Development and evaluation of peptide nucleic acid (PNA) probes for the rapid detection of clinically relevant microorganisms

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Development and evaluation of peptide nucleic acid (PNA) probes for the rapid detection of clinically relevant microorganisms


Cerqueira, L.



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Microbial infections are a major cause of hospitalization leading to high morbidity and mortality. An earlier diagnosis can ensure a more rapid treatment, preventing a worse-case scenario. However, ineffective treatments due to inaccurate microorganism identification can lead to hospitalization extension, undesirable side effects, antimicrobial resistance and increased economic costs. Currently, culturing and serologic methods are the methods of choice in clinical diagnosis, but they lack specificity and are time-consuming. PCR based techniques are frequently used, but they are technically-demanding and need DNA manipulation, being prone to contaminations. Therefore, the development of new molecular techniques more specific, reliable and simpler to use is of great concern. Peptide nucleic acid oligonucleotides can be used with fluorescence in situ hybridization as an alternative molecular method to specifically target the rRNA of microorganisms. In the first part of this thesis, it was intended to design and optimize specific probes for the detection of clinical relevant microorganisms, such as, Helicobacter pylori and its clarithromycin resistance and Aspergillus fumigatus detection. With the application of this technique on different cellular morphologies and in different microbial domains it can be definitely proved the capacity of this method to be used as diagnostic method. H. pylori can colonize the human stomach and cause severe gastric diseases. The standard triple therapy where clarithromycin is the main antibiotic used, is given indiscriminately to all symptomatic patients, leading to antimicrobial resistance. In this study three probes were designed, targeting the three most prevalent clarithromycin resistance point mutations (Hp1, Hp2 and Hp3) in the positions A2143G, A2142G and A2142C of the peptidyltransferase region encoded in domain V of the H. pylori 23S rRNA gene. An additional probe targeting susceptible strains (Hpwt) was also developed and tested simultaneously in H. pylori smears with known clarithromycin resistance profile (specificity and sensitivity of 100%). For method validation in gastric biopsies, a retrospective and prospective cohort studies were made where validity indexes such as sensitivity, specificity and likelihood ratios were assessed to test PNAFISH accuracy. In the retrospective assay, full agreement between PNA-FISH and PCR was achieved, but comparing to the reference method based on culture techniques, the specificity was of 90.9% and sensitivity was of 84.2%. In the prospective cohort study comparing to reference method, PNA-FISH demonstrated that can always detect H. pylori resistant and susceptible genotypes (sensitivity and specificity are 100%). Discrepancies detected between positive PNA-FISH results and negative in the reference method can be explained by some culturing method shortcomings, such as difficulty on H. pylori growth in certain media, contaminations and the viable but non-cultivable state of the bacteria (VBNC). Aspergillus fumigatus is a filamentous fungus that produces conidia. When inhaled, conidia can adhere to epithelial lung cells, leading to pulmonary diseases such as Invasive Aspergillosis. The designed probe could discriminate A. fumigatus from other species, presenting practical specificity and sensitivity of 100%. A germination assay was done to assess probe performance for different cells morphology along mold germination. At 0h and 2h the signal was faint due to lack of ribosome content and cell wall structure. From 4h onwards swelling conidia can be observed, and germ tubes start to appear at 6h. Partial and full germination were detected at 8h and 12h, respectively. This germination process was followed by a signal intensity increase that was not uniform throughout the cell, as more intense fluorescence dots near the nucleus appeared. These dots may correspond to increased ribosomal content. Different concentrations of this fungus were applied on artificially contaminated samples for A. fumigatus detection testing. In blood it was possible to detect this mold as early as 6h at concentrations ranging 1x103 - 1x104 cells ml-1. In artificial sputum media it was only possible to detect A. fumigatus from 16h to 24h at concentrations of 1x102 cells ml-1. In addition, and as a final aim of this work, it was used a commercially available PNA-FISH kit to study Escherichia coli and Pseudomonas aeruginosa biofilm interactions simulating urinary catheters conditions, to demonstrate the potential uses of PNA-FISH. These two bacteria are some of the main responsible for catheters associated urinary tract infections, mainly due to catheter biofilm formation. DAPI staining and CFUs counts in silicon coupons embedded in artificial urine, were used in addition to E. coli/P. aeruginosa PNA-FISH® probes to have an overlook of the behavior in terms of biofilm formation capacity of these two bacteria in single and mixed biofilms. In single-species biofilms it was possible to observe that E. coli appears to form more easily biofilm than P. aeruginosa. From all the results for mixed-biofilms it was possible to infer that some sort of interaction between these two species is happening although P. aeruginosa seems to benefit the most (Log 7 CFU of P. aeruginosa/cm2, comparing to Log 6 CFU/cm2 obtained in pure cultures) comparing to E. coli that decreased cell concentration in presence of the other counterpart. The PNA-FISH combined with CLSM could indeed confirm P. aeruginosa outcompeting E. coli in dual-species biofilms. This probably happens due to P. aeruginosa secreting substances that can be toxic to cultivable E. coli cells, preventing them to proliferate. In fact, although this is the main causative agent of urinary infections for its constant presence in infection places and for its capacity of forming singlespecies biofilm, in the presence of other bacteria seems to lose that capacity. The whole results from this thesis indicate that PNA-FISH is a reliable technique for the specific identification of microorganisms or its genotypic characteristics and it can be used as a method of diagnosis ready to use, in clinical practice or as a mean for studying important microorganism’s mechanisms in research laboratories. It can also be used to discriminate populations in biofilms, as the uncharged backbone of the PNA molecule allows it to diffuse more freely through the biofilm matrix providing a more comprehensive view of the biofilm structure. This feature may allow the more effective development of new eradication therapies.