Employment of 16 S rDNA gene sequencing techniques to identify culturable environmental eubacteria in a tertiary referral hospital

Xu, Jianguo; Smyth, Cian; Buchanan, Janet A.; Dolan, Anthony; Rooney, Paul J.; Millar, B. Cherie; Goldsmith, Colin E.; Elborn, J. Stuart; Moore, John E.

doi:10.1016/j.jhin.2004.01.011

Cited by 25 publications

(11 citation statements)

References 6 publications

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“…Broad-range PCR assay was optimised to amplify ~762 bp region (257–1019 np) of 16S rRNA gene using published primers (FP5’-CAGACTCCTACGGGGAGGCAGCAGT-3’ and RP 5’-ACTTAACCCAACATCTCACGACAC-3)9 using standard strain of E. coli (ATCC 25922). The different parameters for PCR assay were optimised, and amplification from the DNA extracted from vitreous specimen was carried out in 25 µL of reaction mixture containing 10× reaction buffer (Fermentas, USA), 2.5 mM MgCl 2 , 200 µM dNTPs (Fermentas), 0.4 µM forward and reverse primers (IDT, India), 1.25U Taq Polymerase (Fermentas), and Milli-Q water quantum satis .…”

Section: Methodsmentioning

confidence: 99%

Utility of broad-range 16S rRNA PCR assay versus conventional methods for laboratory diagnosis of bacterial endophthalmitis in a tertiary care hospital

et al. 2018

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BackgroundEndophthalmitis, a sight-threatening intraocular infection, can be of postsurgical, post-traumatic or endogenous origin. Laboratory diagnosis-based appropriate therapy can be vision-saving. Conventional culture-based laboratory diagnosis takes time and lacks sensitivity. In this study a broad-range PCR assay was assessed against conventional and automated culture methods in vitreous specimens for accurate microbiological diagnosis.AimsTo use broad-range PCR assay targeting 16S ribosomal RNA (rRNA) region of bacteria and to assess its performance vis-à-vis conventional and automated culture methods in the laboratory diagnosis of endophthalmitis.MethodsVitreous specimens from 195 patients with clinically diagnosed endophthalmitis were processed for classical and automated culture methods, antimicrobial sensitivity and broad-range PCR assay targeting 762 bp region of 16S rRNA followed by nucleotide sequencing by Sanger’s method. Causative agents were identified from the nucleotide sequences analysed against the GenBank database, and organisms were identified using the Clinical and Laboratory Standards Institute (CLSI) MM18A guidelines.ResultsBacteria could be detected from 127 (65.13%) of the 195 vitreous specimens by broad-range PCR assay; bacterial isolation was possible from 17 (8.7%) and 60 (30.76%) of these specimens by conventional and automated culture methods, respectively (p<0.0001). PCR assay could detect two uncultured bacterium, and in five cases the bacterial identity could not be determined from NCBI database matching.ConclusionBroad-range PCR assay could provide definitive microbial diagnosis within 24 hours in significantly more patients (p<0.0001). Some rare organisms could be detected, useful in treatment modalities. Automated culture was significantly more sensitive than conventional culture.

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Section: Methodsmentioning

confidence: 99%

Utility of broad-range 16S rRNA PCR assay versus conventional methods for laboratory diagnosis of bacterial endophthalmitis in a tertiary care hospital

et al. 2018

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“…The identiied representatives were 16 canonical bacterial phyla, members of the phyla Firmicutes mainly Staphylococcus and Streptococcus and Actinobacteria mainly Micrococcaceae, Corynebacteriaceae and Brevibacteriaceae , the phylum Proteobacteria mainly by members of the families Enterobacteriaceae, Methylobacteriaceae and Sphingomonadaceae , the phyla Proteobacteria, Bacteroidetes, Deinococcus-Thermus and Cyanobacteria, Proteobacteria mainly due to the high abundance of Enterobacteriaceae members [74]. 16 S rDNA PCR and sequencing was also employed in study of Xu et al [75], where 53 isolates from environmental water-associated sites in a haematology unit, and the outer surfaces of cleaning lotion containers sited throughout a tertiary referral hospital were investigated. Sequence analysis was able to identify 51 isolates, mostly Gram-positive bacteria.…”

Section: Pcr-based Microbial Complexity Analysesmentioning

confidence: 99%

“…Sequence analysis was able to identify 51 isolates, mostly Gram-positive bacteria. Nine diferent genera were identiied from the haematology unit and 13 from the cleaning lotion containers [75].…”

Section: Pcr-based Microbial Complexity Analysesmentioning

confidence: 99%

PCR Technique for the Microbial Analysis of Inanimate Hospital Environment

Rozman¹,

Turk²

2016

Polymerase Chain Reaction for Biomedical Applications

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Discipline of molecular ecology and molecular techniques such as polymerase chain reaction PCR ofers a possibility to study and reveal the microbial diversity in environmental setings with complicated mixed communities, non-culturable organisms, interfering contaminants and low levels of target DNA. Hospital environment represents a new ecological niche for clinically important nosocomial pathogens and antibiotic-resistant microorganisms, which have been commonly found on various hospital surfaces. Accurate characterization of microbial communities depends on several factors, starting with sample collection and conditional enrichment step. In the step of nucleic acid isolation and puriication, the DNA, as a dominant signature molecule, is extracted followed by removing co-extracted impurities. PCR target sequences are often 16S rDNA gene, functional gene probes or species-speciic probes, depending on the objective of the study. Furthermore, properly prepared PCR amplicons can serve as a basis for characterizing microbial community. The PCR technique is a powerful tool for the analysis of microbial diversity of environmental ecosystems. In a hospital environment, advantages of detecting pathogens and antibiotic-resistant bacteria need to be pointed out.Keywords: microorganisms, hospital environment, inanimate surfaces, DNA extraction, PCR . IntroductionCharacteristics of the hospital environments are very speciic where inanimate environment can be colonized with a wide range of microorganisms [1,2]. The cultured microorganisms represent only a small fraction of natural microbial communities, hence the microbial diversity in terms of species richness and species abundance is grossly underestimated [3]. Therefore, the discipline of molecular ecology and molecular techniques such as polymerase chain reaction PCR ofers a possibility to study and reveal the real-microbial population complexity and a possibility to overcome limitations of culture-based approaches. Due to the power of the PCR to amplify small amounts of DNA, organisms occurring in small numbers in an environment are now detectable [3]. Special challenges in environmental setings are complicated mixed communities, interfering contaminants and low levels of target DNA [4]. The speciics of environmental samples are low to medium concentration of target cells, low sample homogeneity and high degree of PCR inhibition [5].Many types of pathogenic microorganisms have been found on various common hospital surfaces. Most common nosocomial bacteria present and detected on inanimate hospital surfaces, using speciic marker genes, are presented in

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“…PD fluids were subjected to the two (762/598bp) fragment 16S rDNA PCR assay recently described by Jenkins et al [15], which uses the primers described by Xu et al [16] and listed in Table 1. We converted the PCR assay to a real-time format with a high-resolution melt (HRM) point analysis to confirm positive amplification.…”

Section: Real-time 16s Rdna Pcr Assaymentioning

confidence: 99%

The Role of 16s rDNA PCR in the Diagnosis of Peritoneal Dialysis-Associated Peritonitis

Collier¹

2012

J Med Microb Diagn

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Introduction: Despite recent advances in peritoneal dialysis (PD), peritonitis remains the most common and serious complication. In a significant proportion of patients a pathogen is not cultured. In this study we investigated the use of 16S rDNA PCR to make a bacterial diagnosis. Methods:We used an optimised DNA extraction and 16S rDNA PCR with DNA sequencing to detect pathogens in peritoneal dialysis-associated peritonitis (PDAP).Results: Seventy-one PD fluids from twenty-four patients were analysed. In suspected cases of PDAP, thirteen out of twenty-one patients had a bacterial pathogen cultured and 16S rDNA PCR with DNA sequencing identified one additional pathogen. However 16S rDNA PCR only detected the pathogen in five of the culture-positive fluids. All follow-up samples were culture-negative, but possible pathogens were identified in three samples by the 16S rDNA PCR.In suspected cases of PDAP the sensitivity and specificity of the PCR was calculated as 69% and 63%, respectively. Conclusion:The use of 16S rDNA PCR in diagnosing PDAP needs further study and improved sensitivity before widespread introduction.

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Employment of 16 S rDNA gene sequencing techniques to identify culturable environmental eubacteria in a tertiary referral hospital

Cited by 25 publications

References 6 publications

Utility of broad-range 16S rRNA PCR assay versus conventional methods for laboratory diagnosis of bacterial endophthalmitis in a tertiary care hospital

Utility of broad-range 16S rRNA PCR assay versus conventional methods for laboratory diagnosis of bacterial endophthalmitis in a tertiary care hospital

PCR Technique for the Microbial Analysis of Inanimate Hospital Environment

The Role of 16s rDNA PCR in the Diagnosis of Peritoneal Dialysis-Associated Peritonitis

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