Elisabetta Nucleo’s research while affiliated with University of Pavia and other places

Antimicrobial consumption in veterinary medicine has led to the spread of multi drug-resistance in clinically important bacteria, with the companion animals and their environment involved as emerging reservoirs. While CTX-M-15 and CMY-2 acquired β-lactamases have been widely detected in the bacterial population of companion and breeding animals in European area, DHA-1 enzymes have been rarely reported in veterinary medicine. The aim of the study was to characterize the Escherichia coli associated with mortality of a litter of Bulldog puppies in a breeding kennel located in Pesaro area, Central Italy. The E. coli strains O39 serotype were resistant to 3rd/4th generation cephalosporins, chloramphenicol, aminoglycosides, trimethoprim-sulfamethoxazole, and ciprofloxacin, retaining susceptibility to carbapenems, colistin, fosfomycin, and levofloxacin (by Microscan Autoscan4, EUCAST clinical breakpoints). Pulse field gel electrophoreses (PFGE-XbaI) on five E. coli strains revealed the presence of a single profile. Whole genome sequencing (WGS) analysis revealed a complex resistome, harboring blaTEM–1b, blaCTX–M–15, blaOXA–1, aph(6)-Ib, aac(6′)Ib-cr, aac(3)-Ila, aph(6)-Id, aadA1, qnrB1, sul2, catA1, catB3, tetA, and dfrA14 genes located on a 302597 bp IncHI2/HI2A plasmid. Moreover, blaDHA–1, qnrB4, mph(A), sul1, and dfrA17 determinants were carried on an 83,429 bp IncFII plasmid. A blaCMY–2 determinant was carried on a 90,249 bp IncI1 plasmid. Two IncX1 and IncX4 plasmids without antimicrobial resistance genes were also detected. The presence of lpfA, iss, astA, and gad virulence factors was highlighted. This is the first report in Italy on an invasive infection in eight 2-weeks old dogs caused by the same MDR E. coli O39 blaCTX–M–15, blaCMY–2, blaDHA–1, and aac(6′)-Ib-cr positive strain. The above MDR E. coli clone caused the death of the entire litter, despite amoxicillin-clavulanate and enrofloxacin administration. The tank for storage of the water used to prepare the milk-based meal for the litter was the suspected reservoir.

Klebsiella pneumoniae (KP) is an important pathogen involved in serious nosocomial infections all over the world. Here, we describe the first report on a blood-stream infection caused by an OXA- 48/NDM-1 ST101-KP, in Italy. The patient was an Italian woman, transferred from Cairo Hospital to a Neurosurgery ward in Cuneo (IT). The detection described here enhances the need for an effective National infection control strategy in Italy.

Background: The spread of carbapenemase genes, such as blaNDM-1, in Proteus mirabilis poses a public health threat. The aim of the study was to characterize the genome and plasmids sequences of an NDM-1-positive strain (IBCRE14), which was isolated in 2019 from a catheterized patient hospitalized in Italy. Methods: Whole genome sequencing (WGS) of IBCRE14 was performed on extracted genomic DNA using Sequel I platform. Genome assembly was performed using “Microbial Assembly”. Genomic analysis was conducted by uploading the contigs to ResFinder and PlasmidFinder databases from the Center for Genomic Epidemiology. Results: IBCRE14 had a genome size of 4,018,329 bp and harboured genes coding for resistance to aminoglycosides (aadA1), phenicol (cat), tetracycline (tetJ), and trimethoprim (dfrA1). A large plasmid (pIB_NDM_1) harboured antibiotic resistance genes against sulphonamide (sul1), trimethoprim (dfrA14), tetracycline (tetB), rifampicin (arr-2), aminoglycosides (aadA1, aph3-VI), and beta-lactams (blaOXA-10, blaNDM-1). Furthermore, a small plasmid (pIB_COL3M) harboured a qnrD1 gene coding for quinolone resistance. Conclusion: The ability to conjugate and the presence of a composite antibiotic resistance island suggests that pIB_NDM_1 could both acquire more resistance genes and easily disseminate. To our knowledge, this is the first report on an untypable plasmid harbouring blaNDM-1 in P. mirabilis, in Italy.

Aim of this study was to genetically characterize two carbapenemase-producing Escherichia coli strains obtained from a pediatric patient affected by diarrhea, expressing OXA-181 and/or NDM-5 type enzymes. The above microorganisms were collected in the same Desenzano hospital (Northern Italy) where the blaNDM–5 gene was detected for the first time in Italy 3 years ago. One strain (5P), belonged to sequence type ST405/ST477 (according to Pasture/Oxford schemes) and serotype O102:H6. It was characterized by a 130562 bp multi-replicon plasmid IncFII/IncFIA/IncFIB (pVSI_NDM-5) enclosing two main antibiotic resistance islands: (i) ARI-I, 10030 bp in size, carried genes coding for β-lactam- (blaOXA–1, blaCTX–M–15), fluoroquinolone/aminoglycoside- (aac(6′)-lb-cr) and phenicol- resistance (catB3), (ii) ARI-II, 15326 bp in size, carried genes coding for sulfonamide- (sul1), β-lactam- (blaNDM–5, blaTEM–1B), phenicol- (catB3), trimethoprim- (dfrA17), antiseptic- (qacEΔ1), and aminoglycoside- (aadA5, rmtB) resistance. The other isolate (5M), belonged to sequence type ST2659/ST759 and serotype O50/02:H18, and carried four plasmids: a 153866 bp multi-replicon IncFII/IncFIA/IncFIB (pISV_IncFII_NDM-5), an 89866 bp IncI1 plasmid, a 51480 bp IncX3 plasmid (pISV_IncX3_OXA181), and a 41143 bp IncI plasmid (pISV_IncI_CMY-42). pISV_IncFII_NDM-5 carried two main antibiotic resistance islands: (i) ARI-III, 12220 bp in size, carried genes coding for β-lactam- (blaOXA–1), fluoroquinolone/aminoglycoside- (aac(6′)-lb-cr), tetracycline- (tet(B)) and phenicol- resistance (catB3, catA1), and ii) ARI-IV, 26527 bp in size, carried determinants coding for macrolide- (erm(B), mph(A)), sulfonamide- (sul1), beta-lactam- (blaNDM–5, blaTEM–1B), trimethoprim- (dfrA14, dfrA12), antiseptic- (qacEΔ1), and aminoglycoside- resistance (aadA5). pISV_IncI_CMY-42 harbored the blaCMY–42 gene coding for beta-lactam resistance, pISV_IncX3_OXA181 harbored genes encoding fluoroquinolone- (qnrS1) and beta-lactams- resistance (blaOXA–181). In conclusion, the detection of two different NDM-5 E. coli strains from a pediatric patient with a history of travel to the Far East countries strongly highlight an increasing trend and risk of importation from such areas.

Acinetobacter baumannii is a known opportunistic pathogen, dangerous for public health mostly due to its ability to rapidly acquire antibiotic-resistance traits. Its genome was described as characterized by remarkable plasticity, with a high frequency of homologous recombinations and proliferation of Insertion Sequences (IS). The SMAL pulsotype is an A. baumannii strain currently isolated only in Italy, characterized by a low incidence and a high persistence over the years. In this present work, we have conducted a comparative genomic analysis on this clone. The genome of 15 SMAL isolates was obtained and characterized in comparison with 24 other assemblies of evolutionary related isolates. The phylogeny highlighted the presence of a monophyletic clade (named ST78A), which includes the SMAL isolates. ST78A isolates have a low rate of homologous recombination and low gene content variability when compared to two related clades (ST78B and ST49) and to the most common A. baumannii variants worldwide (International Clones I and II). Remarkably, genomes in the ST78A clade present a high number of IS, including classes mostly absent in the other related genomes. Among these IS, one copy of IS66 was found to interrupt the gene comEC/rec2, involved in the acquisition of exogenous DNA. The genomic characterization of SMAL isolates shed light on the surprisingly low genomic plasticity and the high IS proliferation present in this strain. The interruption of the gene comEC/rec2 by an IS in the SMAL genomes brought us to formulate an evolutionary hypothesis according to which the proliferation of IS is slowing the acquisition of exogenous DNA, thus limiting genome plasticity. Such genomic architecture could explain the epidemiological behavior of high persistence and low incidence of the clone and provides an interesting framework to compare ST78 with the highly epidemic International Clones, characterized by high genomic plasticity.

Here we report two KPC-producing Citrobacter freundii isolates from unrelated patients. In one case, blaKPC-2 was harbored on a novel variant of Tn 4401 transposon of an IncN plasmid, conjugated together with a co-resident IncA plasmid, while in the other one, blaKPC-3 was on a Tn 4401a located on a IncX3-IncA self-conjugative plasmid fusion. The interplay occurring among plasmids carrying blaKPC and the co-resident IncA plasmids offers new information on plasmids co-resident within clinically relevant enterobacteria.

Objectives: Genomic characterization of the internationally spread sequence type (ST) 16 carbapenem-resistant Klebsiella pneumoniae. Methods: The complete genomes of three carbapenem producing ST16 K. pneumoniae from Italian patients were analysed by single-nucleotide polymorphism-based phylogeny, core genome multilocus sequence typing, resistance, plasmid, and virulence content and compared with ten genomes of ST16 strains isolated in other countries. Plasmids carrying blaNDM-1 or blaOXA-232 carbapenemase genes were assembled and sequences were analysed. Results: The internationally spread ST16 K. pneumoniae clone showed variability in terms of distribution of NDM-1 and OXA-232 type carbapenemases. In some ST16 strains, up to six plasmids can be simultaneously present in the same cell, including ColE-like plasmids carrying blaOXA-232 and IncF plasmids carrying blaNDM-1. The differences observed in plasmid, resistance, and virulence content and core genome suggested that there is not a unique, highly conserved ST16 clone, but instead different variants of this lineage circulate worldwide. Conclusions: The ST16 K. pneumoniae clone has spread worldwide and may become a high-risk clone.

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. Electronic supplementary material The online version of this article (10.1186/s13756-018-0326-0) contains supplementary material, which is available to authorized users.

To evaluate the water compartment antibiotic-resistance contamination rates, 11 wells, five streams, and four treatment plants located in the Oltrepò Pavese area were screened for the presence of third generation cephalosporins resistant Gram-negative bacteria. Enterobacteriaceae were also characterized for the Extended-Spectrum-β-Lactamases (ESBLs), carbapenemases, and mcr-1 genes presence. From December 2014 to November 2015, 246 water samples were filtered, plated on Plate Count Agar, MacConkey Agar, and MacConkey Agar with cefotaxime. Isolates were species identified using AutoSCAN-4-System and ESBLs, carbapenemases, and colistin resistance determinants were characterized by PCR, sequencing, and microarray. Plasmid conjugative transfer experiments, PCR-based Replicon typing, Pulsed-Field Gel Electrophoresis, Multi-Locus-Sequence-Typing, and in-silico plasmid characterization were performed. A total of 132 enterobacteria isolates grew on MacConkey agar with cefotaxime: 82 (62.1%) were obtained from streams, 41 (31.1%) from treatment plants, and 9 (6.8%) from wells. Thirty out of 132 (22.7%) isolates, mainly belonging to Escherichia coli (n = 15) species, showed a synergic effect with piperacillin-tazobactam. A single ESBL gene of blaCTX−M-type was identified in 19/30 isolates. In further two E. coli strains, a blaCTX−M−1 gene co-existed with a blaSHV-type ESBL determinant. A blaSHV−12 gene was detected in two isolates of E. coli (n = 1) and Klebsiella oxytoca (n = 1), while any ESBL determinant was ascertained in seven Yersinia enterocolitica strains. A blaDHA-type gene was detected in a cefoxitin resistant Y. enterocolitica from a stream. Interestingly, two Klebsiella pneumoniae strains of ST307 and ST258, collected from a well and a wastewater treatment plant, resulted KPC-2, and KPC-3 producers, respectively. Moreover, we report the first detection of mcr-1.2 ST10 E. coli on a conjugative IncX4 plasmid (33.303 bp in size) from a stream of Oltrepò Pavese (Northern Italy). Both ESBLs E. coli and ESBLs/carbapenemase-producing K. pneumoniae strains showed clonal heterogeneity by Pulsed-Field Gel Electrophoresis and Multi-Locus-Sequence-Typing. During one-year study and taking in account the whole Gram-negative bacterial population, an average percentage of cefotaxime resistance of 69, 32, and 10.3% has been obtained for the wastewater treatment plants, streams, and wells, respectively. These results, of concern for public health, highlight the need to improve hygienic measures to reduce the load of discharged bacteria with emerging resistance mechanisms.