Monoclonal Antibody Microarray for Microbe characterisation

Infectious diseases cause substantial suffering of patients combined with the force to complex treatments, thus they belong to the major concerns in clinical routine. Further problems arise due to the steadily increasing occurrence of multiple antibiotic resistances, which get boosted by the application of broad-range antibiotics, which seems unavoidable because of time consuming pathogen identification methods. One of the most frequently isolated pathogens is Staphylococcus aureus, which causes very serious health problems with high mortality rates. Due to its genetic variability the symptoms vary from harmless skin infections to septicemia with high mortality rate. The severity grade of an infection is influenced by antibiotic resistances of the pathogen and the prevalence of virulence factors. Among the most serious illnesses are respiratory tract infections, bloodstream infections, soft tissue- and wound infections. Bloodstream infections are even the major cause of death in intensive care units. Common methods of identification in clinical routine are based on pathogen cultivation, thus lasting at least 2 to 3 days until final pathogen characterization. DNA-based methods do not require high numbers of pathogen cells, but are disadvantageous due to a high contamination risk during DNA isolation and PCR setup. Furthermore the amplification process lasts at least 3 to 4 hours until results are available. A satisfying sensitivity of PCR based methods can only be achieved for singleplex detections. Detection of several molecular targets in parallel ideally needs the set up of several PCR reactions. Microarray technology as such is the method of choice for the highly parallel detection of up to several thousands of targets. Therefore we will combine in this project the highly parallel detection of microarray technology with the speed and specificity of monoclonal antibodies to achieve within one assay the identification of the pathogen as well as antibiotic resistance - and virulence factor characterization of S. aureus, one of the most problematic pathogens causing bloodstream infections. Several measures will be introduced to overcome the shortcomings of antibody- based detection methods. Additionally once the proof-of-principle was shown using S. aureus, further pathogens and their antibiotic resistances can be identified and typed by this method. An attractive focus would then for example be the gram negative bacteria and the large group of beta-lactam resistant strains including ESBLs, ampC and Carbapenemases.

Infectious diseases represent a major issue in clinical routine since they cause substantial suffering of patients, can be life threatening or even lethal and therefore require a fast and efficient treatment e.g. via antibiotics. A great challenge in antibiotic treatment represents the steadily increasing occurrence of multiple antibiotic resistances, which get boosted by the application of broad-range antibiotics, which seems unavoidable because of the currently used, time consuming pathogen identification methods which can last up to five days before the infection causing pathogen can be determined and therefore a pathogen-targeted antibiotic treatment can be started. Among the categories of bacteria most known to infect patients are the MRSA (methicillin resistant strains of S. aureus) which cause serious health problems. Due to its genetic variability the course of Staphylococcus infections varies from harmless skin infections to septicemia with high mortality rates. The severity grade of an infection is influenced by existing antibiotic resistances of the pathogen and the prevalence of virulence factors. As mentioned above current infectious disease diagnostic approaches are pretty slow since they require pathogen enrichment via culturing. Novel and faster detection approaches include PCR-based methods, which nevertheless also have their restrictions such as 1) they often detect only a small selection of pathogens or antibiotic resistances and no virulence factors due to a limited multiplexing capacity, 2) they require multi-step and labour-intensive laboratory procedures from sample to diagnosis and 3) as nucleic acid-based methods they can lead to a false positive diagnosis of antibiotic resistance since detecting antibiotic resistance on the genetic level does not always reflect a functional resistance on the protein level. With respect to the previously mentioned facts protein-based and highly multiplex detection methods, comprising antibiotic resistance, pathogen identification and virulence factors represent the preferred approach even though they typically suffer from low sensitivity, and the need of high amounts of sample material.Along these lines the present joined research project of the AIT- Austrian Institute of Technology and the University of Natural Resources and Life Sciences, Vienna (BOKU) aimed and succeeded in the development and the establishment of a novel antibody-based microarray for MRSA detection, which comprises along the project generated single-chain variable fragment (scFv) Fc fusion antibodies specific for the MRSA-relevant virulence factor Penton-Valentin Leucocidin F (LukF-PV) and S (LukS-PV) as well as for the antibiotic resistance protein Penicillin binding protein 2 (PBP2). The MRSA microarray-based diagnostic assay could be set up and optimized in a way that the sample preparation/Labeling procedure was kept simple and that the limit of detection for each of the above mentioned proteins was between 100-500 pg/ml sample when spiked with recombinant protein. The diagnostic assay was further successfully tested with serum samples spiked with clinical MRSA isolates.