A total of 43 A. baumannii strains, isolated from 43 patients affected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and by bacterial sepsis, were analyzed by antimicrobial susceptibility testing. All strains were resistant to almost three different classes of antibiotics, including carbapenems and colistin. The whole-genome sequencing (WGS) of eight selected A. baumannii isolates showed the presence of different insertion sequences (ISs), such as ISAba13, ISAba26, IS26, ISVsa3, ISEc29, IS6100 and IS17, giving to A. baumannii a high ability to capture and mobilize antibiotic resistance genes. Resistance to carbapenems is mainly mediated by the presence of OXA-23, OXA-66 and OXA-82 oxacillinases belonging to OXA-51-like enzymes. The presence of AmpC cephalosporinase, ADC-25, was identified in all A. baumannii. The pathogenicity of A. baumannii was exacerbated by the presence of several virulence factors. The multi-locus sequence typing (MLST) analysis showed that all strains belong to sequence type 2 (ST) international clone.

K. pneumoniae (KPN) is one of the widest spread bacteria in which combined resistance to several antimicrobial groups is frequent. The most common β-lactamases found in K. pneumoniae are class A carbapenemases, both chromosomal-encoded (i.e., NMCA, IMI-1) and plasmid-encoded (i.e., GES-enzymes, IMI-2), VIM, IMP, NDM, OXA-48, and extended-spectrum β-lactamases (ESBLs) such as CTX-M enzymes. In the present study, a total of 68 carbapenem-resistant KPN were collected from twelve long-term care facilities (LTCFs) in the Northern Italian region. The whole-genome sequencing (WGS) of each KPN strain was determined using a MiSeq Illumina sequencing platform and analysed by a bacterial analysis pipeline (BAP) tool. The WGS analysis showed the prevalence of ST307, ST512, and ST37 as major lineages diffused among the twelve LTCFs. The other lineages found were: ST11, ST16, ST35, ST253, ST273, ST321, ST416, ST1519, ST2623, and ST3227. The bla_KPC-2, bla_KPC-3, bla_KPC-9, bla_SHV-11, bla_SHV-28, bla_CTX-M-15, bla_OXA-1, bla_OXA-9, bla_OXA-23, qnrS1, qnrB19, qnrB66, aac(6′)-Ib-cr, and fosA were the resistance genes widespread in most LTCFs. In this study, we demonstrated the spreading of thirteen KPN lineages among the LTCFs. Additionally, KPC carbapenemases are the most widespread β-lactamase.

In the present study, the in vitro activity of the sulbactam–durlobactam (SUL–DUR) combination was evaluated against 141 carbapenem-resistant A. baumannii (CRAb) clinical strains collected from six Italian laboratories. Over half (54.6%) of these isolates were resistant to colistin. The SUL–DUR combination was active against these CRAb isolates with MIC₅₀ and MIC₉₀ values of 0.5 mg/L and 4 mg/L, respectively. Only eleven isolates were resistant to SUL–DUR with MIC values ranging from 8 to 128 mg/L. The SUL–DUR resistant A. baumannii exhibited several antimicrobial resistance genes (ARGs) such as bla_OXA-20, bla_OXA-58, bla_OXA-66, bla_ADC-25, aac(6′)-Ib3 and aac(6′)-Ib-cr and mutations in gyrA (S81L) and parC (V104I, D105E). However, in these isolates, mutations Q488K and Y528H were found in PBP3. Different determinants were also identified in these CRAb isolates, including adeABC, adeFGH, adeIJK, abeS, abaQ and abaR, which encode multidrug efflux pumps associated with resistance to multiple antibacterial agents. This is the first report on the antimicrobial activity of SUL–DUR against carbapenem-resistant A. baumannii isolates selected from multiple regions in Italy.

The whole-genome sequencing (WGS) of eighteen S. marcescens clinical strains isolated from 18 newborns hospitalized in the Neonatal Intensive Care Unit (NICU) at Pescara Public Hospital, Italy, was compared with that of S. marcescens isolated from cradles surfaces in the same ward. The identical antibiotic resistance genes (ARGs) and virulence factors were found in both clinical and environmental S. marcescens strains. The aac(6′)-Ic, tetA(41), bla_SRT-3, adeFGH, rsmA, and PBP3 (D350N) genes were identified in all strains. The SRT-3 enzyme, which exhibited 10 amino acid substitutions with respect to SST-1, the constitutive AmpC β-lactamase in S. marcescens, was partially purified and tested against some β-lactams. It showed a good activity against cefazolin. Both clinical and environmental S. marcescens strains exhibited susceptibility to all antibiotics tested, with the exception of amoxicillin/clavulanate.

Long-term care facilities (LTCFs) are important reservoirs of antimicrobial-resistant (AMR) bacteria which colonize patients transferred from the hospital, or they may emerge in the facility as a result of mutation or gene transfer. In the present study, we characterized, from a molecular point of view, 43 E. coli strains collected from residents of LTCFs in Northern Italy. The most common lineage found was ST131, followed by sporadic presence of ST12, ST69, ST48, ST95, ST410 and ST1193. All strains were incubators of several virulence factors, with iss, sat, iha and senB being found in 84%, 72%, 63% and 51% of E. coli, respectively. Thirty of the ST131 analyzed were of the O25b:H4 serotype and H30 subclone. The ST131 isolates were found to be mainly associated with IncF plasmids, CTX-M-1, CTX-M-3, CTX-M-15, CTX-M-27 and gyrA/parC/parE mutations. Metallo-β-lactamases were not found in ST131, whereas KPC-3 carbapenemase was found only in two ST131 and one ST1193. In conclusion, we confirmed the spread of extended-spectrum β-lactamase genes in E. coli ST131 isolated from colonized residents living inside LTCFs. The ST131 represents an incubator of fluoroquinolones, aminoglycosides and other antibiotic resistance genes in addition to different virulence factors.

Four NDM-1 mutants (L218T, L221T, L269H and L221T/Y229W) were generated in order to investigate the role of leucines positioned in L10 loop. A detailed kinetic analysis stated that these amino acid substitutions modified the hydrolytic profile of NDM-1 against some β-lactams. Significant reduction of k_cat values of L218T and L221T for carbapenems, cefazolin, cefoxitin and cefepime was observed. The stability of the NDM-1 and its mutants was explored by thermofluor assay in real-time PCR. The determination of T_mB and T_mD demonstrated that NDM-1 and L218T were the most stable enzymes. Molecular dynamic studies were performed to justify the differences observed in the kinetic behavior of the mutants. In particular, L218T fluctuated more than NDM-1 in L10, whereas L221T would seem to cause a drift between residues 75 and 125. L221T/Y229W double mutant exhibited a decrease in the flexibility with respect to L221T, explaining enzyme activity improvement towards some β-lactams. Distances between Zn1-Zn2 and Zn1-OH- or Zn2-OH- remained unaffected in all systems analysed. Significant changes were found between Zn1/Zn2 and first sphere coordination residues.