Antimicrobial susceptibilities, drug resistance gene arrays and epidemic potential of Enterobacteriaceae and other Gram negative clinical isolates: novel diagnostic and therapeutic strategies

Antibiotic resistance can be horizontally acquired through transfer of antibiotic resistance plasmids or determinants between bacteria, or can be due to a mutational event in a target site in response of antibiotics excessive use/misuse for treatment of infections in human and veterinary medicine. Consequently the proliferation of Extended Spectrum β-lactamases (ESBLs)- among Gram negative bacteria, mediated through promiscuous conjugative plasmids, transposons and integrons increased the volume of carbapenems into clinical medicine worldwide. Within this decade, four molecular classes of carbapenemases have emerged throughout the world: Chromosomal encoded Class A carbapenemases (IMI, NMC-A and SME); Non chromosomal encoded Class A such as KPC in Enterobacteriaceae; Class B enzymes MBLs with the most prevalent IMP-and VIM- and NDM-type; Class D acquired, as OXA-23, OXA-58 and OXA-48-like. The thesis work was on: 1.Characterizing ESBLs and Class B carbapenemases: i)CASE STUDY: A 62-year-old patient was transferred to the cardiac rehabilitation unit of the I.R.C.C.S. Fondazione S. Maugeri after undergoing heart transplantation. On 1 August 2013 and during hospitalization in the rehabilitation unit, Klebsiella oxytoca and Citrobacter koseri clinical isolates were simultaneously recovered from the patient’s preputial swab. Molecular characterization confirmed the presence of blaCMY-4 and blaVIM-4 determinants in a 150 Kb transferable plasmid of IncA/C group. This case is the first detection in Italy of the K. oxytoca and C. koseri clinical isolates co-producing the CMY-4 and VIM-4 enzymes. ii) ESBL OUTBREAK by Klebsiella pneumoniae at a Neonatal Intensive Care Unit (NICU).We report an outbreak of an extended spectrum beta-lactamase Klebsiella pneumoniae (ESBL-Kp) clones in a NICU in northern Italy. Colonization of patients was assessed by cultures of rectal swabs sampled once a week. ESBL production was investigated by phenotypic and molecular tests. Plasmid characterization and whole-genome sequencing were performed. Molecular typing was carried out by PFGE and MLST. 2.Characterizing Class A carbapenemases: i)CASE STUDY: Aim of the study was to characterize KPC-producing Escherichia coli clinical isolates among a Northern Italy Long-Term Care and Rehabilitation Facility (LTCRF) residents. Here we report a LTCRF outbreak caused by an ST131-B2 E. coli associated with blaKPC-2 and blaKPC-8 genes, and the emergence of the new ST3948. 3.Characterizing Class B carbapenemases ST405 NDM-5-positive Escherichia coli strains were isolated from two inpatients at an Italian Hospital in 2014-2015. Bacterial typing was performed and the entire plasmid content was fully sequenced. An IncFII plasmid harboring blaNDM-5, rmtB, blaTEM-1b, dfrA12, aadA2, mphA, sul1 genes and an IncI1 plasmid, harboring the blaCMY-42 gene were co-resident within the ST405 strains. The emergence of an hidden ST405 local reservoir cannot be excluded, even if foreign origin of the MDR ST405 clone was hypothesized. 4.Characterizing Class D carbapenemases. The aim was to assess by NGS the Antimicrobial-resistance evolution in a collection of A. baumannii strains belonging to ST78 lineage and responsible for outbreaks in Italy since 2002. The genomic evolution of these strains in 10 years period and the detection of their genomic characterization, also associated with Italian ST78 clone, were also investigated. Our results suggest that isolates belonging to ST78 acquired novel antibiotic resistance genes. To our knowledge, this is the first study on the genomic evolution in an epidemic A. baumannii genotype (i.e. ST78) beside the three main ICs.