Emergence of resistance mechanisms to last-resource antibiotics in multi-drug resistant (MDR) pathogens

Nowadays, the antibiotic treatment of infections caused by multi-drug resistant (MDR) pathogens is a cardinal topic. The MDR bacterial isolates harbour multiple antibiotic resistant genes conferring resistance to antibiotics of different classes, leaving a few options for therapy. In the case of MDR Gram-positive bacteria (methicillin-resistant staphylococci, vancomycin-resistant enterococci) linezolid is a last-resource antibiotic. Linezolid inhibits bacterial protein synthesis by binding to the domain V region of 23S rRNA and the effect of binding to 50S is inhibition of 70S formation. Linezolid resistance is low, and most frequently mediated by mutations at different positions at the binding site of the antibiotic. In the case of Gram-negative pathogens, the carbapenems became frequently administered due to increased frequency of bacterial strains producing extended-spectrum beta-lactamases (ESBLs). Currently, resistance to carbepenems is a global health-care problem. In Enterobacteriaceae strains, variety of carbapenemases hydrolysing beta-lactam antibiotic confer resistance to carbapenems most frequently. The goal of the thesis was to characterize the molecular genetic background of Gram-positive, linezolid-resistant staphylococci and to define antibiotic resistant genes and plasmid profile of Gram-negative, carbapenem-resistant Enterobacteriaceae strains. In the Gram-positive MDR project, 11 linezolid-resistant Staphylococcus haemolyticus from the University Hospital of Verona and 14 coagulase-negative staphylococci (CoNS) (10 S. epidermidis, 2 S. capitis and 2 S. hominis) from Vicenza Hospital were investigated. Investigating linezolid-resistant CoNS from Verona and Vicenza, point mutations in domain V of the 23S rRNA gene were revealed. The linezolid-resistant S. haemolyticus strains from Verona uniformly harboured alteration at position G2576T, while the collection of CoNS strains from Vicenza carried either G2576T or G2447T mutations. The alteration at G2447T was associated with S. epidermidis, while the classical G2576T mutation was found to be related to CoNS other than S. epidermidis, namely S. capitis and S. hominis. The phenotypic effect of these mutations was increased linezolid MICs of 16 – 32 mg/L, a high level of resistance that, at least for mutation G2576T, was explained by the documented nucleotide change in every copy of the 23S rRNA. In the staphylococci collection from Vicenza, to the best of our knowledge, this is the first report in which 2-2 different CoNS (S. epidermidis - S. capitis and S. epidermidis - S. hominis) species with different mutations in the 23S rRNA gene were isolated from the same patients. In the Gram-negative MDR study, 10 carbapenem-resistant Enterobacteriaceae strains (8 Klebsiella pneumoniae, 1 Escherichia coli and 1 Enterobacter aerogenes) from University Hospital of Verona, Negrar Hospital and Treviso Hospital were included. From Croatia 5 carbapenem-resistant Enterobacteriaceae strains (4 K. pneumoniae and 1 Citrobacter koseri) were included. Among the 15 carbapenem-resistant Enterobacteriaceae strains, 9 strains carried blaKPC-3, 5 strains were positive for blaNDM-1 and 1 K. pneumoniae harboured blaOXA-48 carbapenemase gene. The clinical isolates hosted different plasmid profiles and several additional antibiotic resistant determinants (beta-lactamases and plasmid-mediated quionolone resistant determinants, namely, CTX-M-15, TEM-1, SHV-1, SHV-11, SHV-12, OXA-1, CMY-4, AAC(6’)-Ib-cr and QnrB1). These carbapenemase genes were localised on conjugative plasmids, namely, blaKPC-3 on IncFIIk, blaNDM-1 on IncR and on A/C and blaOXA-48 on L/M. The KPC-3 positive IncFIIk plasmid co-expressed of TEM-1 and OXA-9 enzymes. Beside blaNDM-1, the IncR plasmid co-carried the genes of TEM-1, OXA-1, CTX-M-15, SHV-12, AAC(6’)-Ib-cr, while A/C plasmid co-harboured genes of SHV-1, CMY-4 and AAC(6’)-Ib-cr. Although the K. pneumoniae clinical isolate expressing OXA-48 produced many associated antibiotic resistant determinants, the L/M plasmid responsible for the carbapenemase carried only blaOXA-48. Multilocus sequence typing (MLST) revealed that, among the KPC-3 expressing strains, 2 K. pneumoniae belonged to the widespread ST258 clone and 5 strains to ST512 and E. coli was ST43. In the NDM-1 positive collection, 3 K. pneumoniae strains were member of ST15 and one K. pneumoniae was ST16. The OXA-48 producing K. pneumoniae belonged to ST101, international clone. Noteworthy, that the transmission of blaKPC-3 gene between K. pneumoniae strain ST512 and E. coli ST43 in a single patient was confirmed. The epidemiological relevance of our findings is that beside the ST258 international clone, the ST512 clone is also spreading among K. pneumoniae strains. Among NDM-1 producing Enterobacteriaceae, the detection of IncR plasmid is of particular relevance, to the best of our knowledge so far not reported in the literature. These results confirmed that Enterobacteriaceae strains can acquire different antibiotic resistance determinants, thus resulting in multi-drug resistant phenotypes leaving few therapeutic options. The phenotypic antimicrobial susceptibility testing should be confirmed by molecular genetic analysis to follow the spread of the different carbapenemase genes and the responsible plasmid types. MLST is recommended for epidemiological purposes, so as to determine the bacterial clones and their spreading even between different countries.