Aurora Piazza

Carbapenem-resistant Klebsiella pneumoniae carrying New Delhi metallo-β-lactamase (NDM) enzymes poses a major clinical and public health challenge due to their ability to spread rapidly in hospitals, and their resistance to most β-lactam antibiotics. Following the first report of the emerging NDM-producing K. pneumoniae lineage ST6668 in Pavia, Italy, we conducted a retrospective analysis at San Raffaele Hospital (Milan). We identified 87 ST6668 isolates, several of which were collected prior to the initial report from Pavia. Whole-genome sequencing data were used to compare the Milan isolates with the 52 genomes from Pavia, in order to characterize a total of 139 ST6668 isolates collected between 2020 and 2025 from three hospitals in northern Italy. Phylogenetic and comparative genomic analyses were performed based on single-nucleotide polymorphisms (core SNPs), including 310 ST147 genomes for comparison. Accessory genome profiling was further validated by hybrid assemblies of three representative strains. A phylogenetic cluster containing isolates from different hospitals suggested possible interhospital transmission. Although the origin of ST6668 remains unknown, it clustered closely with KL64-ST147. The hybrid assemblies revealed that bla _NDM-1 is carried by the IncFIB(pQil) or IncHI1BIncFIB(Mar) plasmids. ST6668 lacks virulence markers such as iuc and rmpADC . All ST6668 isolates analyzed here carry a prophage identical to vB_Kpn_147Tu, which has been previously reported in ST147 isolates from the Tuscany outbreak. Together, these findings suggest either recent divergence from a common ancestor or convergence via mobile genetic elements. IMPORTANCE: Carbapenem-resistant Klebsiella pneumoniae producing New Delhi metallo-β-lactamase (NDM)-type enzymes represent a serious threat to infection control and patient safety. Understanding how novel resistant lineages emerge and spread is essential to limit hospital outbreaks. Our study traced the early presence of the recently identified ST6668 lineage in Milan, showing that it was circulating before its first description in Pavia. By linking this clone to the well-known high-risk lineage ST147, we demonstrate how resistance and genetic elements move between successful bacterial populations. These insights highlight how genomic surveillance can uncover hidden transmission pathways and guide interventions to prevent further dissemination of multidrug-resistant K. pneumoniae in healthcare settings.

The spread of extended-spectrum beta-lactamase (ESBL)- and carbapenemase-producing Escherichia coli clones in humans, animals, and the environment is of great concern worldwide. In this study, we characterized four carbapenemase-producing E. coli (CP-Ec) isolated from human samples. Two isolates of ST401, rarely associated with carbapenemase and/or ESBL resistance genes, harbored blaKPC-3 and blaVIM-1 genes, respectively, and were genetically distant from each other. One CP-Ec isolate belonging to ST355, typically found in poultry and environmental sources and not associated with carbapenemases, was blaKPC-3 positive and showed a wide range of virulence genes. The last CP-Ec strain belonged to ST3564, previously described in livestock with a large virulome but no carbapenemase. The CP-Ec ST3564 isolate co-harbored blaVIM-1 and blaNDM-7 genes, which, to our knowledge, have not been previously reported in Italy. These results emphasize the crucial role of a genomic-based surveillance program to intercept the emergence of critical E. coli clones.

Carbapenemase-producing Enterobacterales (CPE) represent an important threat to global health, resulting in an urgent issue in clinical settings. CPE often exhibit a multidrug-resistant (MDR) phenotype, thus reducing the antimicrobial armamentarium, with few antibiotics retaining residual antimicrobial activity against these pathogens. Carbapenemases are divided into three classes (A, B, and D) according to the Ambler classification system. Among these, KPC (class A), NDM, VIM, IMP (class B), and OXA-48-like (class D) represent the most important carbapenemases in terms of diffusion and clinical impact. CPE diffusion has been observed worldwide, with current endemicity in multiple territories around the world. In this context, the clonal spread and plasmid-mediated transmission of carbapenemases have contributed to the global spread of CPE worldwide and to the diffusion of carbapenemases among different Enterobacterales species. In recent years, novel molecules showing excellent in vitro and in vivo activity have been developed against CPE. However, the recent emergence of novel traits of resistance to these molecules has already been reported in several cases, mitigating the initial promising results. This review aims to provide an updated description of the major classes of carbapenemases, their global distribution, and future perspectives to limit the diffusion of CPEs.

Enterobacter asburiae, member of the Enterobacter cloacae complex (ECC) group, shows an increasing clinical relevance being responsible for infections like pneumonia, urinary tract infections and septicemia. The aim of the present study was the investigation of the genomic features of two XDR E. asburiae ST229 clinical strains co-carrying blaNDM-1 and blaVIM-1 determinants, collected in October 2021 and in June 2022, respectively. Two E. asburiae strains were collected from rectal swabs of as many patients admitted to the cardiopulmonary intensive care unit of Fondazione I.R.C.C.S. “Policlinico San Matteo” in Pavia, Italy. Based on the antibiotic susceptibility profile results, both isolates showed an XDR phenotype, retaining susceptibility only to fluoroquinolones. Both isolates shared identical resistome, virulome, plasmid content, and belonged to ST229, a rarely reported sequence type. They co-harbored blaNDM-1 and blaVIM-1 genes, that resulted located on transferable plasmids by conjugation and transformation. Moreover, both strains differed in 24 SNPs and showed genetic relatedness with E. asburiae ST709 and ST27. We described the first case of ST229 E. asburiae co-harboring blaNDM-1 and blaVIM-1 in Italy. This study points out the emergence of carbapenemases in low-risk pathogens, representing a novel challenge for public health, that should include such types of strains in dedicated surveillance programs. Antimicrobial susceptibility testing was carried out using Thermo Scientific™ Sensititre™ Gram Negative MIC Plates DKMGN. Both strains underwent whole-genome sequencing (WGS) using Illumina Miseq platform. Resistome, plasmidome, virulome, MLST, plasmid MLST and a SNPs-based phylogenetic tree were in silico determined.

In the last few years, Acinetobacter baumannii has ranked as a number one priority due to its Multi Drug Resistant phenotype. The different metabolic states, such as the one adopted when growing as biofilm, help the bacterium to resist a wide variety of compounds, placing the discovery of new molecules able to counteract this pathogen as a topic of utmost importance. In this context, bacterial cell division machinery and the conserved protein FtsZ are considered very interesting cellular targets. The benzothiadiazole compound C109 is able to inhibit bacterial growth and to block FtsZ GTPase and polymerization activities in Burkholderia cenocepacia, Pseudomonas aeruginosa, and Staphylococcus aureus. In this work, the activity of C109 was tested against a panel of antibiotic sensitive and resistant A. baumannii strains. Its ability to inhibit biofilm formation was explored, together with its activity against the A. baumannii FtsZ purified protein. Our results indicated that C109 has good MIC values against A. baumannii clinical isolates. Moreover, its antibiofilm activity makes it an interesting alternative treatment, effective against diverse metabolic states. Finally, its activity was confirmed against A. baumannii FtsZ.