Genomic Insight of VIM-harboring IncA Plasmid from a Clinical ST69 Escherichia coli Strain in Italy

Background: VIM (Verona Integron-encoded Metallo-beta-lactamase) is a member of the Metallo-Beta-Lactamases (MBLs), and is able to hydrolyze all beta-lactams antibiotics, except for monobactams, and including carbapenems. Here we characterize a VIM-producing IncA plasmid isolated from a clinical ST69 Escherichia coli strain from an Italian Long-Term Care Facility (LTCF) inpatient. Methods: An antimicrobial susceptibility test and conjugation assay were carried out, and the transferability of the blaVIM-type gene was confirmed in the transconjugant. Whole-genome sequencing (WGS) of the strain 550 was performed using the Sequel I platform. Genome assembly was performed using "Microbial Assembly". Genomic analysis was conducted by uploading the contigs to ResFinder and PlasmidFinder databases. Results: Assembly resulted in three complete circular contigs: the chromosome (4,962,700 bp), an IncA plasmid (p550_IncA_VIM_1; 162,608 bp), harboring genes coding for aminoglycoside resistance (aac(6')-Ib4, ant(3″)-Ia, aph(3″)-Ib, aph(3')-XV, aph(6)-Id), beta-lactam resistance (blaSHV-12, blaVIM-1), macrolides resistance (mph(A)), phenicol resistance (catB2), quinolones resistance (qnrS1), sulphonamide resistance (sul1, sul2), and trimethoprim resistance (dfrA14), and an IncK/Z plasmid (p550_IncB_O_K_Z; 100,306 bp), free of antibiotic resistance genes. Conclusions: The increase in reports of IncA plasmids bearing different antimicrobial resistance genes highlights the overall important role of IncA plasmids in disseminating carbapenemase genes, with a preference for the blaVIM-1 gene in Italy

Emergence of Escherichia coli Sequence Type 131 (ST131) and ST3948 with KPC-2, KPC-3 and KPC-8 carbapenemases from a Long-Term Care and Rehabilitation Facility (LTCRF) in Northern Italy

Aim of the study was to characterize KPC-producing Escherichia coli (KPC-Ec) clinical isolates among a Northern Italy Long-Term Care and Rehabilitation Facility (LTCRF) residents. Thirteen consecutive non repeated MDR E. coli isolates showing ertapenem Minimum Inhibitory Concentrations (MICs) >0.5 mg/L, collected during the period March 2011 - May 2013 from ASP "Redaelli" inpatients, were investigated. The bla KPC/CTX-M/SHV/TEM/OXA genes were identified by PCR and sequencing. KPC-Ec isolates underwent phylotyping, Pulsed-Field Gel Electrophoresis (PFGE), multilocus sequence typing (MLST) and repetitive sequence-based PCR (rep-PCR) profiling. Incompatibility groups analysis and conjugation were also performed. Eleven out of 13 isolates, resulted bla KPC-type positive, were consistently resistant to third generation cephalosporins, fluoroquinolones and trimethoprim-sulphametoxazole (84.6 %), retaining susceptibility to colistin (EUCAST guidelines). At least n = 4/11 of KPC-Ec patients received ≥48 h of meropenem therapy. Sequencing identified 9 bla KPC-2, 1 bla KPC-3 and 1 bla KPC-8 determinants. KPC-Ec plasmids belonged to IncF group (FIIk replicon); conjugation confirmed bla KPC/TEM-1/OXA-9 genes transferability for 10 KPC-Ec. Although three pulsotypes (A, B, C) were identified, all KPC-Ec belonged to phylogenetic group B2. Clone B (B-B5) caused an outbreak of infection involving nine inpatients at five wards. Rep-PCR showed relatedness for seven representative KPC-Ec isolates. Here we report a LTCRF outbreak caused by a ST131-B2 E. coli associated with bla KPC-2 and bla KPC-8 genes, and the emergence of the new ST3948. Elderly people with co-morbidities are at risk for ST131 colonization. KPC-Ec clones local monitoring appears essential both to avoid their spreading among healthcare settings, and to improve therapeutic choices for LTCRF residents

Genomic Characterization of VIM and MCR Co-Producers: The First Two Clinical Cases, in Italy

Background: the co-production of carbapenemases and mcr-genes represents a worrisome event in the treatment of Enterobacteriaceae infections. The aim of the study was to characterize the genomic features of two clinical Enterobacter cloacae complex (ECC) isolates, co-producing VIM and MCR enzymes, in Italy. Methods: species identification and antibiotic susceptibility profiling were performed using MALDI-TOF and broth microdilution methods, respectively. Transferability of the bla(VIM-) and mcr- type genes was verified through conjugation experiment. Extracted DNA was sequenced using long reads sequencing technology on the Sequel I platform (PacBio). Results: the first isolate showed clinical resistance against ertapenem yet was colistin susceptible (EUCAST 2020 breakpoints). The mcr-9.2 gene was harbored on a conjugative IncHI2 plasmid, while the bla(VIM-1) determinant was harbored on a conjugative IncN plasmid. The second isolate, resistant to both carbapenems and colistin, harbored: mcr-9 gene and its two component regulatory genes for increased expression on the chromosome, mcr-4.3 on non-conjugative (yet co-transferable) ColE plasmid, and bla(VIM-1) on a non-conjugative IncA plasmid. Conclusions: to our knowledge, this is the first report of co-production of VIM and MCR in ECC isolates in Italy

Emergence of KPC-type producing Escherichia coli ST131 from three acute care hospitals and two long term and rehabilitation facilities in northern Italy

Background: Although carbapenem resistant Escherichia coli remains uncommon in Europe, Italy is one of the four countries showing the highest resistance percentages, with 2010-2013 increasing trends reported. The aim of the study was to investigate the population structure and beta-lactamases (BLs) content of nine KPC-2 and three KPC-3 E. coli strains from five hospitals in Northern Italy. Material/Methods: In the period April 2011-May 2013, 12 MDR E. coli isolates obtained from five Northern Italy Facilities were studied using PFGE, DiversiLab automated rep-PCR system, MLST and Phylogenetic group analysis. Conjugation and replicon typing were also performed. Carbapenem MIC values were evaluated by Etest. Other BLs presence was assessed by blaCTX-M/SHV/TEM/OXA genes amplification and sequencing. Results: Out of the 12 strains, six were from rehabilitation wards, four from intensive care units (ICUs), and two from nephrology and neurosurgery wards, respectively. Nine KPC-E. coli were from urine, two from sputum samples and one from blood. Two isolates retained susceptibility to ertapenem, eight to meropenem and nine to imipenem (EUCAST 2015 breakpoints). blaCTX-M-group 1 (blaCTX-M-15) and blaCTX-M-group 9 genes (blaCTX-M-27) were detected in 4/12 strains only, while blaTEM-1 and blaOXA-9 determinants were detected in 9/12 and 10/12 isolates respectively. All but one strain resulted of phylogenetic group B2. Eight different PFGE profiles (A-H) and four rep-PCR profiles were distinguishable, while five MLSTs (ST131, ST3948, ST3426, ST5839, ST3861) were detected. All KPC-2 strains, detected in each hospital, belonged to ST131 Complex (Cplx), being 8/9 of ST131 and 1/9 of ST3948, while five different pulsotypes were distinguishable. The three KPC-3 E. coli showed unique PFGE fingerprints and belonged to the here newly identified ST5839 and ST3681, in addition to the already reported ST3426, occurring in 2012 in UK. IncF, IncFII, groups were detected in all but one cases; the ST3426 KPC-3 strain showed, in addition to IncF, IncU and IncX1 plasmids. Carbapenem resistance gene transfer was obtained in three cases; for two of the above cases, the acquisition of the conjugative plasmid from a Klebsiella pneumoniae previously responsible for a patient’s infection, was ascertained. The PFGE clone F, that caused an intra-hospital outbreak during the period June 2012-May 2013 at a Long Term and Rehabilitation Facility (LTCF), showed the highest inter-hospital spreading potential, being identified also in an ICU of an Acute Care Hospital (ACH), over the same period. Conclusions: Here we report the spread of several clones of KPC-positive E: coli isolates both from ACHs and LTCFs in Northern Italy. While KPC-2 E. coli resulted linked to the ST131 Cplx, the KPC-3 strains showed a multi-clonal dissemination. The low meropenem and/or imipenem MIC values contributed to the inter-hospital dissemination and an underestimation of the real presence of such pathogen in these settings

Whole Genome Sequencing and Molecular Analysis of Carbapenemase-Producing Escherichia coli from Intestinal Carriage in Elderly Inpatients

The spread of carbapenemase-producing (CP) Enterobacterales is currently a worldwide concern, especially in the elderly. Twelve CP-E. coli isolated from rectal swabs of colonized inpatients aged ≥65 years from four hospitals in two Italian cities (Milan and Rome) were analyzed by whole genome sequencing (WGS) to obtain multi-locus sequence typing (MLST), identification of carbapenemase-encoding genes, resistome, plasmid content, and virulence genes. MLST analysis showed the presence of 10 unrelated lineages: ST410 (three isolates from three different hospitals in two cities) and ST12, ST38, ST69, ST95, ST131, ST189, ST648, ST1288, and ST1598 (one isolate each). Most isolates (9/12, 75%) contained a serine-β-lactamase gene (5 blaKPC-3, 2 blaKPC-2, and 2 blaOXA-181), while three isolates harbored a metallo-β-lactamase gene (two blaNDM-5 and one blaVIM-1). In most CP-E. coli, the presence of more than one plasmid was observed, with the predominance of IncF. Several virulence genes were detected. All isolates contained genes enhancing the bacterial fitness, such as gad and terC, and all isolates but one, fimH, encoding type 1 fimbriae. In conclusion, CP-E. coli clones colonizing elderly patients showed heterogeneous genetic backgrounds. We recommend strict surveillance to monitor and prevent the spread of successful, high-risk clones in healthcare settings

Colonization of long-term care facility residents in three Italian Provinces by multidrug-resistant bacteria

Abstract Background Rationale and aims of the study were to compare colonization frequencies with MDR bacteria isolated from LTCF residents in three different Northern Italian regions, to investigate risk factors for colonization and the genotypic characteristics of isolates. The screening included Enterobacteriaceae expressing extended-spectrum β-lactamases (ESβLs) and high-level AmpC cephalosporinases, carbapenemase-producing Enterobacteriaceae, Pseudomonas aeruginosa or Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Methods Urine samples and rectal, inguinal, oropharyngeal and nasal swabs were plated on selective agar; resistance genes were sought by PCR and sequencing. Demographic and clinical data were collected. Results Among the LTCF residents, 75.0% (78/104), 69.4% (84/121) and 66.1% (76/115) were colonized with at least one of the target organisms in LTCFs located in Milan, Piacenza and Bolzano, respectively. ESβL producers (60.5, 66.1 and 53.0%) were highly predominant, mainly belonging to Escherichia coli expressing CTX-M group-1 enzymes. Carbapenemase-producing enterobacteria were found in 7.6, 0.0 and 1.6% of residents; carbapemenase-producing P. aeruginosa and A. baumannii were also detected. Colonization by MRSA (24.0, 5.7 and 14.8%) and VRE (20.2, 0.8 and 0.8%) was highly variable. Several risk factors for colonization by ESβL-producing Enterobacteriaceae and MRSA were found and compared among LTCFs in the three Provinces. Colonization differences among the enrolled LTCFs can be partially explained by variation in risk factors, resident populations and staff/resident ratios, applied hygiene measures and especially the local antibiotic resistance epidemiology. Conclusions The widespread diffusion of MDR bacteria in LTCFs within three Italian Provinces confirms that LTCFs are an important reservoir of MDR organisms in Italy and suggests that future efforts should focus on MDR screening, improved implementation of infection control strategies and antibiotic stewardship programs targeting the complex aspects of LTCFs