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Peptide Nucleic Acid-Fluorescence In Situ Hybridization for Detection of Staphylococci From Endophthalmitis Isolates: A Proof-of-Concept Study.

ABSTRACT: Rapid identification of pathogens causing endophthalmitis may improve treatment outcomes through early administration of species-specific medication. The current study reports a new molecular application of peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) with Staphylococcus-specific molecular PNA probes for the potential rapid detection of common pathogens causing endophthalmitis.An experimental study was designed to evaluate the proof of concept at the microbiology laboratory of the Bascom Palmer Eye Institute. Stored culture-positive staphylococci endophthalmitis isolates obtained from prior vitreous samples (n = 15), along with broth as negative controls (n = 5) were used. Inoculum was prepared to a final concentration of 1 × 105 colony-forming units/mL to ensure that the isolates were viable. Smears of samples were fixed and hybridized using QuickFISH protocol with probes for Staphylococcus.With PNA-FISH technique, Staphylococcus aureus was identified in 9 of 10 samples and coagulase-negative staphylococci were identified in 10 of 10 samples. Detection time was 20 minutes.This study serves a proof of concept using a new microbial detection system with FISH probes, and may have the potential for clinical use in the rapid and accurate identification of isolates from patients with endophthalmitis.

SUBMITTER: Patel N

PROVIDER: S-EPMC5555249 | biostudies-other | 2017 Aug

REPOSITORIES: biostudies-other

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Project description:BackgroundOur current understanding of biofilms indicates that these structures are typically composed of many different microbial species. However, the lack of reliable techniques for the discrimination of each population has meant that studies focusing on multi-species biofilms are scarce and typically generate qualitative rather than quantitative data.Methodology/principal findingsWe employ peptide nucleic acid fluorescence in situ hybridization (PNA FISH) methods to quantify and visualize mixed biofilm populations. As a case study, we present the characterization of Salmonella enterica/Listeria monocytogenes/Escherichia coli single, dual and tri-species biofilms in seven different support materials. Ex-situ, we were able to monitor quantitatively the populations of ∼56 mixed species biofilms up to 48 h, regardless of the support material. In situ, a correct quantification remained more elusive, but a qualitative understanding of biofilm structure and composition is clearly possible by confocal laser scanning microscopy (CLSM) at least up to 192 h. Combining the data obtained from PNA FISH/CLSM with data from other established techniques and from calculated microbial parameters, we were able to develop a model for this tri-species biofilm. The higher growth rate and exopolymer production ability of E. coli probably led this microorganism to outcompete the other two [average cell numbers (cells/cm(2)) for 48 h biofilm: E. coli 2,1 × 10(8) (± 2,4 × 10(7)); L. monocytogenes 6,8 × 10(7) (± 9,4 × 10(6)); and S. enterica 1,4 × 10(6) (± 4,1 × 10(5))]. This overgrowth was confirmed by CSLM, with two well-defined layers being easily identified: the top one with E. coli, and the bottom one with mixed regions of L. monocytogenes and S. enterica.SignificanceWhile PNA FISH has been described previously for the qualitative study of biofilm populations, the present investigation demonstrates that it can also be used for the accurate quantification and spatial distribution of species in polymicrobial communities. Thus, it facilitates the understanding of interspecies interactions and how these are affected by changes in the surrounding environment.

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Peptide Nucleic Acid-Fluorescence In Situ Hybridization for Detection of Staphylococci From Endophthalmitis Isolates: A Proof-of-Concept Study.

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