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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), blaSRT-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. [ABSTRACT FROM AUTHOR]

Antibiotic-resistant bacteria (ARB) are widespread in nature and represent a serious public and environmental problem. In the present study, we report for the first time the presence of bacterial β-lactamases in two macroinvertebrate species with different feeding traits. The class A β-lactamases, SHV-1 and TEM-1, were found in Citrobacter freundii isolated from Gammarus elvirae and Escherichia coli from water samples, respectively. The metallo-β-lactamase CphA was found in Aeromonas veronii and Aeromonas hydrophila strains isolated from the predator Dina lineata. The presence of a large plasmid was ascertained only in E. coli strains isolated from water. In all strains studied, an integrase I typical of class I integrin was found. In contaminated freshwater habitats, ARB and antibiotic resistance genes could be disseminated through trophic links with important ecological implications. Transmission through the food chain may contribute to spreading and transferring antibiotic resistance not only in freshwater ecosystems but also outside the aquatic habitat. [ABSTRACT FROM AUTHOR]

The injudicious usage of antibiotics during infections caused by Gram-negative bacteria leads to the emergence of β-lactamases. Among them, the NDM-1 enzyme poses a serious threat to human health. Developing new antibiotics or inhibiting β-lactamases might become essential to reduce and prevent bacterial infections. Nanobodies (Nbs), the smallest antigen-binding single-domain fragments derived from Camelidae heavy-chain-only antibodies, targeting enzymes, are innovative alternatives to develop effective inhibitors. The biopanning of an immune VHH library after phage display has helped to retrieve recombinant antibody fragments with high inhibitory activity against recombinant-NDM-1 enzyme. Nb02NDM-1, Nb12NDM-1, and Nb17NDM-1 behaved as uncompetitive inhibitors against NDM-1 with Ki values in the nM range. Remarkably, IC50 values of 25.0 nM and 8.5 nM were noted for Nb02NDM-1 and Nb17NDM-1, respectively. The promising inhibition of NDM-1 by Nbs highlights their potential application in combating particular Gram-negative infections. [ABSTRACT FROM AUTHOR]

The main goal of this study was to identify Gram-negative bacteria resistant to antibiotics, in particular β-lactams, in stream waters and effluents from urban wastewater treatment plants draining into Fino, Tavo, and Saline rivers of the Abruzzo region, Italy. Eight sampling sites were selected because they were the most contaminated by coliforms during previous sampling campaign. One sample for each site was collected for the detection of total and fecal coliforms, Escherichia coli and Enterococcus species by Colilert-18 and Enterolert-E Quanti-Tray/2000. Antibiotic-resistant bacteria, selected on ampicillin and cefotaxime-supplemented agar plates, were identified by EnteroPluri test systems and then confirmed by MALDI-TOF. The resistant determinants were identified and characterized by PCR and sequencing. The microbiological analysis allowed to detect E. coli, total coliforms, fecal coliforms, and enterococci with a coefficient of variation of 215.7%, 212.8%, 242.5%, and 188.5%, respectively. Several Gram-negative bacteria were identified: Serratia liquefaciens, E. coli, Enterobacter cloacae, Citrobacter freundii, Raoultella ornithinolytica, Acinetobacter johnsonii, Aeromonas veronii, Aeromonas hydrophila, and Pseudomonas koreensis. All strains possessed class 1 integrons, insertion sequences, and genes encoding for serin- and metallo-β-lactamases. Extended-spectrum β-lactamases, such as CTX-M-15 and CTX-M-27, were found in Enterobacteriaceae, whereas CphA metallo-β-lactamase was found in A. veronii and A. hydrophila. The main resistance's mechanism to β-lactams observed among the analyzed strains is represented by the production of serin β-lactamases (CTX-M-15, CTX-M-27, and SHV-1) and metallo β-lactamase (CphA). [ABSTRACT FROM AUTHOR]

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 kcat 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 TmB and TmD 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. [ABSTRACT FROM AUTHOR]

• Taniborbactam is a novel broad-spectrum bicyclic boronate able to inhibit metallo-β-lactamases (MBLs) • Taniborbactam potentiated cefepime MICs in 26 MBL-producing Enterobacterales • Taniborbactam behaved as competitive inhibitor against NDM-1 and engineered NDM-1 variants • Taniborbactam protected antibacterial activity of cefepime from MBLs, except IMP-variants Objective: This study aimed to investigate the in vitro activity of taniborbactam (VNRX-5133), a novel broad-spectrum bicyclic boronate, against NDM-1 and Q119E, Q119K, Q119C, Q119F, Q119V, and Q119Y NDM-1 variants, which showed an increased activity towards some β-lactams, including cefepime. Methods: Inhibition kinetic assays were spectrophotometrically performed using cefepime (50 μM) as the reporter substrate and 80 nM of each enzyme. Taniborbactam behaves as a competitive inhibitor towards NDM-1 and NDM-1 Q119 variants with lower Ki values (range 3–16 nM). The phenotypic profile was assessed in both Enterobacterales clinical isolates and engineered Escherichia coli BL21(DE3) strains by conventional broth microdilution procedures according to the Clinical and Laboratory Standards Institute (CLSI). Results: Taniborbactam at a fixed concentration of 4 mg/L was able to restore activity of cefepime in 24 of 26 Enterobacterales clinical isolates harbouring metallo-β-lactamases with MIC 50 /MIC 90 values of 14 mg/L. Cefepime MICs were drastically reduced in all clinical isolates and in NDM-1 and Q119X producing Escherichia coli BL21(DE3). Taniborbactam was unable to restore susceptibility to cefepime in two IMP variants producing clinical isolates. Conclusion: The inhibition level of NDM enzymes provided by taniborbactam protects the antibacterial activity of cefepime from this important metallo-β-lactamase. [ABSTRACT FROM AUTHOR]

KPC enzymes are the most common class A carbapenemases globally diffused. The peculiarity of this family of β-lactamases is represented by their ability to hydrolyse all classes of β-lactams, including carbapenems, posing a serious problem to public health. In the present study, seven laboratory mutants of KPC-3 (D228S, D228W, D228M, D228K, D228L, D228I and D228G) were generated by site-saturation mutagenesis to explore the role of residue 228, a non-active site residue. Compared to KPC-3, the seven mutants showed evident differences in kcat and Km values calculated for some penicillins, cephalosporins and carbapenems. In particular, D228S and D228M showed a significant increase of Km values for cefotaxime and ceftazidime. Circular dichroism (CD) experiments have demonstrated that substitution at position 228 does not affect the secondary structure of the mutants. Molecular dynamics (MD) simulations were performed on KPC-3, D228S and D228M uncomplexed and complexed with cefotaxime (substrate). Although the residue 228 is located far from the active site, between α11 helix and β7 sheet in the opposite site of the Ω-loop, amino acid substitution at this position generates mechanical effects in the active site resulting in enzyme activity changes. [ABSTRACT FROM AUTHOR]

The design of novel chelators for therapeutic applications has been the subject of extensive research to address various diseases. Many chelators can manipulate the levels of metal ions within cells and effectively modulate the metal excess. In some cases, chelators show significant toxicity to cells. We investigated polyimidazole ligands by potentiometry and UV–Vis spectroscopy for their ability to form copper(II) complexes. We also compared the antiproliferative activity of the polyimidazole ligands and their copper(II) complexes with polypyridine ligands in CaCo-2 (colorectal adenocarcinoma), SH-SY5Y (neuroblastoma) and K562 (chronic myelogenous leukemia) cells and normal HaCaT (keratinocyte) cells. Polyimidazole ligands are less cytotoxic than their analogous polypyridine ligands. All polyimidazole ligands, except the tetraimidazole ligand for K562 cells, did not show any significant effect on the viability of cancer and normal cells. In contrast, the cytotoxic activity of polypiridine ligands was also observed in normal cells with IC 50 values similar to those of cancer cells. Tetraimidazole ligand, the only ligand active on the leukemic K562 cell line, induced caspase-dependent apoptosis and increased intracellular reactive oxygen species production with mitochondrial damage. The low cytotoxicity of the polyimidazole ligands, even if it limits their use as anticancer agents, could make them useful in other medical applications, such as in the treatment of metal overload, microbial infections, inflammation or neurodegenerative disorders. The low cytotoxicity of imidazole ligands makes them promising for medical applications, including the treatment of metal overload. [Display omitted] • Chelators can modulate the metal ion homeostasis inside cells. • Imidazole is a binding site in biological systems. • Polyimidazole ligands can bind copper(II). • Imidazole ligands show a low antiproliferative effect. • The low cytotoxicity could make them promising for a variety of medical applications. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae strain is an important opportunistic pathogen that causes severe nosocomial infections. In the present study a molecular characterization of carbapenem resistant K. pneumoniae , isolated from blood samples of hospitalized patients of Verona University Hospital, was performed. The simultaneous presence of SHV-1/CTX-M-15/KPC-3 and SHV-1/CTX-M-15/OXA-48 serin-β-lactamases was ascertained in the 89% and 11% of K. pneumoniae ST512 and K. pneumoniae ST14, respectively. Molecular characterization of bla genes showed that bla KPC-3 was found in Tn 4401a transposon with the tnp R, tnp A, IS Kpn6 , and IS Kpn7 mobile elements whereas bla CTX-M-15 was detected downstream IS Ecp1 genetic element. A class 1 integron with a gene cassette of 780 bp corresponding to aad A2 gene was identified in 33 K. pneumoniae ST512 isolates. [ABSTRACT FROM AUTHOR]

The New Delhi metallo-β-lactamase-1 (NDM-1) is a typical carbapenemase and plays a crucial role in antibiotic-resistance bacterial infection. Phylogenetic analysis, performed on known NDM-variants, classified NDM enzymes in seven clusters. Three of them include a major number of NDM-variants. In this study, we evaluated the role of the V88L substitution in NDM-24 by kinetical and structural analysis. Functional results showed that V88L did not significantly increase the resistance level in the NDM-24 transformant toward penicillins, cephalosporins, meropenem, and imipenem. Concerning ertapenem, E. coli DH5α/NDM-24 showed a MIC value 4-fold higher than that of E. coli DH5α/NDM-1. The determination of the kcat, Km, and kcat/Km values for NDM-24, compared with NDM-1 and NDM-5, demonstrated an increase of the substrate hydrolysis compared to all the β-lactams tested, except penicillins. The thermostability testing revealed that V88L generated a destabilized effect on NDM-24. The V88L substitution occurred in the β-strand and low β-sheet content in the secondary structure, as evidenced by the CD analysis data. In conclusion, the V88L substitution increases the enzyme activity and decreases the protein stability. This study characterizes the role of the V88L substitution in NDM-24 and provides insight about the NDM variants evolution. [ABSTRACT FROM AUTHOR]

Antibiotic resistance is a public health problem with increasingly alarming data being reported. Gram-positive bacteria are among the protagonists of severe nosocomial and community infections. The objective of this review is to conduct an extensive examination of emerging treatments for Gram-positive infections including ceftobiprole, ceftaroline, dalbavancin, oritavancin, omadacycline, tedizolid, and delafloxacin. From a methodological standpoint, a comprehensive analysis on clinical trials, molecular structure, mechanism of action, microbiological targeting, clinical use, pharmacokinetic/pharmacodynamic features, and potential for therapeutic drug monitoring will be addressed. Each antibiotic paragraph is divided into specialized microbiological, clinical, and pharmacological sections, including detailed and appropriate tables. A better understanding of the latest promising advances in the field of therapeutic options could lead to the development of a better approach in managing antimicrobial therapy for multidrug-resistant Gram-positive pathogens, which increasingly needs to be better stratified and targeted. [ABSTRACT FROM AUTHOR]

Beta-lactam antibiotics are one of the most commonly used drug classes in managing bacterial infections. However, their use is threatened by the alarming phenomenon of antimicrobial resistance, which represents a worldwide health concern. Given the continuous spread of metallo-β-lactamases (MBLs) producing pathogens, the need to discover broad-spectrum β-lactamase inhibitors is increasingly growing. A series of zinc chelators have been synthesized and investigated for their ability to hamper the Zn-ion network of interactions in the active site of MBLs. We assessed the inhibitory activity of new polyimidazole ligands N , N ′-bis((imidazol-4-yl)methyl)-ethylenediamine, N,N,N′-tris((imidazol-4-yl)methyl)-ethylenediamine, N,N,N,N′-tetra((imidazol-4-yl-methyl)-ethylenediamine toward three different subclasses B1 MBLs: VIM-1, NDM-1 and IMP-1 by in vitro assays. The activity of known zinc chelators such as 1,4,7,10,13-Pentaazacyclopentadecane, 1,4,8,11-Tetraazacyclotetradecane and 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid was also assessed. Moreover, a molecular docking study was carried to gain insight into the interaction mode of the most active ligands. All imidazole rings of tetra-imidazole ligand are accommodated into the active site of VIM-1. The compound showed the best inhibitory activity against metallo-β-lactamase. [Display omitted] • Bacteria have developed antibiotic resistance mechanisms producing β-lactamases. • Zn chelators can inhibit metallo-β-lactamase. • Polyimidazole ligands were synthesized as metallo-beta-lactamase inhibitors. • The tetraimidazole ligand showed efficient activity against NDM-1 and VIM-1. [ABSTRACT FROM AUTHOR]

Drug repurposing is an attractive strategy for developing new antibacterial molecules. Herein, we evaluated the in vitro antibacterial, antibiofilm, and antivirulence activities of eight FDA-approved "non-antibiotic" drugs, comparatively to tobramycin, against selected Pseudomonas aeruginosa strains from cystic fibrosis patients. MIC and MBC values were measured by broth microdilution method. Time–kill kinetics was studied by the macro dilution method, and synergy studies were performed by checkerboard microdilution assay. The activity against preformed biofilms was measured by crystal violet and viable cell count assays. The effects on gene expression were studied by real-time quantitative PCR, while the cytotoxic potential was evaluated against IB3-1 bronchial CF cells. Ciclopirox, 5-fluorouracil, and actinomycin D showed the best activity against P. aeruginosa planktonic cells and therefore underwent further evaluation. Time–kill assays indicated actinomycin D and ciclopirox, contrarily to 5-fluorouracil and tobramycin, have the potential for bacterial eradication, although with strain-dependent efficacy. Ciclopirox was the most effective against the viability of the preformed biofilm. A similar activity was observed for other drugs, although they stimulate extracellular polymeric substance production. Ribavirin showed a specific antibiofilm effect, not dependent on bacterial killing. Exposure to drugs and tobramycin generally caused hyperexpression of the virulence traits tested, except for actinomycin D, which downregulated the expression of alkaline protease and alginate polymerization. Ciclopirox and actinomycin D revealed high cytotoxic potential. Ciclopirox and ribavirin might provide chemical scaffolds for anti-P. aeruginosa drugs. Further studies are warranted to decrease ciclopirox cytotoxicity and evaluate the in vivo protective effects. [ABSTRACT FROM AUTHOR]

Although lymphopenia is currently considered a good predictor for the prognosis of COVID-19, it must be critically evaluated in patients with CLL, where other clinical markers should be considered to define the prognosis and treatment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

New Delhi Metallo-β-lactamase-1 (NDM-1) is the most prevalent type of metallo-β-lactamase, able to hydrolyze almost all antibiotics of the β-lactam group, leading to multidrug-resistant bacteria. To date, there are no clinically relevant inhibitors to fight NDM-1. The use of dromedary polyclonal antibody inhibitors against NDM-1 represents a promising new class of molecules with inhibitory activity. In the current study, immunoreactivities of dromedary Immunoglobulin G (IgG) isotypes containing heavy-chain and conventional antibodies were tested after successful immunization of dromedary using increasing amounts of the recombinant NDM-1 enzyme. Inhibition kinetic assays, performed using a spectrophotometric method with nitrocefin as a reporter substrate, demonstrated that IgG1, IgG2, and IgG3 were able to inhibit not only the hydrolytic activity of NDM-1 but also Verona integron-encoded metallo-β-lactamase (VIM-1) (subclass B1) and L1 metallo-β-lactamase (L1) (subclass B3) with inhibitory concentration (IC50) values ranging from 100 to 0.04 μM. Investigations on the ability of IgG subclasses to reduce the growth of recombinant Escherichia coli BL21(DE3)/codon plus cells containing the recombinant plasmid expressing NDM-1, L1, or VIM-1 showed that the addition of IgGs (4 and 8 mg/L) to the cell culture was unable to restore the susceptibility of carbapenems. Interestingly, IgGs were able to interact with NDM-1, L1, and VIM-1 when tested on the periplasm extract of each cultured strain. The inhibitory concentration was in the micromolar range for all β-lactams tested. A visualization of the 3D structural basis using the three enzyme Protein Data Bank (PDB) files supports preliminarily the recorded inhibition of the three MBLs. [ABSTRACT FROM AUTHOR]