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Master student project in the DeNeWa framework Genetic (and phenotypic) characterization of ESBL producing Escherichia coli and Klebsiella pneumoniae , Methicillin-Resistant Staphylococcus aureus and enterococci VRE antibiotic resistances in strains collected from various hospital wastewater streams Running title: Setup of ESBL, MRSA and VRE PCR Background The global increase of antibiotic resistance poses a problem for hospitals and other medical institutions. In the framework of the DeNeWa project Extended Spectrum Beta-Lactamases (ESBL)producing Enterobacteriaceae (with focus on ESBL Escherichia coli and Klebsiella pneumoniae) are collected from different wastewater streams of the hospital in Sneek. ESBLs are β-lactamases which are formed as a result of mutations and show an extended activity. They belong to different types of β-lactamases gene familiys (such as TEM, SHV, CTX-M) and are mostly plasmid-coded and thus more easily horizontally transmissible. ESBL´s hydrolyze different beta-lactam antibiotics which cause resistances to penicillins, cephalosporins and aztreonam. During the same sampling also Methicillin-Resistant Staphylococcus aureus (MRSA) are collected from the different wastewater streams. Resistance to methicillin is mediated by the production of a penicillin-binding protein (PBP2) with low binding affinity not only for methicillin, but for all licensed β-lactams. It is encoded by the mecA structural gene that is part of a larger mobile genetic element, the staphylococcal chromosomal cassette mec (SCCmec). Methicillin-susceptible S. aureus (MSSA) which lack the mecA gene, evolved into MRSA by acquisition of the gene most likely from coagulase-negative staphylococci. Additionally also Vancomycine Resistant Enterococci (VRE) are collected. Six different types of vancomycin resistance are known: Van-A, Van-B, Van-C, Van-D, Van-E and Van-F. Of these, only Van-A, B and C are known to cause outbreaks. Van-A VRE is resistant to both vancomycin and teicoplanin, Van-B VRE is resistant to vancomycin but susceptible to teicoplanin, and Van-C is only partly resistant to vancomycin and susceptible to teicoplanin. The resistance-mechanism to vancomycin in enterococcus involves the alteration to the terminal amino acid residues of the NAM/NAG-peptide subunits to which vancomycin binds. A loss of just one point of interaction results in a 1000-fold decrease in affinity Project The ESBL Escherichia coli and Klebsiella pneumoniae, MRSA and VRE strains will be typed according to their genetic profiles using different PCR methods which need to be established in our laboratory. Additionally, phenotyping for some questions should be implemented. For ESBL the determination of β-lactamase families will be carried out using PCR and subsequent sequencing. Focus will be made on the following five β-lactamase gene families: blaTEM, blaSHV, blaCTX-M(1,2,9),. For additional phenotyping the PhP - EC kit will be used. 1 Master student project in the DeNeWa framework For MRSA a mecA structural gene PCR needs to be performed. If this PCR shows positive results spa-typing and PVL-toxin gene prevalence need to be executed. VRE enterococci will be typed using gene specific (multiplex) PCR and/or 16S ribosomal DNA sequencing. Data analysis will be performed using the necessary software for sequence analysis, statistics and plotting. Reports and a thesis will be written. Supervision The project will be supervised by Sanne van den Hengel and Astrid Paulitsch–Fuchs at Wetsus. Additional supervision will be provided by Gernot Zarfel (Medical University of Graz, Austria). 2