Laura Cerqueira

DNA mimics for the rapid identification of microorganisms by fluorescence in situ hybridization (FISH).

Fluorescence in situ hybridization (FISH) is a well-established technique that is used for a variety of purposes, ranging from pathogen detection in clinical diagnostics to the determination of chromosomal stability in stem cell research. The key step of FISH involves the detection of a nucleic acid region and as such, DNA molecules have typically been used to probe for the sequences of interest. However, since the turn of the century, an increasing number of laboratories have started to move on to the more robust DNA mimics methods, most notably peptide and locked nucleic acids (PNA and LNA). In this review, we will cover the state-of-the-art of the different DNA mimics in regard to their application as efficient markers for the presence of individual microbial cells, and consider their potential advantages and pitfalls. Available PNA probes are then reassessed in terms of sensitivity and specificity using rRNA databases. In addition, we also attempt to predict the applicability of DNA mimics in well-known techniques attempting to detect in situ low number of copies of specific nucleic acid sequences such as catalyzed reporter deposition (CARD) and recognition of individual genes (RING) FISH.

PNA-FISH as a new diagnostic method for the determination of clarithromycin resistance of Helicobacter pylori

BackgroundTriple therapy is the gold standard treatment for Helicobacter pylori eradication from the human stomach, but increased resistance to clarithromycin became the main factor of treatment failure. Until now, fastidious culturing methods are generally the method of choice to assess resistance status. In this study, a new genotypic method to detect clarithromycin resistance in clinical samples, based on fluorescent in situ hybridization (FISH) using a set of peptide nucleic acid probes (PNA), is proposed.ResultsThe set of probes targeting the point mutations responsible for clarithromycin resistance was applied to H. pylori suspensions and showed 100% sensitivity and specificity (95% CI, 79.9-100 and 95% CI, 71.6-100 respectively). This method can also be amenable for application to gastric biopsy samples, as resistance to clarithromycin was also detected when histological slides were tested.ConclusionsThe optimized PNA-FISH based diagnostic method to detect H. pylori clarithromycin resistance shown to be a very sensitive and specific method for the detection of clarithromycin resistance in the H. pylori smears and also proved to be a reliable method for the diagnosis of this pathogen in clinical samples and an alternative to existing plating methods.

Biofilm formation with mixed cultures of Pseudomonas aeruginosa/Escherichia coli on silicone using artificial urine to mimic urinary catheters

In this study, single and dual species biofilms of Pseudomonas aeruginosa and Escherichia coli, two common bacteria associated with urinary tract infections, were formed in silicon coupons immersed in artificial urine medium. In single species experiments, E. coli appeared to form biofilms more easily than P. aeruginosa. In mixed biofilms, both species apparently benefited from the presence of the other, as the average Log total cells cm−2 of mixed biofilms (7.29 cells cm−2) was higher than obtained for single cultures (6.99 cells cm−2). However, the use of selective media seemed to indicate that P. aeruginosa was the only microorganism to benefit in mixed biofilms (Log 7 CFU of P. aeruginosa cm−2, compared to Log 6 CFU cm−2 obtained in pure cultures). Peptide nucleic acid-fluorescence in situ hybridization combined with confocal laser scanning microscopy confirmed that E. coli was indeed being outnumbered by P. aeruginosa at 48 h. Whereas E. coli is the main causative agent of catheter-associated urinary tract infections, the results from this study indicate that the reason for the higher prevalence of this microorganism is not related to an enhanced ability to form biofilm and outcompete other species that may also be present, but rather to a better ability to form single-species biofilms possibly due to a more frequent access to the catheter surface.

Validation of a Fluorescence In Situ Hybridization Method Using Peptide Nucleic Acid Probes for Detection of Helicobacter pylori Clarithromycin Resistance in Gastric Biopsy Specimens

ABSTRACT Here, we evaluated a previously established peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method as a new diagnostic test for Helicobacter pylori clarithromycin resistance detection in paraffin-embedded gastric biopsy specimens. Both a retrospective study and a prospective cohort study were conducted to evaluate the specificity and sensitivity of a PNA-FISH method to determine H. pylori clarithromycin resistance. In the retrospective study (n = 30 patients), full agreement between PNA-FISH and PCR-sequencing was observed. Compared to the reference method (culture followed by Etest), the specificity and sensitivity of PNA-FISH were 90.9% (95% confidence interval [CI], 57.1% to 99.5%) and 84.2% (95% CI, 59.5% to 95.8%), respectively. In the prospective cohort (n = 93 patients), 21 cases were positive by culture. For the patients harboring clarithromycin-resistant H. pylori, the method showed sensitivity of 80.0% (95% CI, 29.9% to 98.9%) and specificity of 93.8% (95% CI, 67.7% to 99.7%). These values likely represent underestimations, as some of the discrepant results corresponded to patients infected by more than one strain. PNA-FISH appears to be a simple, quick, and accurate method for detecting H. pylori clarithromycin resistance in paraffin-embedded biopsy specimens. It is also the only one of the methods assessed here that allows direct and specific visualization of this microorganism within the biopsy specimens, a characteristic that allowed the observation that cells of different H. pylori strains can subsist in very close proximity in the stomach.

Proposal for a method to estimate nutrient shock effects in bacteria

BackgroundPlating methods are still the golden standard in microbiology; however, some studies have shown that these techniques can underestimate the microbial concentrations and diversity. A nutrient shock is one of the mechanisms proposed to explain this phenomenon. In this study, a tentative method to assess nutrient shock effects was tested.FindingsTo estimate the extent of nutrient shock effects, two strains isolated from tap water (Sphingomonas capsulata and Methylobacterium sp.) and two culture collection strains (E. coli CECT 434 and Pseudomonas fluorescens ATCC 13525) were exposed both to low and high nutrient conditions for different times and then placed in low nutrient medium (R2A) and rich nutrient medium (TSA).The average improvement (A.I.) of recovery between R2A and TSA for the different times was calculated to more simply assess the difference obtained in culturability between each medium. As expected, A.I. was higher when cells were plated after the exposition to water than when they were recovered from high-nutrient medium showing the existence of a nutrient shock for the diverse bacteria used. S. capsulata was the species most affected by this phenomenon.ConclusionsThis work provides a method to consistently determine the extent of nutrient shock effects on different microorganisms and hence quantify the ability of each species to deal with sudden increases in substrate concentration.

Yeasts identification in microfluidic devices using peptide nucleic acid fluorescence in situ hybridization (PNA-FISH)

Peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) is a highly specific molecular method widely used for microbial identification. Nonetheless, and due to the detection limit of this technique, a time-consuming pre-enrichment step is typically required before identification. In here we have developed a lab-on-a-chip device to concentrate cell suspensions and speed up the identification process in yeasts. The PNA-FISH protocol was optimized to target Saccharomyces cerevisiae, a common yeast that is very relevant for several types of food industries. Then, several coin-sized microfluidic devices with different geometries were developed. Using Computational fluid dynamics (CFD), we modeled the hydrodynamics inside the microchannels and selected the most promising options. SU-8 structures were fabricated based on the selected designs and used to produce polydimethylsiloxane-based microchips by soft lithography. As a result, an integrated approach combining microfluidics and PNA-FISH for the rapid identification of S. cerevisiae was achieved. To improve fluid flow inside microchannels and the PNA-FISH labeling, oxygen plasma treatment was applied to the microfluidic devices and a new methodology to introduce the cell suspension and solutions into the microchannels was devised. A strong PNA-FISH signal was observed in cells trapped inside the microchannels, proving that the proposed methodology works as intended. The microfluidic designs and PNA-FISH procedure described in here should be easily adaptable for detection of other microorganisms of similar size.

Anti-miRNA oligonucleotides: A comprehensive guide for design

ABSTRACT MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression post-transcriptionally. As a consequence of their function towards mRNA, miRNAs are widely associated with the pathogenesis of several human diseases, making miRNAs a target for new therapeutic strategies based on the control of their expression. Indeed, numerous works were published in the past decades showing the potential use of antisense oligonucleotides to target aberrant miRNAs (AMOs) involved in several human pathologies. New classes of chemical-modified-AMOs, including locked nucleic acid oligonucleotides, have recently proved their worth in silencing miRNAs. A correct design of a specific AMOs can help to improve their performance and potency towards the target miRNA by increasing for instance nuclease resistance and target affinity. This review outlines the technologies involved to suppress aberrant miRNAs. From the design strategies used in AMOs to its application in novel miRNA-based therapeutics and detection methodologies.

A new colorimetric peptide nucleic acid-based assay for the specific detection of bacteria

AIM Developments on synthetic molecules, such as peptide nucleic acid (PNA), make FISH procedures more robust for microbial identification. Fluorochromes use might hinder a broader implementation of PNA-FISH, but colorimetric applications are inexistent so far. METHODS A biotin-labeled eubacteria probe was used to develop a colorimetric PNA-in situ hybridization (ISH) assay. An enzymatic-conjugate, targeting biotin, was introduced. The procedure was optimized and evaluated regarding sensitivity, specificity and detection limit. RESULTS RESULTS have shown strong ISH signals. The method was specific, but permeabilization problems were observed for Gram-positive bacteria. Detection limit was 5 × 10(7) CFU/ml, limiting current applications to pre-enriched samples. CONCLUSION The PNA-ISH procedure described here is a simple alternative to other detection methods, and is also the base for the development of other PNA colorimetric systems.