Multidrug-resistant (MDR) and Extensively Drug-resistant (XDR) Gram-negative bacteria of clinical interest: laboratory diagnosis, clinical management and infection control

Antimicrobial resistance (AMR) represents one of the major 21st century public health tasks: its increasing has reduced the effective prevention and treatment strategies against infection events. The problem of AMR is especially urgent for Gram-negative bacteria, on which an over-/inappropriate use (incorrect choices, inadequate dosing, and poor adherence to treatment guidelines) of antibiotics have acted as a selective pressure, favouring the survival and multiplication of strains with acquired antibiotic-resistant traits. The aim of my PhD research were: i) to perform an evaluation and characterization of the antibiotic-resistance determinants circulating in Gram-negative bacteria collected from both human and veterinary settings; ii) to obtain, through NGS analysis, a complete picture of the structure of replicons and plasmids harbouring such resistance genes arrays, and iii) to get the own genetic characteristics of the most spread MDR successful clones/plasmids collected. The first project provides the characterization of two carbapenemase-producing Citrobacter freundii strains from two Italian patients without any apparent epidemiological link. The isolates were susceptible only to gentamycin, colistin, tigecycline and trimethoprim-sulfamethoxazole. Illumina sequencing revealed the presence of two different KPC variants: blaKPC-2 gene in a novel variant of Tn4401 trasposon, inserted in an IncN plasmid; blaKPC-3 gene inserted in an IncX3-IncA self-conjugative plasmid fusion. The two isolates resulted clonally unrelated belonging to different sequence types (ST46 and ST19). The second project was on two carbapenem-resistant E. coli strains collected in Italy from a three years-old outpatient affected by diarrhea, after a travel in India. The strains resulted clonally unreleated and showed an MDR profile, retaining susceptibility only to colistin, tigecycline and fosfomycin. WGS analysis identified an ST2659 and an ST405 E. coli. One strain contained a 130,562 bp multi-replicon plasmid IncFII/IncFIA/IncFIB (pVSI_NDM-5) coding blaOXA-, blaNDM-5, blaTEM-1B, blaCTX-M-15, aac(6’)-lb-cr, aadA5, rmtB, sul1, dfrA17, qacEΔ1 and catB3. The other isolate carried four plasmids: a 153,866 bp multi-replicon plasmid IncFII/IncFIA/IncFIB (pISV_IncFII_NDM-5), a 41143 bp IncI plasmid (pISV_IncI_CMY-42), a 51,480 bp IncX3 plasmid (pISV_IncX3_OXA-181) and an 89,866 bp IncI1 plasmid. pISV_IncFII_NDM-5 carried genes coding blaOXA-1, blaNDM-5, blaTEM-1B, aac(6’)-lb-cr, aadA5, tet(B), catB3 and catA1. The third project describes a deadly infection events occurred in a bulldog breeding located in Central Italy. A total of five E. coli strains were collected from biopsy specimens of a two-weeks old dog, belonged to an eight-puppy litter. The entire litter contracted haemorrhagic enteritis with renal involvement and cerebral vessels congestion. All the strains were clonally releated, belonging to ST58. WGS analysis, performed on a representative strain, revealed the presence of: i) an IncHI2α replicon plasmid (pMBC_CTX-M-15, 302,597 bp) harboured dfrA14, catA1, sul2, aac(6’)-Ib-cr, qnrB1, tet(A), blaOXA-1, blaTEM-1b, blaCTX-M-15, aac(3)-Iia and aph(6)-Id resistance genes; ii) an IncI1 replicon plasmid (pMBC_CMY-2, 83,077 bp) harboured the blaCMY-2 determinat only; iii) an IncFII replicon plasmid (pMBC_DHA-1, 83,429 bp) harboured sul1, qnrB4 and blaDHA-1 resistance genes. The source of contamination and infection still remains unknown. The results show that a worldwide spread of antibiotic-resistance genes is occurring in different Gram-negative microorganisms, through MDR hyper-epidemic plasmids or successful clone’s dissemination. The molecular characterization of the antibiotic-resistance genes represents an essential support to explain the epidemiology, modes and high speed rate ability to spread of certain bacterial clones or their resistance determinants.Antimicrobial resistance (AMR) represents one of the major 21st century public health tasks: its increasing has reduced the effective prevention and treatment strategies against infection events. The problem of AMR is especially urgent for Gram-negative bacteria, on which an over-/inappropriate use (incorrect choices, inadequate dosing, and poor adherence to treatment guidelines) of antibiotics have acted as a selective pressure, favouring the survival and multiplication of strains with acquired antibiotic-resistant traits. The aim of my PhD research were: i) to perform an evaluation and characterization of the antibiotic-resistance determinants circulating in Gram-negative bacteria collected from both human and veterinary settings; ii) to obtain, through NGS analysis, a complete picture of the structure of replicons and plasmids harbouring such resistance genes arrays, and iii) to get the own genetic characteristics of the most spread MDR successful clones/plasmids collected. The first project provides the characterization of two carbapenemase-producing Citrobacter freundii strains from two Italian patients without any apparent epidemiological link. The isolates were susceptible only to gentamycin, colistin, tigecycline and trimethoprim-sulfamethoxazole. Illumina sequencing revealed the presence of two different KPC variants: blaKPC-2 gene in a novel variant of Tn4401 trasposon, inserted in an IncN plasmid; blaKPC-3 gene inserted in an IncX3-IncA self-conjugative plasmid fusion. The two isolates resulted clonally unrelated belonging to different sequence types (ST46 and ST19). The second project was on two carbapenem-resistant E. coli strains collected in Italy from a three years-old outpatient affected by diarrhea, after a travel in India. The strains resulted clonally unreleated and showed an MDR profile, retaining susceptibility only to colistin, tigecycline and fosfomycin. WGS analysis identified an ST2659 and an ST405 E. coli. One strain contained a 130,562 bp multi-replicon plasmid IncFII/IncFIA/IncFIB (pVSI_NDM-5) coding blaOXA-, blaNDM-5, blaTEM-1B, blaCTX-M-15, aac(6’)-lb-cr, aadA5, rmtB, sul1, dfrA17, qacEΔ1 and catB3. The other isolate carried four plasmids: a 153,866 bp multi-replicon plasmid IncFII/IncFIA/IncFIB (pISV_IncFII_NDM-5), a 41143 bp IncI plasmid (pISV_IncI_CMY-42), a 51,480 bp IncX3 plasmid (pISV_IncX3_OXA-181) and an 89,866 bp IncI1 plasmid. pISV_IncFII_NDM-5 carried genes coding blaOXA-1, blaNDM-5, blaTEM-1B, aac(6’)-lb-cr, aadA5, tet(B), catB3 and catA1. The third project describes a deadly infection events occurred in a bulldog breeding located in Central Italy. A total of five E. coli strains were collected from biopsy specimens of a two-weeks old dog, belonged to an eight-puppy litter. The entire litter contracted haemorrhagic enteritis with renal involvement and cerebral vessels congestion. All the strains were clonally releated, belonging to ST58. WGS analysis, performed on a representative strain, revealed the presence of: i) an IncHI2α replicon plasmid (pMBC_CTX-M-15, 302,597 bp) harboured dfrA14, catA1, sul2, aac(6’)-Ib-cr, qnrB1, tet(A), blaOXA-1, blaTEM-1b, blaCTX-M-15, aac(3)-Iia and aph(6)-Id resistance genes; ii) an IncI1 replicon plasmid (pMBC_CMY-2, 83,077 bp) harboured the blaCMY-2 determinat only; iii) an IncFII replicon plasmid (pMBC_DHA-1, 83,429 bp) harboured sul1, qnrB4 and blaDHA-1 resistance genes. The source of contamination and infection still remains unknown. The results show that a worldwide spread of antibiotic-resistance genes is occurring in different Gram-negative microorganisms, through MDR hyper-epidemic plasmids or successful clone’s dissemination. The molecular characterization of the antibiotic-resistance genes represents an essential support to explain the epidemiology, modes and high speed rate ability to spread of certain bacterial clones or their resistance determinants