| Summary: |
More than 4 million patients are estimated to acquire a healthcare-associated infection in the EU every year, resulting in approximately 37000 deaths. With the widespread use of empiric antibiotic treatments, the selective pressure for resistances has resulted in a high prevalence of multidrug resistant bacteria. As an example, surveillance programs (such as SCOPE and SENTRY), indicate that 70 to 80% of the coagulase-negative staphylococci are resistant to methicillin. This data highlights both the need for rapid and accurate diagnostics that can direct the therapy.
In the last decade, nucleic acid mimics-based assays, specially Peptide Nucleic Acid Fluorescence in situ Hybridization (PNA FISH) assays, have been developed for the identification of clinical pathogens, in an expedite way of unparallel accuracy. More recently, some studies have shown that its implementation as a routine method in clinical laboratories allows for a rational use of medicines, reduces hospitalization times and mortality, consequently resulting in considerable reduction of the health costs for institutions. Another important advantage of these methods is its capacity to detect antibiotic resistances resulting from point mutations in the ribosomal RNA. These point mutations have been extensively found in several bacteria exhibiting resistance to different macrolides and to linezolid. Our group took advantage of this property and successfully developed a PNA FISH method to detect clarithromycin-resistant strains of H. pylori in gastric biopsies. This high accurate method was in fact the basis for the creation of a start-up company (Biomode), which has received a 1.6M¤ investment.
Despite the success of this method in detecting point-mutations associated with antibiotic resistance, there is still a gap in the detection of other resistance mechanisms. These latter ones are associated with the presence of specific, single (or low copy genes) in the bacterial chromosome or plasmids. This la  |
Summary
More than 4 million patients are estimated to acquire a healthcare-associated infection in the EU every year, resulting in approximately 37000 deaths. With the widespread use of empiric antibiotic treatments, the selective pressure for resistances has resulted in a high prevalence of multidrug resistant bacteria. As an example, surveillance programs (such as SCOPE and SENTRY), indicate that 70 to 80% of the coagulase-negative staphylococci are resistant to methicillin. This data highlights both the need for rapid and accurate diagnostics that can direct the therapy.
In the last decade, nucleic acid mimics-based assays, specially Peptide Nucleic Acid Fluorescence in situ Hybridization (PNA FISH) assays, have been developed for the identification of clinical pathogens, in an expedite way of unparallel accuracy. More recently, some studies have shown that its implementation as a routine method in clinical laboratories allows for a rational use of medicines, reduces hospitalization times and mortality, consequently resulting in considerable reduction of the health costs for institutions. Another important advantage of these methods is its capacity to detect antibiotic resistances resulting from point mutations in the ribosomal RNA. These point mutations have been extensively found in several bacteria exhibiting resistance to different macrolides and to linezolid. Our group took advantage of this property and successfully developed a PNA FISH method to detect clarithromycin-resistant strains of H. pylori in gastric biopsies. This high accurate method was in fact the basis for the creation of a start-up company (Biomode), which has received a 1.6M¤ investment.
Despite the success of this method in detecting point-mutations associated with antibiotic resistance, there is still a gap in the detection of other resistance mechanisms. These latter ones are associated with the presence of specific, single (or low copy genes) in the bacterial chromosome or plasmids. This lack remains because FISH signal is the result of the hundreds/thousands of probes bound to the rRNA copies present in each cell. This means that, in an hybridization directed to the genome there is only a probe-target complex, which is insufficient to emit a detectable fluorescent signal. Also an important drawback of FISH-based techniques (and of other molecular techniques) is the low number of distinguishable targets, which usually is limited to 2 or 3 targets - a limitation related with the number of reporter fluorochromes that can be discriminated.
To address these challenges, new FISH variations have been developed that allow (i) de detection of single copy genes or (ii) the detection of multiple targets simultaneously. Thanks to the recent advances in the development of biological labeling and fluorescence image analysis (Combinatorial Labeling and Spectral Imaging, CLASI) devised by Dr. Borisy team, it is now possible to identify tens to hundreds of different microorganisms through overlapping spectra in a single microscopic image - which will provide a colour code for each characteristic. In other hand, Recognition of Individual Genes FISH (RING FISH) technique has recently used polyribonucleotide probes to allow a broader insight into the bacterial genomic information, including single-copy genes located in the bacterial chromosome or plasmids. However, no technique exists that combines these different advantages. Also, these two FISH techniques typically rely on traditional DNA probes and on long hybridization steps (which usually compromise the procedure robustness an |