Dataset Information

Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci.

ABSTRACT: To compare commonly used phenotypic methods with genotypic identification methods 47 clinical isolates of coagulase-negative staphylococci (CONS), 10 CONS ATCC strains, and a Staphylococcus aureus clinical isolate were identified using the API Staph ID test, BD Phoenix Automated Microbiology System, and 16S rRNA gene and tuf gene sequencing. When necessary part of the sodA gene was sequenced for definitive identification. The results show that tuf gene sequencing is the best method for identification of CONS, but the API Staph ID test is a reasonably reliable phenotypic alternative. The performance of the BD Phoenix Automated Microbiology System for identification of CONS is poor. The present study also showed that although genotypic methods are clearly superior to phenotypic identifications, a drawback of sequence-based genotypic methods may be a lack of quality of deposited sequences in data banks. In particular, 16S rRNA gene sequencing suffers from the lack of high quality among sequences deposited in GenBank. Furthermore, genotypic identification based on 16S rRNA sequences has limited discriminating power for closely related Staphylococcus species. We propose partial sequencing of the tuf gene as a reliable and reproducible method for identification of CONS species.

SUBMITTER: Heikens E

PROVIDER: S-EPMC1153770 | biostudies-literature | 2005 May

REPOSITORIES: biostudies-literature

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Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci.

Heikens E E Fleer A A Paauw A A Florijn A A Fluit A C AC

Journal of clinical microbiology 20050501 5

To compare commonly used phenotypic methods with genotypic identification methods 47 clinical isolates of coagulase-negative staphylococci (CONS), 10 CONS ATCC strains, and a Staphylococcus aureus clinical isolate were identified using the API Staph ID test, BD Phoenix Automated Microbiology System, and 16S rRNA gene and tuf gene sequencing. When necessary part of the sodA gene was sequenced for definitive identification. The results show that tuf gene sequencing is the best method for identific ...[more]

PMID: 15872257

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Project description:BackgroundCoagulase-negative staphylococci (CNS) survive as commensals of skin, anterior nares and external canals of human and were regarded as non-infectious pathogens. However, they are emerging as a major cause of nosocomial infectious due to their ability to form biofilms and high resistance to several classes of antibiotics. This study examines the biofilm forming abilities of 214 clinical CNS isolates using phenotypic and genotypic methods, and determines their antibiotic susceptibility patterns.MethodsA total of 214 clinical isolates collected from different clinical samples were identified as CNS and their antibiotic susceptibility determined by CLSI guidelines. The biofilm forming ability of all isolates was determined by three phenotypic methods; Congo red agar (CRA) method, tube adherence method (TM) and tissue culture plate (TCP) method and by genotypic method for the detection of icaAD genes.ResultsAmong all the isolates, S. epidermidis (57.5%) was found the most frequently, followed by S. saprophyticus (18.7%), S. haemolyticus (11.2%), S. hominis (7%), and S. capitis (5.6%). Antibiotic susceptibility pattern demonstrated 91.6% isolates were resistant to penicillin and 66.8% to cefoxitin while 91.1% isolates were susceptible to chloramphenicol. Constitutive and inducible clindamycin resistant phenotype as measured by D-test was seen among 28% and 14.5% of isolates respectively. Tissue culture plate method detected biofilm production in 42.1% isolate followed by 31.8% through tube method while 20.1% isolates were found to produce slime in Congo red agar method. The genotypic assay revealed presence of icaA and icaD genes in 19.2% isolates.ConclusionThe study shows a high prevalence of biofilm formation and inducible clindamycin resistance in CNS isolates, indicating the importance of in-vitro biofilm production test and D-test in routine laboratory diagnostics. Implementation of efficient diagnostic techniques for detection of biofilm production in clinical samples can help manage staphylococcal infections and minimize risks of treatment failures in hospitals.

Project description:Purpose: The aim of the study was to compare the miRNA expression in non-infected (H) mammary gland parenchyma samples with that of glands infected with coagulase-positive staphy lococci (CoPS) or coagulase-negative staphylococci (CoNS) using next-generation sequencing. Methods: miRNA-seq analysis was performed on mammary gland parenchyma samples collected from non-infected cows and those infected with coagulase-positive or -negative staphylococci. The miRNA libraries were constructed from total RNA using NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs) according to the manufacturer protocol. The quantification of the obtained libraries was performed on a Qubit 2.0 spectrophotometer (Invitrogen, Life Technologies), while a quality control on a TapeStation 2200 instrument (D1000 ScreenTape; Agilent). Single-end cycle sequencing was performed on the HiScanSQ platform (Illumina) with the use of TruSeq SR Cluster Kit v3- CBOT-HS and TruSeq SBS Kit v 3 - HS (Illumina). MicroRNA differentially expressed between investigated groups were identified with the DESeq2 software. Results: Comparing the CoPS and H groups, 256 known and 260 potentially new miRNAs were identifed, including 32 that were diferentially expressed (p≤0.05), of which 27 were upregulated and 5 downregulated. Comparing the CoNS and H groups, 242 known and 171 new unique miRNAs were identifed: 10 were upregulated (p≤0.05), and 2 downregulated (p≤0.05). Comparing CoPS with H and CoNS with H, 5 Kyoto Encyclopedia of Genes and Genomes pathways were identifed; in both comparisons, diferentially-expressed miRNAs were associated with the bacterial invasion of epithelial cells and focal adhesion pathways. Four gene ontology terms were identifed in each comparison, with 2 being common to both immune system processes and signal transduction. Conclusions: Obtained results enabled us to characterize the miRNA profile of the mammary gland parenchyma tissue, not only the healthy one but also the tissue infected with coagulase-positive and negative staphylococci as well as allowed identification of miRNAs differing the examined groups and characteristic for the staphylococci infection. They also indicated that miRNAs, especially miR-99 and miR-182, play an essential role in the epigenetic regulation of a range of cellular processes, including immunological systems bacterial growth in dendritic cells and disease pathogenesis (miR-99), DNA repair and tumor progression (miR-182).

Dataset Information

Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci.

Publications

Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci.

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