Development and application of PNA probes for the detection of specific bacteria by FISH

The gold standard for microbial detection, in clinical settings or in biofilm studies, is the culture method, which is time-consuming, technical demanding and can give inaccurate results. These limitations have driven scientist to the development of new molecular techniques such as fluorescence in situ hybridization (FISH). This technique has been combined with peptide nucleic acid (PNA) molecules, allowing a faster and specific detection of microorganism. In this work, we attempted to evaluate this technology on the detection of relevant bacterial pathogens on food and clinical samples, and on the characterization of multi-species biofilms. As such, the first goal of this thesis was to design, optimize and test for specificity and sensitivity, three new PNA probes to detect Cronobacter spp., Salmonella spp. and Proteus spp. in different samples. Afterwards, the PNA FISH method was compared with the standard methods used to analyze each type of sample. Cronobacter spp. are clinically relevant bacteria because they can cause infections in new-born infants manly due to the consumption of contaminated powdered infant formula (PIF). A PNA probe for the rapid detection of Cronobacter species in PIF was developed and showed experimental specificity and sensitivity both of 100%. Salmonella spp. are well known enteropathogenic bacteria that cause diseases ranging from a mild gastroenteritis to septicaemia, and infection usually occurs due to the consumption of contaminated products. The Salmonella probe was applied to artificially contaminated samples (powdered infant formula and blood) and to natural samples (water and faeces) and probe testing showed specificity and sensitivity values of 100 and 97,6%, respectively. Proteus species are related with the emergence of complicated urinary tract infection (UTI) mainly for catheterized patients (catheter associated UTI’s *CAUTI’s+), which are the most common nosocomial infection. The PNA probe developed for Proteus spp. detection in urine samples showed experimental specificity and sensitivity both of 100%. Regarding the total time required to obtain the results, considering a preenrichment step before the PNA FISH application, this method allowed detection in less than 20 hours for Salmonella spp. and less than 12 hours for Cronobacter spp., even for detection levels of 1 CFU per 10 g or ml and when the target bacteria are outnumbered by other microorganisms. Comparing to the corresponding standard procedures, this represented time savings for both microorganisms of at least 3 days. For Proteus spp. detection, as an enrichment step is not needed, the PNA FISH method was able to detect in approximately 2 hours, as low as 1×104 CFU/mL (a concentration considered indicative of infection for CAUTI’s), which represents time saving of at least 24 hours. The second main goal of this work was to evaluate the PNA FISH performance, using a multiplex approach, on mixed biofilm samples. As such, the PNA FISH method was applied to the characterization of Salmonella enterica/ Listeria monocytogenes/Escherichia coli single, dual and tri-species biofilms in seven different support materials. Results showed that PNA FISH can bring important information not accessed by the other established techniques (culture and crystal violet) especially regarding the spatial distribution (when combined with confocal laser scanning microscopy) and viable but non cultivable stages. Moreover, the PNA FISH data in combination with data from other established techniques, allowed the development of a model for the tri-species biofilm. It was observed that the higher growth rate and exopolysacharide production ability of E. coli led this microorganism to outcompete the other two species resulting on two well defined layers: the top one only with E. coli, and the bottom one with mixed regions of L. monocytogenes and S. enterica. Our results indicate that PNA FISH could be a reliable alternative or complement to the currently used culture-based techniques as it is a very sensitive, specific and rapid method for the detection and location of specific microorganisms. It can be adapted with little effort to different types of samples, even using multiplex approaches, and to microorganisms with different cell wall properties. Finally, it was also demonstrated that PNA FISH is powerful tool for the characterization of multi-species biofilms and that its application can bring important information about inter-species interactions within these structures.

Name thesis:degree_name

Tese de doutoramento em Engenharia Biomédica

Year dc:date.issued

2010

Author dc:creator

• Almeida, Carina

Advisors dc:contributor.advisor

• Vieira, M. J.
• Keevil, C. W.

Statement dc:rights

• restrictedAccess

Language dc:language.iso

por

Handle dc:identifier.uri

https://hdl.handle.net/1822/11782

OAI record oai:identifier

http://repositorium.sdum.uminho.pt/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Arepositorium.uminho.pt%3A1822%2F11782