scholarly journals Identification of Clinically Relevant Streptococcus and Enterococcus Species Based on Biochemical Methods and 16S rRNA, sodA, tuf, rpoB, and recA Gene Sequencing

Pathogens ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 939
Author(s):  
Maja Kosecka-Strojek ◽  
Mariola Wolska ◽  
Dorota Żabicka ◽  
Ewa Sadowy ◽  
Jacek Międzobrodzki
Keyword(s):  
16S Rrna ◽  
Reca Gene ◽  
Clinical Samples ◽  
Enterococcus Species ◽  
Opportunistic Pathogens ◽  
Species Identity ◽  
First Choice ◽  
Streptococcal Species ◽  
Enterococcal Species ◽  
Phenotypic Methods

Streptococci and enterococci are significant opportunistic pathogens in epidemiology and infectious medicine. High genetic and taxonomic similarities and several reclassifications within genera are the most challenging in species identification. The aim of this study was to identify Streptococcus and Enterococcus species using genetic and phenotypic methods and to determine the most discriminatory identification method. Thirty strains recovered from clinical samples representing 15 streptococcal species, five enterococcal species, and four nonstreptococcal species were subjected to bacterial identification by the Vitek® 2 system and Sanger-based sequencing methods targeting the 16S rRNA, sodA, tuf, rpoB, and recA genes. Phenotypic methods allowed the identification of 10 streptococcal strains, five enterococcal strains, and four nonstreptococcal strains (Leuconostoc, Granulicatella, and Globicatella genera). The combination of sequencing methods allowed the identification of 21 streptococcal strains, five enterococcal strains, and four nonstreptococcal strains. The 16S rRNA and rpoB genes had the highest identification potential. Only a combination of several molecular methods was sufficient for unambiguous confirmation of species identity. This study will be useful for comparison of several identification methods, both those used as a first choice in routine microbiology and those used for final confirmation.

scholarly journals Development of a reference data set for assigning Streptococcus and Enterococcus species based on next generation sequencing of the 16S–23S rRNA region

2019 ◽  
Vol 8 (1) ◽  
Author(s):  
Maja Kosecka-Strojek ◽  
Artur J. Sabat ◽  
Viktoria Akkerboom ◽  
Anna M. D. Kooistra-Smid ◽  
Jacek Miedzobrodzki ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Sequence Analysis ◽  
16S Rrna ◽  
Species Identification ◽  
23S Rrna ◽  
Clinical Samples ◽  
Next Generation ◽  
Enterococcal Species ◽  
Reference Sequences ◽  
Generation Sequencing

Abstract Background Many members of Streptococcus and Enterococcus genera are clinically relevant opportunistic pathogens warranting accurate and rapid identification for targeted therapy. Currently, the developed method based on next generation sequencing (NGS) of the 16S–23S rRNA region proved to be a rapid, reliable and precise approach for species identification directly from polymicrobial and challenging clinical samples. The introduction of this new method to routine diagnostics is hindered by a lack of the reference sequences for the 16S–23S rRNA region for many bacterial species. The aim of this study was to develop a careful assignment for streptococcal and enterococcal species based on NGS of the 16S–23S rRNA region. Methods Thirty two strains recovered from clinical samples and 19 reference strains representing 42 streptococcal species and nine enterococcal species were subjected to bacterial identification by four Sanger-based sequencing methods targeting the genes encoding (i) 16S rRNA, (ii) sodA, (iii) tuf and (iv) rpoB; and NGS of the 16S–23S rRNA region. Results This study allowed obtainment and deposition of reference sequences of the 16S–23S rRNA region for 15 streptococcal and 3 enterococcal species followed by enrichment for 27 and 6 species, respectively, for which reference sequences were available in the databases. For Streptococcus, NGS of the 16S–23S rRNA region was as discriminative as Sanger sequencing of the tuf and rpoB genes allowing for an unambiguous identification of 93% of analyzed species. For Enterococcus, sodA, tuf and rpoB genes sequencing allowed for identification of all species, while the NGS-based method did not allow for identification of only one enterococcal species. For both genera, the sequence analysis of the 16S rRNA gene was endowed with a low identification potential and was inferior to that of other tested identification methods. Moreover, in case of phylogenetically related species the sequence analysis of only the intergenic spacer region was not sufficient enough to precisely identify Streptococcus strains at the species level. Conclusions Based on the developed reference dataset, clinically relevant streptococcal and enterococcal species can now be reliably identified by 16S–23S rRNA sequences in samples. This study will be useful for introduction of a novel diagnostic tool, NGS of the 16S–23S rRNA region, which undoubtedly is an improvement for reliable culture-independent species identification directly from polymicrobially constituted clinical samples.

scholarly journals Comparison of Different Phenotypic Methods for Detection of Vancomycin Drug Resistance in Enterococcus Species

2021 ◽  
Vol 8 (9) ◽  
pp. 289-293
Author(s):  
Deepa Pramod Devhare ◽  
Sae Pol
Keyword(s):  
Drug Resistance ◽  
Tertiary Care ◽  
Vancomycin Resistance ◽  
Automated System ◽  
Disk Diffusion ◽  
Clinical Samples ◽  
Care Hospital ◽  
Enterococcus Species ◽  
Vancomycin Resistant ◽  
Phenotypic Methods

Introduction: Vancomycin resistant enterococci have emerged as an important cause of nosocomial infection worldwide. Vancomycin drug resistance needs to be detected accurately in all Enterococcus species in order to prevent its spread in health care setting. Present study was conducted to compare three different phenotypic methods for detection of vancomycin resistance in enterococci. Material and methods: Study was conducted in a tertiary care hospital over a period of one year. Enterococcus species isolated from clinical samples like urine, pus, blood and sterile body fluids were tested by three different methods namely disk diffusion, E-strip and Phoenix automated system for detection of vancomycin resistance. Results: 400 Enterococcus species were isolated from clinical samples. 19(4.8%) Enterococcus species were found to be vancomycin resistant and one (0.25%) strain was found to be intermediate resistant to vancomycin by all three methods resulting in 100% sensitivity and 100%specificity. Conclusion: Present study recommends vancomycin disk diffusion as screening and E-strip as good confirmatory tests in resource poor settings for detection of vancomycin drug resistance. Keywords: VRE, vancomycin, disk diffusion, E strip, Phoenix automated system..

Isolation of Stenotrophomonas maltophilia from Clinical Samples in Some Hospitals in Shiraz, Iran

2020 ◽  
pp. 1-3
Author(s):  
Majid Baserisalehi ◽  
Samira Zarezadeh ◽  
Majid Baserisalehi ◽  
Saeed Shoa
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Enterobacter Aerogenes ◽  
Late Summer ◽  
Early Spring ◽  
Clinical Samples ◽  
Bacillus Species ◽  
Microbiological Analysis ◽  
Blood Samples ◽  
Healthcare Facilities ◽  
Phenotypic Methods

Stenotrophomonas maltophilia is an emerging pathogenic non-fermentative Gram-negative Bacillus species. It has caused many nosocomial infections and can be isolated from various hospital wards and healthcare facilities. Research has shown that most of its strains are inherently resistant to many antibiotics and have multidrug resistance. This research intended to determine its occurrence frequency at some Hospitals in shiraz, Iran. The present study was conducted in six months (from early spring to late summer 2019). Clinical samples (Blood, Urine and cerebrospinal fluid (CSF)) collected from 120 patients afflicted with various infections. The samples were transferred to the Laboratory and subjected to microbiological analysis. Identification of the isolates was carried out by phenotypic methods and Stenotrophomonas maltophilia isolates verified using molecular methods. In total, various bacteria were isolated from 84 clinical samples. The isolates were Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Stenotrophomonas maltophilia, Staphylococcus aureus and Pseudomonas aeruginosa. Stenotrophomonas maltophilia was isolated from 17 (20.2%) positive samples and most of them were isolated from blood samples. Our finding indicated that Stenotrophomonas maltophilia isolated more from blood samples follow by CSF sample. In addition, our finding illustrated that Stenotrophomonas maltophilia can be considered as the common nosocomial agent at hospitals in Shiraz, Iran.

scholarly journals 16S rRNA gene pyrosequencing of reference and clinical samples and investigation of the temperature stability of microbiome profiles

Microbiome ◽  
2014 ◽  
Vol 2 (1) ◽  
pp. 31 ◽  
Author(s):  
Jun Hang ◽  
Valmik Desai ◽  
Nela Zavaljevski ◽  
Yu Yang ◽  
Xiaoxu Lin ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Temperature Stability ◽  
Clinical Samples ◽  
Rrna Gene ◽  
16S Rrna Gene Pyrosequencing

scholarly journals Whole-Genome Relationships among Francisella Bacteria of Diverse Origins Define New Species and Provide Specific Regions for Detection

2016 ◽  
Vol 83 (3) ◽  
Author(s):  
Jean F. Challacombe ◽  
Jeannine M. Petersen ◽  
La Verne Gallegos-Graves ◽  
David Hodge ◽  
Segaran Pillai ◽  
...  
Keyword(s):  
New Species ◽  
16S Rrna ◽  
Response Times ◽  
Amino Acid Sequences ◽  
Clinical Samples ◽  
Whole Genome ◽  
Multiple Gene ◽  
Content Type ◽  
Environmental Surveys ◽  
Genome Comparisons

ABSTRACT Francisella tularensis is a highly virulent zoonotic pathogen that causes tularemia and, because of weaponization efforts in past world wars, is considered a tier 1 biothreat agent. Detection and surveillance of F. tularensis may be confounded by the presence of uncharacterized, closely related organisms. Through DNA-based diagnostics and environmental surveys, novel clinical and environmental Francisella isolates have been obtained in recent years. Here we present 7 new Francisella genomes and a comparison of their characteristics to each other and to 24 publicly available genomes as well as a comparative analysis of 16S rRNA and sdhA genes from over 90 Francisella strains. Delineation of new species in bacteria is challenging, especially when isolates having very close genomic characteristics exhibit different physiological features—for example, when some are virulent pathogens in humans and animals while others are nonpathogenic or are opportunistic pathogens. Species resolution within Francisella varies with analyses of single genes, multiple gene or protein sets, or whole-genome comparisons of nucleic acid and amino acid sequences. Analyses focusing on single genes (16S rRNA, sdhA), multiple gene sets (virulence genes, lipopolysaccharide [LPS] biosynthesis genes, pathogenicity island), and whole-genome comparisons (nucleotide and protein) gave congruent results, but with different levels of discrimination confidence. We designate four new species within the genus; Francisella opportunistica sp. nov. (MA06-7296), Francisella salina sp. nov. (TX07-7308), Francisella uliginis sp. nov. (TX07-7310), and Francisella frigiditurris sp. nov. (CA97-1460). This study provides a robust comparative framework to discern species and virulence features of newly detected Francisella bacteria. IMPORTANCE DNA-based detection and sequencing methods have identified thousands of new bacteria in the human body and the environment. In most cases, there are no cultured isolates that correspond to these sequences. While DNA-based approaches are highly sensitive, accurately assigning species is difficult without known near relatives for comparison. This ambiguity poses challenges for clinical cases, disease epidemics, and environmental surveillance, for which response times must be short. Many new Francisella isolates have been identified globally. However, their species designations and potential for causing human disease remain ambiguous. Through detailed genome comparisons, we identified features that differentiate F. tularensis from clinical and environmental Francisella isolates and provide a knowledge base for future comparison of Francisella organisms identified in clinical samples or environmental surveys.

scholarly journals Is Staphylococcus lugdunensis Significant in Clinical Samples?

2017 ◽  
Vol 55 (11) ◽  
pp. 3167-3174 ◽  
Author(s):  
Xavier Argemi ◽  
Yves Hansmann ◽  
Philippe Riegel ◽  
Gilles Prévost
Keyword(s):  
Pathogenic Bacteria ◽  
Clinical Manifestations ◽  
Clinical Samples ◽  
Opportunistic Pathogens ◽  
Coagulase Negative Staphylococci ◽  
Staphylococcus Lugdunensis ◽  
Content Type ◽  
Severe Infections ◽  
Microbiological Data

ABSTRACTThe implication of coagulase-negative staphylococci in human diseases is a major issue, particularly in hospital settings wherein these species often act as opportunistic pathogens. In addition, some coagulase-negative staphylococci such asS. lugdunensishave emerged as pathogenic bacteria, implicated in severe infections, particularly, osteoarticular infections, foreign-body-associated infections, bacteremia, and endocarditis.In vitrostudies have shown the presence of several putative virulence factors such as adhesion factors, biofilm production, and proteolytic factors that might explain clinical manifestations. Taken together, the clinical and microbiological data might change the way clinicians and microbiologists look atS. lugdunensisin clinical samples.

scholarly journals Alteration of Bacterial Communities in Anterior Nares and Skin Sites of Patients Undergoing Arthroplasty Surgery: Analysis by 16S rRNA and Staphylococcal-Specific tuf Gene Sequencing

2020 ◽  
Vol 8 (12) ◽  
pp. 1977
Author(s):  
Søren Iversen ◽  
Thor Bech Johannesen ◽  
Anna Cäcilia Ingham ◽  
Sofie Marie Edslev ◽  
Staffan Tevell ◽  
...  
Keyword(s):  
16S Rrna ◽  
Bacterial Communities ◽  
Gene Sequencing ◽  
Alpha Diversity ◽  
Clinical Samples ◽  
Staphylococcal Species ◽  
Tuf Gene ◽  
Staphylococcus Hominis ◽  
The Impact ◽  
Arthroplasty Surgery

The aim was to study alterations of bacterial communities in patients undergoing hip or knee arthroplasty to assess the impact of chlorhexidine gluconate soap decolonisation and systemic antibiotic prophylaxis. A Swedish multicentre, prospective collection of samples obtained from elective arthroplasty patients (n = 83) by swabbing anterior nares, skin sites in the groin and the site of planned surgery, before and after arthroplasty surgery, was analysed by 16S rRNA (V3-V4) gene sequencing and a complementary targeted tuf gene sequencing approach to comprehensively characterise alterations in staphylococcal communities. Significant reductions in alpha diversity was detected for both bacterial (p = 0.04) and staphylococcal (p = 0.03) groin communities after arthroplasty surgery with significant reductions in relative Corynebacterium (p = 0.001) abundance and Staphylococcus hominis (p = 0.01) relative staphylococcal abundance. In nares, significant reductions occurred for Staphylococcus hominis (p = 0.02), Staphylococcus haemolyticus (p = 0.02), and Staphylococcus pasteuri (p = 0.003) relative to other staphylococci. Staphylococcus aureus colonised 35% of anterior nares before and 26% after arthroplasty surgery. Staphylococcus epidermidis was the most abundant staphylococcal species at all sampling sites. No bacterial genus or staphylococcal species increased significantly after arthroplasty surgery. Application of a targeted tuf gene sequencing approach provided auxiliary staphylococcal community profiles and allowed species-level characterisation directly from low biomass clinical samples.

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

2022 ◽  
Vol 11 ◽  
Author(s):  
Juanjuan Ding ◽  
Bing Ma ◽  
Xupeng Wei ◽  
Ying Li
Keyword(s):  
16S Rrna ◽  
Ribosomal Rna ◽  
Human Herpesvirus ◽  
Antibiotic Sensitivity ◽  
Common Species ◽  
16S Ribosomal Rna ◽  
Clinical Samples ◽  
Klebsiella Pneumonia ◽  
Antimicrobial Drugs ◽  
Detection And Identification

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.

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