Cultivation-independent approach for the direct detection of bacteria in human clinical specimens as a tool for analysing culture-negative samples: a prospective study

Abstract

Administration of empirical antibiotic therapy prior to microbiological diagnosis is thought to be associated the failure of subsequent bacterial growth in culture. The aim of this study was to detect bacterial pathogens via direct amplification and sequencing of the 16S rDNA gene in samples showing negative culture results as alternative diagnostic tools to troubleshoot difficult samples. Twenty-three (7.66 %) positive samples were detected, most of which were monomicrobial infections; 15 of the cases were identified as HAIs, 6 had catheter colonisation, and 2 had sample colonisation. The pathogens identified included Escherichia, Salmonella, Pseudomonas spp., Enterococcus spp. and coagulase-negative staphylococci (CoNS). The most frequent infections were bacteraemia and urinary tract infection, but meningitis, warm infection and soft tissue infection were also documented. These findings emphasise the efficacy and usefulness of molecular diagnosis, thus 16S rDNA gene analysis is strongly indicated by HAIs diagnostics.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-1949-3) contains supplementary material, which is available to authorized users.

Correct Identification of Ochrobactrum anthropi From Blood Culture Using 16rRNA Sequencing: A First Case Report in an Immunocompromised Patient in Mexico

“…The accurate identification of microorganisms involved in infectious diseases is paramount for clinical microbiology, since it can lead to specific and effective treatments and help prevent antibiotic resistance ( 15 ).…”

“…One of the most commonly used methods to identify bacteria is 16S rRNA gene sequencing. Universal primers designed to amplify the hypervariable V1, V2 and V3 regions of the 16S rRNA gene have been used to identify uncommon clinical bacteria as well as environmental strains that are not included in the databases of automated systems ( 15 ). Following this method, proper analysis of the sequences can lead to better identification of many organisms, including several that have rarely been studied ( 22 ).…”

“…Following this method, proper analysis of the sequences can lead to better identification of many organisms, including several that have rarely been studied ( 22 ). In this work, a broad-range PCR that includes the V1-V3 regions of 16S rRNA ( 15 ) was useful to discriminate between Brucella and Ochrobactrum despite their close phylogenetic relationship. When the presence of Brucella is suspected in a blood culture, the recommendation is to avoid manipulation of the bacterium outside of a class 3 laboratory.…”

“…The results of this test were negative, indicating that the isolated bacterium did not belong to the Brucella species. These contradicting results called for further testing, therefore a broad-range PCR reaction focused on the amplification of a segment of the 16S ribosomal gene (510 bp) was performed following the conditions described by Aguilera-Arreola et al ( 15 ). The PCR product was sequenced, and the 16S assembled sequence (16S_B1) was uploaded into GenBank under accession number KY982960 .…”

The changing face of microbial quality control practices in the brewing industry: Introducing mass spectrometry proteomic fingerprinting for microbial identification

“…Additionally, culture‐free methods have been developed to increase the speed of identification and to allow the sequencing of microorganisms that prove difficult or impossible to culture . The major advantage of this method is that it is not targeted to a specific microorganism, meaning that (a) it can identify any microorganism and requires no prior ‘guesswork’ since primers are broadly amplifying and (b) it can identify novel species.…”