A Two Amino Acid Duplication, L167E168, in the Ω-Loop Drastically Decreases Carbapenemase Activity of KPC-53, a Natural Class A ß-Lactamase.

KPC-53 enzyme is a natural KPC variant which showed a duplication of L167E168 residues in the Ω-loop structure. The blaKPC-53 gene was cloned both into pBC-SK and pET-24a vectors, and the recombinant plasmids were transferred by transformation in Escherichia coli competent cells to evaluate the antimicrobial susceptibility and to produce the enzyme. Compared to KPC-3, the KPC-53 was less stable and showed a dramatic reduction of kcat and kcat/Km versus several ß-lactams, in particular carbapenems. Indeed, a 2,000-fold reduction was observed in the kcat values of KPC-53 for imipenem and meropenem. Concerning inhibitors, KPC-53 was susceptible to tazobactam and clavulanic acid but maintained resistance to avibactam. The molecular modeling indicates that the L167E168 duplication in KPC-53 modifies the interactions between residues involved in the catalytic pocket, changing the flexibility of the Ω-loop, which is directly coupled with the catalytic properties of the KPC enzymes.

Laboratory Variants GESG170L, GESG170K, and GESG170H Increase Carbapenem Hydrolysis and Confer Resistance to Clavulanic Acid.

The Guiana extended-spectrum (GES) ß-lactamase GESG170H, GESG170L, and GESG170K mutants showed kcat, Km , and kcat/Km values very dissimilar to those of GES-1 and GES-5. The enhancement of the hydrolytic activity against carbapenems is potentially due to a shift of the substrate in the active site that provides better positioning of the deacylating water molecule caused by the presence of the imidazole ring of H170 and of the long side chain of K170 and L170.

Exploring the Role of L10 Loop in New Delhi Metallo-ß-lactamase (NDM-1): Kinetic and Dynamic Studies.

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 ß-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 ß-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.

Inhibitory Potential of Polyclonal Camel Antibodies against New Delhi Metallo-ß-lactamase-1 (NDM-1).

New Delhi Metallo-ß-lactamase-1 (NDM-1) is the most prevalent type of metallo-ß-lactamase, able to hydrolyze almost all antibiotics of the ß-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-ß-lactamase (VIM-1) (subclass B1) and L1 metallo-ß-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 ß-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.

Kinetic Profile and Molecular Dynamic Studies Show that Y229W Substitution in an NDM-1/L209F Variant Restores the Hydrolytic Activity of the Enzyme toward Penicillins, Cephalosporins, and Carbapenems.

The New Delhi metallo-ß-lactamase-1 (NDM-1) enzyme is the most common metallo-ß-lactamase identified in many Gram-negative bacteria causing severe nosocomial infections. The aim of this study was to focus the attention on non-active-site residues L209 and Y229 of NDM-1 and to investigate their role in the catalytic mechanism. Specifically, the effect of the Y229W substitution in the L209F variant was evaluated by antimicrobial susceptibility testing, kinetic, and molecular dynamic (MD) studies. The Y229W single mutant and L209F-Y229W double mutant were generated by site-directed mutagenesis. The Km , kcat, and kcat/Km kinetic constants, calculated for the two mutants, were compared with those of (wild-type) NDM-1 and the L209F variant. Compared to the L209F single mutant, the L209F-Y229W double mutant showed a remarkable increase in kcat values of 100-, 240-, 250-, and 420-fold for imipenem, meropenem, benzylpenicillin, and cefepime, respectively. In the L209F-Y229W enzyme, we observed a remarkable increase in kcat/Km of 370-, 140-, and 80-fold for cefepime, meropenem, and cefazolin, respectively. The same behavior was noted using the antimicrobial susceptibility test. MD simulations were carried out on both L209F and L209F-Y229W enzymes complexed with benzylpenicillin, focusing attention on the overall mechanical features and on the differences between the two systems. With respect to the L209F variant, the L209F-Y229W double mutant showed mechanical stabilization of loop 10 and the N-terminal region. In addition, Y229W substitution destabilized both the C-terminal region and the region from residues 149 to 154. The epistatic effect of the Y229W mutation jointly with the stabilization of loop 10 led to a better catalytic efficiency of ß-lactams. NDM numbering is used in order to facilitate the comparison with other NDM-1 studies.

TEM-184, a Novel TEM-Derived Extended-Spectrum ß-Lactamase with Enhanced Activity against Aztreonam.

TEM-184, a novel TEM-derived extended-spectrum ß-lactamase (ESBL), was isolated from an Escherichia coli ST354 clinical strain. Compared to TEM-1, TEM-184 contains the mutations Q6K, E104K, I127V, R164S, and M182T. Kinetic analysis of this enzyme revealed extended-spectrum activity against aztreonam in particular. TEM-184 was also susceptible to inhibitors, including clavulanic acid, tazobactam, and avibactam.

A Kinetic Study of the Replacement by Site Saturation Mutagenesis of Residue 119 in NDM-1 Metallo-ß-Lactamase.

New Delhi metallo-ß-lactamase 1 (NDM-1) is a subclass B1 metallo-ß-lactamase that exhibits a broad spectrum of activity against ß-lactam antibiotics. Here we report the kinetic study of 6 Q119X variants obtained by site-directed mutagenesis of NDM-1. All Q119X variants were able to hydrolyze carbapenems, penicillins and first-, second-, third-, and fourth-generation cephalosporins very efficiently. In particular, Q119E, Q119Y, Q119V, and Q119K mutants showed improvements in kcat/Km values for penicillins, compared with NDM-1. The catalytic efficiencies of the Q119K variant for benzylpenicillin and carbenicillin were about 65- and 70-fold higher, respectively, than those of NDM-1. The Q119K and Q119Y enzymes had kcat/Km values for ceftazidime about 25- and 89-fold higher, respectively, than that of NDM-1.

P174E Substitution in GES-1 and GES-5 ß-Lactamases Improves Catalytic Efficiency toward Carbapenems.

GES-type ß-lactamases are a group of enzymes that have evolved their hydrolytic activity against carbapenems. In this study, the role of residue 174 inside the Ω-loop of GES-1 and GES-5 was investigated. GES-1P174E and GES-5P174E mutants, selected by site saturation mutagenesis, were purified and kinetically characterized. In comparison with GES-1 and GES-5 wild-type enzymes, GES-1P174E and GES-5P174E mutants exhibited lower kcat and kcat/Km values for cephalosporins and penicillins. Concerning carbapenems, GES-1P174E shared higher kcat values but lower Km values than those calculated for GES-1. The GES-1P174E and GES-5P174E mutants showed high hydrolytic efficiency for imipenem, with kcat/Km values 100- and 660-fold higher, respectively, than those of GES-1. Clavulanic acid and tazobactam are good inhibitors for both GES-1P174E and GES-5P174E Molecular dynamic (MD) simulations carried out for GES-1, GES-5, GES-1P174E, and GES-5P174E complexed with imipenem and meropenem have shown that mutation at position 174 induces a drastic increase of enzyme flexibility, in particular in the Ω-loop. The circular dichroism (CD) spectroscopy spectra of the four enzymes indicate that the P174E substitution in GES-1 and GES-5 does not affect the secondary structural content of the enzymes.

Kinetic Study of Laboratory Mutants of NDM-1 Metallo-ß-Lactamase and the Importance of an Isoleucine at Position 35.

Two laboratory mutants of NDM-1 were generated by replacing the isoleucine at position 35 with threonine and serine residues: the NDM-1(I35T)and NDM-1(I35S)enzymes. These mutants were well characterized, and their kinetic parameters were compared with those of the NDM-1 wild type. Thekcat,Km, andkcat/Kmvalues calculated for the two mutants were slightly different from those of the wild-type enzyme. Interestingly, thekcat/Kmof NDM-1(I35S)for loracarbef was about 14-fold higher than that of NDM-1. Far-UV circular dichroism (CD) spectra of NDM-1 and NDM-1(I35T)and NDM-1(I35S)enzymes suggest local structural rearrangements in the secondary structure with a marked reduction of α-helix content in the mutants.

Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems.

Site-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-ß-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

Identification of New Natural CphA Metallo-ß-Lactamases CphA4 and CphA5 in Aeromonas veronii and Aeromonas hydrophila Isolates from Municipal Sewage in Central Italy.

Two new natural CphA metallo-ß-lactamases, the CphA4 and CphA5 enzymes, were identified in water samples from municipal sewage in central Italy. Compared to CphA, the CphA4 and CphA5 enzymes showed numerous point mutations. These enzymes have a narrow spectrum of substrates focused on carbapenems only. CphA5 showed kcat values about 40-, 12-, and 97-fold higher than those observed for CphA4 versus imipenem, ertapenem, and biapenem, respectively.

Kinetic study of the effect of histidines 240 and 164 on TEM-149 enzyme probed by ß-lactam inhibitors.

In the present study, we performed a detailed kinetic analysis of the enzymes TEM-149, TEM-149(H240), and TEM-149(H164-H240) versus a large panel of inhibitors/inactivators, including penicillins, penems, carbapenems, monobactams, cephamycin, and carbacephem. These compounds behaved as poor substrates versus TEM-149, TEM-149(H240), and TEM-149(H164-H240) ß-lactamases, and the Ki (inhibition constant), K (dissociation constant of the Henri-Michaelis complex), k+2 and k+3 (first-order acylation and deacylation constants, respectively), and k+2/K values were calculated.

R164H and V240H replacements by site-directed mutagenesis of TEM-149 extended-spectrum ß-lactamase: kinetic analysis of TEM-149H240 and TEM-149H164-H240 laboratory mutants.

Two laboratory mutant forms, TEM-149(H240) and TEM-149(H164-H240), of the TEM-149 extended-spectrum ß-lactamase enzyme were constructed by site-directed mutagenesis. TEM-149(H240) and TEM-149(H164-H240) were similar in kinetic behavior, except with respect to benzylpenicillin and ceftazidime. Molecular modeling of the two mutant enzymes demonstrated the role of histidine at position 240 in the reduction of the affinity of the enzyme for ceftazidime.

On the structural affinity of macromolecules with different biological properties: molecular dynamics simulations of a series of TEM-1 mutants.

Molecular Dynamics simulations have been carried out in order to provide a molecular rationalization of the biological and thermodynamic differences observed for a class of TEM ß-lactamases. In particular we have considered the TEM-1(wt), the single point mutants TEM-40 and TEM-19 representative of IRT and ESBL classes respectively, and TEM-1 mutant M182T, TEM-32 and TEM-20 which differ from the first three for the additional of M182T mutation. Results indicate that most of the thermodynamic, and probably biological behaviour of these systems arise from subtle effects which, starting from the alterations of the local interactions, produce drastic modifications of the conformational space spanned by the enzymes. The present study suggests that systems showing essentially the same secondary and tertiary structure may differentiate their chemical-biological activity essentially (and probably exclusively) on the basis of the thermal fluctuations occurring in their physiological environment.

Prevalence of quinolone resistance determinant qnrA6 among broad- and extended-spectrum beta-lactam-resistant Proteus mirabilis and Morganella morganii clinical isolates with sul1-type class 1 integron association in a Tunisian Hospital.

OBJECTIVES: The aim of this study was to investigate the prevalence and the emergence of plasmid-mediated quinolone resistance among broad-spectrum beta-lactam-resistant Proteus mirabilis and Morganella morganii clinical isolates recovered in the Military Hospital in Tunisia. METHODS: Of 200 strains examined, 50 exhibited resistance to quinolones. Quinolone resistance determinants (qnr and aac(6')-Ib-cr) were characterized by multiplex PCR and sequencing. Chromosomal quinolone resistance mutations in the quinolone resistance-determining region (QRDR) and class 1 integron characterization were analysed by PCR and sequencing. The clonal relationship between the isolates was studied by pulsed-field gel electrophoresis (PFGE). RESULTS: Fourteen isolates harboured qnrA6 and among them 8 (57%) were extended-spectrum beta-lactamase (ESBL) producers, whilst 12 (85%) isolates harboured blaDHA-1. Mutations in the QRDR were detected in gyrA (Ser83Ile, Glu87Lys), gyrB (Ser464Phe), and parC (Ser80Ile). qnrA6 and blaDHA-1 genes were found embedded in complex sul1-type class 1 integrons. A gene cassette carrying aac(6')-Ib-cr was found located in the class 1 integron upstream of the qacEΔ1 gene. According to the PFGE analysis, the isolates were clonally unrelated. CONCLUSIONS: This is the first description in North Africa of class 1 integrons carrying blaDHA-1, qnrA6 gene, and aac(6')-Ib-cr determinants in clinical strains of Proteus mirabilis and Morganella morganii.

Cytotoxic activity and antioxidant capacity of purified lichen metabolites: an in vitro study.

The purpose of this study was to investigate the effects of six lichen metabolites (diffractaic acid, lobaric acid, usnic acid, vicanicin, variolaric acid, protolichesterinic acid) on proliferation, viability and reactive oxygen species (ROS) level towards three human cancer cell lines, MCF-7 (breast adenocarcinoma), HeLa (cervix adenocarcinoma) and HCT-116 (colon carcinoma). Cells were treated with different concentrations (2.5-100 µM) of these compounds for 48 h. In this comparative study, our lichen metabolites showed various cytotoxic effects in a concentration-dependent manner, and usnic acid was the most potent cytotoxic agent, while variolaric acid did not inhibit the proliferation of any of the three cell lines used. All tested lichen compounds did not exhibit free radical scavenging activity using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The lichen metabolites did not significantly increase the intracellular ROS level and did not prevent oxidative injury induced by t-butylhydroperoxide in HeLa cells. To better clarify the mechanism(s) of cytotoxic effect induced by protolichesterinic acid in HeLa cells, we investigated apoptotic markers such as condensation and fragmentation of nuclear chromatin and activation of caspase-3, 8 and 9. Our results revealed that the antiproliferative activity of 40 µM protolichesterinic acid in HeLa cells is related to its ability to induce programmed cell death involving caspase-3, 8 and 9 activation.

Whole-Genome Sequencing of ST2 A. baumannii Causing Bloodstream Infections in COVID-19 Patients.

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.

Resistome and Virulome of Multi-Drug Resistant E. coli ST131 Isolated from Residents of Long-Term Care Facilities in the Northern Italian Region.

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-ß-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 ß-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.

Evaluation of the Analytical Performances of the Biolabo SOLEA 100 Optical Coagulometer and Comparison with the Stago STA-R MAX Analyser in the Determination of PT, APTT, and Fibrinogen.

INTRODUCTION: The Biolabo Solea 100 is a fully automated coagulation analyser using an optical system to detect coagulation designed to meet the needs of small- and medium-sized laboratories. This study aimed to evaluate the analytical performance in terms of bias, precision, and interference of the Biolabo Solea 100 coagulometer under routine laboratory conditions. In addition, a comparison was made with Stago STA-R MAX. MATERIALS AND METHODS: Imprecision and bias were evaluated for activated partial thromboplastin time (APTT), fibrinogen (FIB), and prothrombin time (PT) at the medical decision levels. The results of 200, 181, and 206 plasma samples for APTT, FIB, and PT, respectively, were compared with those obtained by Stago STA-R MAX. In addition, the interference level of bilirubin, haemoglobin, triglycerides, and fractionated heparin was evaluated. RESULTS: Repeatability, intermediate imprecision, bias, and total error are overall below the defined limits of acceptability. Of interest is the high degree of agreement between Solea 100 and STA-R MAX with respect to PT (s), which fits perfectly with the theoretical line of identity (y = 0 + 1.00x). No interferences were found within the limits stated by the manufacturer, with some exceptions for APTT with heparin and APTT and PT for higher bilirubin concentrations. CONCLUSIONS: In conclusion, the performance of the Solea 100 optical analyser is satisfactory and adequate for the determination of routine coagulation tests. Moreover, they are perfectly comparable to mechanical systems, such as STA-R MAX and other upper-level analysers, even considering the low interference levels under routine conditions.

Protocetraric and Salazinic Acids as Potential Inhibitors of SARS-CoV-2 3CL Protease: Biochemical, Cytotoxic, and Computational Characterization of Depsidones as Slow-Binding Inactivators.

The study investigated the inhibitory activity of protocetraric and salazinic acids against SARS-CoV-2 3CLpro. The kinetic parameters were determined by microtiter plate-reading fluorimeter using a fluorogenic substrate. The cytotoxic activity was tested on murine Sertoli TM4 cells. In silico analysis was performed to ascertain the nature of the binding with the 3CLpro. The compounds are slow-binding inactivators of 3CLpro with a Ki of 3.95 µM and 3.77 µM for protocetraric and salazinic acid, respectively, and inhibitory efficiency kinact/Ki at about 3 × 10-5 s-1µM-1. The mechanism of inhibition shows that both compounds act as competitive inhibitors with the formation of a stable covalent adduct. The viability assay on epithelial cells revealed that none of them shows cytotoxicity up to 80 µM, which is well below the Ki values. By molecular modelling, we predicted that the catalytic Cys145 makes a nucleophilic attack on the carbonyl carbon of the cyclic ester common to both inhibitors, forming a stably acyl-enzyme complex. The computational and kinetic analyses confirm the formation of a stable acyl-enzyme complex with 3CLpro. The results obtained enrich the knowledge of the already numerous biological activities exhibited by lichen secondary metabolites, paving the way for developing promising scaffolds for the design of cysteine enzyme inhibitors.