was 16%. A 10-fold increase in the expression of the hsp-60 gene was induced by exposure to a 10°C heat shock (37 to 47°C) for 10 min. During in vitro growth, the expression of all five housekeeping genes showed rapid up-regulation after inoculation of the bacteria in brain heart infusion medum and started to decline during the mid-exponential-growth phase. Maximal gene expression was 110- to 300-fold higher than gene expression during stationary phase. This indicates that housekeeping metabolism is a very dynamic process that is extremely capable of adapting to different growth conditions. Expression of the 16S rRNA gene decreases significantly earlier than that of other housekeeping genes. This confirms earlier findings forEscherichia coli that a decline in bacterial ribosomal content (measured by 16S rRNA gene expression) precedes the decline in protein synthesis (measured by mRNA expression).

scholarly journals Development and Evaluation of a PCR-Based Assay for Detection of Haemobartonella felis in Cats and Differentiation of H. felis from Related Bacteria by Restriction Fragment Length Polymorphism Analysis

1998 ◽  
Vol 36 (2) ◽  
pp. 462-466 ◽  
Author(s):  
Joanne B. Messick ◽  
Linda M. Berent ◽  
Sandra K. Cooper
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Sequence Data ◽  
Mycoplasma Genitalium ◽  
Restriction Enzymes ◽  
Rrna Gene ◽  
Polymorphism Analysis ◽  
Doxycycline Treatment ◽  
Pcr Products ◽  
The 16S Rrna Gene

The 16S rRNA gene of Haemobartonella felis was amplified by using universal eubacterial primers and was subsequently cloned and sequenced. Based on this sequence data, we designed a set ofH. felis-specific primers. These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of H. felis from each of four experimentally infected cats at peak parasitemia. No PCR product was amplified from purified DNA ofEperythrozoon suis, Mycoplasma genitalium, andBartonella bacilliformis. Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment. The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats. By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data. In this PCR assay, the minimum detectable number of H. felis organisms was determined to be between 50 and 704. The potential usefulness of restriction enzymes DdeI andMnlI for distinguishing H. felis from closely related bacteria was examined. This is the first report of the utility of PCR-facilitated diagnosis and discrimination of H. felisinfection in cats.

scholarly journals Handling of spurious sequences affects the outcome of high-throughput 16S rRNA gene amplicon profiling

2020 ◽  
Author(s):  
Sandra Reitmeier ◽  
Thomas CA Hitch ◽  
Nikolaos Fikas ◽  
Bela Hausmann ◽  
Amanda E Ramer-Tait ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Data Processing ◽  
Relative Abundance ◽  
Large Scale ◽  
Peat Soil ◽  
Rrna Gene ◽  
The Impact

Abstract Background: 16S rRNA gene amplicon sequencing is a very popular approach for studying microbiomes. However, varying standards exist for sample and data processing and some basic concepts such as the occurrence of spurious sequences have not been investigated in a comprehensive manner, which was done in the present study. Methods: Using defined communities of bacteria in vitro and in vivo , we searched for sequences not matching the expected species ( i.e. , spurious taxa) and determine a threshold of occurrence relevant for adequate data analysis. The origin of spurious taxa was then investigated via large-scale amplicon queries. We also assessed the impact of varying sequence filtering stringency on diversity readouts in human fecal and peat soil communities. Results: 16S rRNA gene amplicon data processing based on Operational Taxonomic Units (OTUs) clustering and singleton removal, a commonly used approach that discards any taxa represented by only one sequence across all samples, delivered approx. 50% (mock communities) to 80% (gnotobiotic mice) spurious taxa on average. This spurious fraction of taxa was lower based on amplicon sequence variants (ASVs) analysis but varied depending on the gene region targeted and the barcoding system used. A relative abundance of 0.25% was identified as a threshold below which the analysis of spurious taxa can be prevented to a large extent. Most spurious taxa (approx. 70%) detected in simplified communities occurred in samples multiplexed in the same sequencing run and were present in only one of ten runs. Use of the 0.25% relative abundance threshold decreased the coefficient of variations calculated on richness in the same six human fecal samples across seven sequencing runs by 38% compared with singleton filtering. The output of beta -diversity analyses of human fecal communities was markedly affected by both the filtering strategy and the type of phylogenetic distances used for comparing samples. Importantly, major findings were confirmed by using data generated in a second sequencing facility. Conclusions: Handling of artifact sequences during bioinformatic processing of 16S rRNA gene amplicon data requires careful attention to avoid the generation of misleading findings. A threshold of relative abundance of 0.25% is more appropriate than singleton removal, although study-specific analysis strategies are mandatory. We propose the concept of effective richness, which will help comparing results across studies.

In vitro analysis of the pea chloroplast 16S rRNA gene promoter

1989 ◽  
Vol 9 (12) ◽  
pp. 5650-5659
Author(s):  
E Sun ◽  
B W Wu ◽  
K K Tewari
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Topoisomerase I ◽  
Transcriptional Start Site ◽  
Gene Promoter ◽  
In Vitro Transcription ◽  
Rrna Gene ◽  
Start Site

A cloned pea chloroplast 16S rRNA gene promoter has been characterized in detail by use of a homologous in vitro transcription system that contains a highly purified chloroplast RNA polymerase. The in vivo and in vitro 16S rRNA transcriptional start site has been identified to be a T on the plus strand, 158 bases upstream of the mature 5' end of the gene. BAL 31 deletions of the 16S rRNA leader region demonstrated that the bases between -66 to +30 relative to the transcriptional start site (+1) are necessary for specific 16S transcription. Disruption of canonical TTGACA or TATAAT elements within this region caused complete transcriptional inactivation and prevented protein binding. The topological requirement for 16S transcription was examined by using a construct that synthesized a transcript from the 16S promoter and released it from a pea plastid putative terminator sequence. This minigene was relaxed in vitro with a topoisomerase I from pea chloroplast. It was shown that the 16S promoter was most active when the minigene plasmid was supercoiled.

A spiroplasma associated with tremor disease in the Chinese mitten crab (Eriocheir sinensis)

Microbiology ◽  
2004 ◽  
Vol 150 (9) ◽  
pp. 3035-3040 ◽  
Author(s):  
Wen Wang ◽  
Bohai Wen ◽  
Gail E. Gasparich ◽  
Ningning Zhu ◽  
Liwen Rong ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Light And Electron Microscopy ◽  
Eriocheir Sinensis ◽  
Rrna Gene ◽  
Chinese Mitten Crab ◽  
Mitten Crab ◽  
The 16S Rrna Gene

An epidemic of tremor disease has been a serious problem in Chinese mitten crabs, Eriocheir sinensis, in China in recent years. The disease-causing agent was previously considered to be a rickettsia-like organism. Here, analysis of the 16S rRNA gene sequence, light and electron microscopy and cultivation in vitro were used to identify the agent. Sequence analysis of the 16S rRNA gene found it to have 98 % identity with that of Spiroplasma mirum. The agent was able to be passed through membrane filters with pores 220 nm in diameter and could be cultivated by inoculating the yolk sac of embryonated chicken eggs and M1D medium. Rotary motion and flexional movement were seen by light microscopy, and electron microscopy showed that the organism had a helical morphology and lacked a cell wall. The organism produced small colonies with a diameter of 40–50 μm after 17–25 days of incubation on solid M1D medium. The agent was found in blood cells, muscles, nerves and connective tissues of crabs inoculated with a filtrate of yolk sacs or with cultures grown in M1D medium, and it was similar in structure to those grown in eggs and cultivation broth. Disease was reproduced by experimental infection with the cultivated organisms. This study has demonstrated that the causative agent of tremor disease in the Chinese mitten crab is a member of the genus Spiroplasma. This is believed to be the first time a spiroplasma has been found in a crustacean. These findings are not only significant for studies on pathogenic spiroplasmas, but also have implications for studies of freshwater ecology.

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