Optimization of a peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the detection of bacteria and disclosure of a formamide effect

Abstract

Despite the fact that fluorescence in situ hybridization (FISH) is a wellestablished technique to identify microorganisms, there is a lack of understanding concerning the interaction of the different factors affecting the obtained fluorescence. In here, we used flow cytometry to study the influence of three essential factors in hybridization -temperature, time and formamide concentration -in an effort to optimize the performance of a Peptide Nucleic Acid (PNA) probe targeting bacteria (EUB338). The PNA-FISH optimization was performed with bacteria representing different families employing response surface methodology. Surprisingly, the optimum concentration of formamide varied according to the bacterium tested. While hybridization on the bacteria possessing the thickest peptidoglycan was more successful at nearly 50% (v/v) formamide, hybridization on all other microorganisms appeared to improve with much lower formamide concentrations. Gram staining and transmission electron microscopy allowed us to confirm that the overall effect of formamide concentration on the fluorescence intensity is a balance between a harmful effect on the bacterial cell envelope, affecting cellular integrity, and the beneficial denaturant effect in the hybridization process. We also conclude that microorganisms belonging to different families will require different hybridization parameters for the same FISH probe, meaning that an optimum universal PNA-FISH procedure is non-existent for these situations.2

Towards Fluorescence In Vivo Hybridization (FIVH) Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes

“…Therefore, the first analysis encompassed the evaluation of the performance of this probe at a range of pH values using different hybridization times applying the FISH protocol in suspension, as previously described [ 17 ]. For this analysis we used the response surface methodology (RSM) [ 29 , 30 ] to quantify the relationship between the response (or output variable, i.e. fluorescence intensity upon probe hybridization) and the independent variable (input variable, i.e.…”

Nanomaterials and molecular transporters to overcome the bacterial envelope barrier: Towards advanced delivery of antibiotics

“…However, this might not be the case for all bacteria. Studies using PNA to hybridize in different bacteria suggested that thick peptidoglycan layers from Grampositive bacteria, such as Bacillus cereus (which has a peptidoglycan 6-7 times thicker than that of E. coli [67]), may significantly retard PNA penetration (Figure 3a) [104,105].…”

Effect of Native Gastric Mucus on in vivo Hybridization Therapies Directed at Helicobacter pylori

“…Paraformaldehyde and ethanol are among the most common permeabilization and fixation agents. 1,21,30,31,41,42,43 While paraformaldehyde is a fixative, ethanol acts both as a fixative and permeabilizer of the bacterial membrane lipids. 44,45 Fixation and permeabilization agents cannot, however, be used for in vivo applications due to their toxicity.…”

“…FISH ( Figure 1c ) is based on specific base pairing, at high temperature, of an oligonucleotide with rRNA or mRNA target sequences, obeying to Watson-Crick hydrogen-bonding. 28,29,30,31 Recently, Fontenete et al 32 have proven that oligonucleotides composed of both LNA and 2'OMe (which we term LNA/2'OMe), with either phosphodiester (PO) or phosphorothioate (PS) as backbone linkages, allow specific detection of H. pylori . In this work, well-reported agents to permeabilize the bacterial cell envelope (like paraformaldehyde and ethanol) were used; H. pylori both grown in culture as well as in gastric biopsies could be detected in this way at 37 °C.…”