Caratterizzazione molecolare del genoma di patogeni Gram-negativi: identificazione e analisi biochimica di una nuova β-lattamasi di classe A, l’enzima KPC-53

Abstract The β-lactamases are the most common mechanism of resistance to β-lactam antibiotics in Gramnegative bacteria. Two major families, extended-spectrum-β-lactamases (ESBLs) and metallo-βlactamases, represent a serious risk for human health. β-lactam compounds are bactericidal agents that inhibit cell wall synthesis. Moreover, bacteria escape to their action by three different ways: 1) altered PBPs that exhibit low affinity for β-lactam antibiotics; 2) lack or diminished expression of outer membrane proteins; 3) production of β-lactamases which is the most common mechanism of bacterial resistance to β-lactams. β-lactamases are bacterial enzymes able to hydrolyse the β-lactam ring making the antibiotic inactive before it reaches the PBP target. These enzymes have been categorized into four classes based on their sequence homology (Ambler classes A, B, C and D). The enzymes belonging to classes A, C and D possess in their catalytic site an active residue of serine while class B enzymes (MBLs) require zinc ions for their activity. The production of ESBLs is one of the most important responses of the bacteria to the excessive use of oxyimino-cephalosporins for the treatment of serious infection. MBLs confer resistance to a wide range of β-lactams, including carbapenems. The genes encoding serin- and metallo β-lactamases can be located on the bacterial chromosome, on plasmids, on transposons, or on integrons. The presence of resistance genes on Mobile Genetic Elements allows the spread of these genes among bacterial species by conjugation, transduction, or transformation. The genetic environment of the β-lactamase (bla) genes dictate whether the β-lactamases are produced in a constitutive or inducible manner. Transposable elements were identified as major contributors to bacterial genomic fluidity. Plasmids harbor antibiotic resistance genes to most classes of antibiotics currently used in clinical practice including fluoroquinolones and aminoglycosides. Various antibiotic treatments, as well as a total dependence on nurse care of Long Term Care Facilities (LTCFs) residents or hospitalized patients, expose them both to the selection and horizontal transmission of antibiotic resistant organisms. As a consequence, these facilities represent important reservoirs of antibiotic resistant strains, mainly ESBL- and carbapenemase-producing Enterobacterales. In our study we decided to investigate the dissemination of antibiotic resistant genes and virulence factors in Gram-negative bacterial pathogens by Next Generation Sequencing (NGS) technology. In detail, this thesis draws attention on draft genome analysis of carbapenem resistant Klebsiella pneumoniae and Escherichia coli strains isolated from rectal swabs of residents in LTCFs located in Northern Italian Region. In addition, molecular analysis of draft genome analysis of Acinetobacter baumannii isolated from COVID-19 patients affected by sepsis admitted to the intensive care unit (ICU) of Spirito Santo Hospital in a Central Italian Region, was also performed. The massive sequencing process of the whole genome performed on all these clinical strains has given the possibility to identify new β-lactamase variants: KPC-53. KPC-type enzymes are class A serine β-lactamases which have powerful carbapenemase activity. These enzymes are usually encoded by transferable plasmids. KPC β-lactamases are enzymes in continuous evolution: the new enzyme KPC-53 was identified in a K. pneumoniae resistant to ceftazidime-avibactam combination. The new enzyme showed a duplication of L167E168 residues in the Ω-loop. New mutations in the catalytic pocket of these enzymes, have changed the antibiotic profile hydrolysis of KPC natural variants.