scholarly journals Detection of Nocardia by 16S Ribosomal RNA Gene PCR and Metagenomic Next-Generation Sequencing (mNGS)

Author(s):  
Juanjuan Ding ◽  
Bing Ma ◽  
Xupeng Wei ◽  
Ying Li

In this study, the aim was to investigate the discriminatory power of molecular diagnostics based on mNGS and traditional 16S ribosomal RNA PCR among Nocardia species. A total of fourteen clinical isolates from patients with positive Nocardia cultures and clinical evidence were included between January 2017 and June 2020 in HeNan Provincial People’s Hospital. DNA extraction and 16S rRNA PCR were performed on positive cultures, and pathogens were detected by mNGS in these same samples directly. Among the 14 Nocardia isolates, four species were identified, and N. cyriacigeorgica (8 cases) is the most common species. Twelve of the 14 Nocardia spp. isolates were identified by the two methods, while two strains of N. cyriacigeorgica were not identified by mNGS. All tested isolates showed susceptibility to trimethoprim-sulfamethoxazole (SXT), amikacin and linezolid. Apart from Nocardia species, other pathogens such as Acinetobacter baumannii, Klebsiella pneumonia, Aspergillus, Enterococcus faecalis, Human herpesvirus, etc., were detected from the same clinical samples by mNGS. However, these different pathogens were considered as colonization or contamination. We found that it is essential to accurately identify species for determining antibiotic sensitivity and, consequently, choosing antibiotic treatment. 16S rRNA PCR was useful for identification of nocardial infection among species, while this technique needs the clinicians to make the pre-considerations of nocardiosis. However, mNGS may be a putative tool for rapid and accurate detection and identification of Nocardia, beneficial for applications of antimicrobial drugs and timely adjustments of medication.

1999 ◽  
Vol 122 (2) ◽  
pp. 323-328 ◽  
Author(s):  
M. T. E. P. ALLSOPP ◽  
C. M. HATTINGH ◽  
S. W. VOGEL ◽  
B. A. ALLSOPP

A panel of 16S ribosomal RNA gene probes has been developed for the study of the epidemiology of heartwater; five of these detect different cowdria genotypes, one detects five distinct genotypes; one detects any Group III Ehrlichia species other than Cowdria and one detects any Group II Ehrlichia species. These probes have been used on PCR-amplified rickettsial 16S rRNA genes from over 200 Amblyomma hebraeum ticks. Control ticks were laboratory-reared and either uninfected or fed on sheep experimentally infected with different cowdria isolates, field ticks were collected from animals in heartwater-endemic areas. All tick-derived DNA samples were also examined by PCR amplification and probing for two other cowdria genes (map1 and pCS20) which have previously been used for heartwater epidemiology. This paper describes the first direct comparison of all currently available DNA probes for heartwater-associated organisms.


2007 ◽  
Vol 53 (1) ◽  
pp. 116-128 ◽  
Author(s):  
Richard Villemur ◽  
Philippe Constant ◽  
Annie Gauthier ◽  
Martine Shareck ◽  
Réjean Beaudet

Strains of Desulfitobacterium hafniense, such as strains PCP-1, DP7, TCE1, and TCP-A, have unusual long 16S ribosomal RNA (rRNA) genes due to an insertion of approximately 100 bp in the 5' region. In this report, we analyzed the 16S rRNA genes of different Desulfitobacterium strains to determine if such an insertion is a common feature of desulfitobacteria. We amplified this region by polymerase chain reaction (PCR) from eight Desulfitobacterium strains (D. hafniense strains PCP-1, DP7, TCP-A, TCE1, and DCB-2; D. dehalogenans; D. chlororespirans; and Desulfitobacterium sp. PCE1) and resolved each PCR product by denaturing gradient gel electrophoresis (DGGE). All strains had from two to seven DGGE- migrating bands, suggesting heterogeneity in their 16S rRNA gene copies. For each strain, the 5' region of the 16S rRNA genes was amplified and a clone library was derived. Clones corresponding to most PCR–DGGE migration bands were isolated. Sequencing of representative clones revealed that the heterogeneity was generated by insertions of 100–200 bp. An insertion was found in at least one copy of the 16S rRNA gene in all examined strains. In total, we found eight different types of insertions (INS1–INS8) that varied from 123 to 193 nt in length. Two-dimensional structural analyses of transcribed sequences predicted that all insertions would form an energetically stable loop. Reverse transcriptase – PCR experiments revealed that most of the observed insertions in the Desulfitobacterium strains were excised from the mature 16S rRNA transcripts. Insertions were not commonly found in bacterial 16S rRNA genes, and having a different insertion in several 16S rRNA gene copies borne by a single bacterial species was rarely observed. The function of these insertions is not known, but their occurrence can have an important impact in deriving 16S rRNA oligonucleotidic fluorescence in situ hybridization probes, as these insertions can be excised from 16S rRNA transcripts.Key words: Desulfitobacterium, 16S ribosomal RNA genes, heterogeneity, gene insertions, fluorescence in situ hybridization.


Buletin Palma ◽  
2016 ◽  
Vol 16 (2) ◽  
pp. 147
Author(s):  
JELFINA C. ALOUW ◽  
DIANA NOVIANTI ◽  
MELDY L.A. HOSANG

<p><span style="font-size: medium;">ABSTRACT </span></p><p>Many species of microorganisms can cause diseases and mortality of insect pests. Accurate detection and identification of the entomophatogens are essential for development of biological control agent to the pest. Brontispa longissima, a serious and invasive pest of coconut, was infected by bacterium causing mortality of the larvae and pupae in coconut field. Objective of the research was to identify bacterium as a causal agent of the field-infected B. longissima using molecular  technique.  Research  was  conducted  between  April  and  August 2011.  Molecular  identification  using polymerase chain reaction (PCR) amplification of 16s ribosomal RNA of the infected larvae and sequencing of the gene showed that Serratia marcescens is the causal agent of the disease.</p><p>Keywords: Brontispa longissima, coconut, 16s rRNA, Serratia marcescens.</p><p> </p><p><span style="font-size: medium;">Identifikasi Molekular Bakteri Pathogen yang Menginfeksi Hama Daun Kelapa <br />Brontispa longissima(Coleoptera:Chrysomelidae)</span></p><p><span style="font-size: medium;">ABSTRAK </span></p><p>Banyak mikroorganisme dapat menimbulkan penyakit pada serangga hama.  Deteksi dan identifikasi yang akurat dari  pathogen  penyebab  penyakit  pada  serangga (entomopathogen)  hama  merupakan  tahap  yang  penting  dalam  pengembangan pengendalian biologi untuk hama tersebut.  Brontispa longissima sebagai hama penting dan bersifat  invasif pada tanaman kelapa diinfeksi oleh sejenis bakteri yang menyebabkan kematian larva dan pupa dari serangga  tersebut di lapangan. Penelitian ini bertujuan untuk mengidentifikasi organisme penyebab penyakit pada hama B. longissima dengan menggunakan teknik molekuler. Penelitian dilaksanakan pada bulan April sampai dengan Agustus  2011. Identifikasi bakteri dilakukan dengan mengamplifikasi 16s ribosomal RNA dari larva yang terinfeksi dengan menggunakan PCR (polymerase chain reaction).  Hasil analisis sekuens nukleotida 16s ribosomal RNA dari larva yang terinfeksi menunjukkan bahwa Serratia marcescens adalah bakteri penyebab dari penyakit tersebut.</p><p>Kata kunci: Brontispa longissima, kelapa, 16s rRNA, Serratia marcescens.</p>


1995 ◽  
Vol 73 (11-12) ◽  
pp. 899-905 ◽  
Author(s):  
Seth Stern ◽  
Prakash Purohit

Despite the passage of about 30 years since the discovery of the translational activities of ribosomes and the outlining of the roles of the large and small subunits, the actual molecular basis for the mRNA decoding activities of the small subunit has remained essentially obscure. In this paper, we describe a new approach using oligonucleotide analogs of 16S ribosomal RNA, in which the small ribosomal subunit is effectively deconstructed into a smaller more experimentally tractable form. Specifically, we review the results of experiments using an oligonucleotide analog of the decoding region of 16S ribosomal RNA, suggesting that the decoding region is the functional core of the small subunit, that it contacts both mRNA codons and tRNA anticodons, and that it mediates and probably enhances codon–anticodon base pairing, that is, decoding.Key words: translation, ribosome, 30S, 16S, RNA, decoding, antibiotic.


2013 ◽  
Vol 2013 ◽  
pp. 1-4 ◽  
Author(s):  
Mel S. Lee ◽  
Wen-Hsin Chang ◽  
Su-Chin Chen ◽  
Pang-Hsin Hsieh ◽  
Hsin-Nung Shih ◽  
...  

The diagnosis of periprosthetic joint infection is sometimes straightforward with purulent discharge from the fistula tract communicating to the joint prosthesis. However it is often difficult to differentiate septic from aseptic loosening of prosthesis because of the high culture-negative rates in conventional microbiologic culture. This study used quantitative reverse transcription polymerase chain reaction (RT-qPCR) to amplify bacterial 16S ribosomal RNA in vitro and in 11 clinical samples. The in vitro analysis demonstrated that the RT-qPCR method was highly sensitive with the detection limit of bacterial 16S rRNA being 0.148 pg/μl. Clinical specimens were analyzed using the same protocol. The RT-qPCR was positive for bacterial detection in 8 culture-positive cases (including aerobic, anaerobic, and mycobacteria) and 2 culture-negative cases. It was negative in one case that the final diagnosis was confirmed without infection. The molecular diagnosis of bacterial infection using RT-qPCR to detect bacterial 16S rRNA around a prosthesis correlated well with the clinical findings. Based on the promising clinical results, we were attempting to differentiate bacterial species or drug-resistant strains by using species-specific primers and to detect the persistence of bacteria during the interim period before the second stage reimplantation in a larger scale of clinical subjects.


2021 ◽  
Vol 14 (5) ◽  
pp. 43-50
Author(s):  
ARTEM M. MOROZOV ◽  
◽  
ALEXEY N. SERGEEV ◽  
VICTOR A. KADYKOV ◽  
ELSHAD M. ASKEROV ◽  
...  

Background. Antibiotic resistance is a worldwide problem that is the main cause of the increase in the number of purulent-inflammatory diseases and postsurgical complications. Aim. The aim of the present study was to monitor antibiotic resistance of microorganisms isolated in the course of microbiological study in otorhinolaryngological and surgical patients of the outpatient department of a single preventive health institution. Material and methods. A statistical analysis of the results of microbiological studies, excretions of patients undergoing outpatient treatment in surgical and otorhinolaryngological departments of outpatient clinic No1 of the state budget institution «City Clinical Hospital No 7» of Tver is presented. We processed 280 findings of microbiological studies of ear, nose, pharynx and wound surface cultures for 2019. Results and discussion. It was found that the spectrum of dominant microorganism species in 2019 detected during swabbing of surgical and otorhinolaryngological patients treated in the outpatient clinic included gram-negative Escherichia coli and Klebsiella pneumonia and gram-positive Staphylococcus aureus and Streptococcus pyogenes microorganisms. Among the microorganisms presented, strains with multiple resistance to the action of antimicrobial drugs were identified. According to the total number of resistant microorganisms it was found that among antibacterial drugs, antibiotics of Aminopenicillin class, including those protected by protease inhibitors, as well as fluoroquinolones of the 2nd generation have the lowest clinical efficacy in the given medical institution. Conclusion. The findings of the present study indicate a rapid rate of spread of resistance genes among nosocomial strains of microorganisms, which necessitates continuous monitoring of antibiotic sensitivity by various specific laboratory methods. The results of laboratory studies should be used when prescribing antibiotic therapy for a particular patient, and the results of this study should be considered when selecting an empirical antibiotic, since they reflect the resistance of the most common pathogens. Key words: antibiotic resistance, antibiotics, cephalosporins, fluoroquinolones.


2013 ◽  
Vol 24 (2) ◽  
pp. 85-88 ◽  
Author(s):  
B Alraddadi ◽  
S Al-Azri ◽  
KR Forward

INTRODUCTION: Amplification of the 16S ribosomal RNA gene by polymerase chain reaction (PCR) followed by analysis of generated sequences can be an important adjunct to conventional cultures.OBJECTIVE: To determine how the results of this approach influence physicians’ decisions regarding the management of bone and joint infections.METHOD: Clinical and laboratory findings of patients seen at the Queen Elizabeth II Health Sciences Centre (Halifax, Nova Scotia) between December 2005 and September 2009 were reviewed. Patients who had negative cultures but likely or possible bone and joint infections were further evaluated using 16S rRNA PCR. The impact of the 16S rRNA PCR result on antibiotic management was evaluated and it was assessed whether untreated patients with negative 16S rRNA PCR subsequently presented with infections, suggesting a false-negative result.RESULT: A total of 36 patients (mean age 62 years) were reviewed. Thirty-two patients were evaluated by infectious disease consultants; of these, 20 were considered likely to have infections. Seventeen patients were admitted with suspected prosthetic joint infections. Twenty-nine patients received antimicrobial treatment before the sample for the 16S rRNA PCR assay was obtained. Of the 36 patients, 26 (72.2%) were treated appropriately with modifications to their antibiotic regimen in response to the 16S rRNA PCR assay results. Antimicrobials were discontinued for 19 patients based on negative PCR assay and, in seven patients, antibiotics were changed based on a positive result. There were no relapses among patients with negative PCR assay in whom antibiotics were discontinued.CONCLUSION: 16S ribosomal RNA gene PCR and sequencing is a valuable tool in the guidance of antimicrobial therapy for bone and joint infections.


2017 ◽  
Author(s):  
Robert C. Edgar

AbstractThe 16S ribosomal RNA (rRNA) gene is widely used to survey microbial communities. Sequences are often clustered into Operational Taxonomic Units (OTUs) as proxies for species. The canonical clustering threshold is 97% identity, which was proposed in 1994 when few 16S rRNA sequences were available, motivating a reassessment on current data. Using a large set of high-quality 16S rRNA sequences from finished genomes, I assessed the correspondence of OTUs to species for five representative clustering algorithms using four accuracy metrics. All algorithms had comparable accuracy when tuned to a given metric. Optimal identity thresholds that best approximated species were ∼99% for full-length sequences and ∼100% for the V4 hypervariable region.


2021 ◽  
Author(s):  
Zhuo Bi ◽  
Hong-Wei Su ◽  
Jia-Yao Hong ◽  
Babak Javid

SummaryDespite redundant cellular pathways to minimize translational errors, errors in protein synthesis are common. Pathways and mechanisms to minimize errors are classified as pre-ribosomal or ribosomal. Pre-ribosomal pathways are primarily concerned with appropriate pairing of tRNAs with their cognate amino acid, whereas to date, ribosomal proof-reading has been thought to only be concerned with minimizing decoding errors, since it has been assumed that the ribosomal decoding centre is blind to mischarged tRNAs. Here, we identified that in mycobacteria, deletion of the 16S ribosomal RNA methyltransferase gidB led to increased discrimination of mischarged tRNAs. GidB deletion was necessary but not sufficient for reducing mistranslation due to misacylation. Discrimination only occurred in mycobacteria enriched from environments or genetic backgrounds with high rates of mistranslation. Our data suggest that mycobacterial ribosomes are capable of discriminating mischarged tRNAs and that 16S rRNA methylation by GidB may act as a capacitor for moderating translational error.


Sign in / Sign up

Export Citation Format

Share Document