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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
The global increase of antibiotic resistance poses a problem for hospitals and other medical
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
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.
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
Data analysis will be performed using the necessary software for sequence analysis, statistics and
plotting. Reports and a thesis will be written.
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).