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Functional and structural damage of the intestinal mucosal barrier significantly contribute to translocation of gut microbial products into the bloodstream and are largely involved in HIV-1 associated chronic immune activation. This microbial translocation is largely due to a progressive exhaustion of intestinal macrophage phagocytic function, which leads to extracellular accumulation of microbial derived components and results in HIV-1 disease progression. This study aims to better understand whether the modulation of gut microbiota promotes an intestinal immune restoration in people living with HIV (PLWH). Long-term virologically suppressed PLWH underwent blood, colonic, and fecal sampling before (T0) and after 6 months (T6) of oral bacteriotherapy. Age- and gender-matched uninfected controls (UC) were also included. 16S rRNA gene sequencing was applied to all participants' fecal microbiota. Apoptosis machinery, mitochondria, and apical junctional complex (AJC) morphology and physiological functions were analyzed in gut biopsies. At T0, PLWH showed a different pattern of gut microbial flora composition, lower levels of occludin (p = 0.002) and zonulin (p = 0.01), higher claudin-2 levels (p = 0.002), a reduction of mitochondria number (p = 0.002), and diameter (p = 0.002), as well as increased levels of lipopolysaccharide (LPS) (p = 0.018) and cCK18 (p = 0.011), compared to UC. At T6, an increase in size (p = 0.005) and number (p = 0.008) of mitochondria, as well as amelioration in AJC structures (p 0.0001) were observed. Restoration of bacterial richness (Simpson index) and biodiversity (Shannon index) was observed in all PLWH receiving oral bacteriotherapy (p 0.05). Increased mitochondria size (p = 0.005) and number (p = 0.008) and amelioration of AJC structure (p 0.0001) were found at T6 compared to T0. Moreover, increased occludin and zonulin concentration were observed in PLWH intestinal tracts and decreased levels of claudin-2, LPS, and cCK18 were found after oral bacteriotherapy (T0 vs. T6, p 0.05 for all these measures). Oral bacteriotherapy supplementation might restore the balance of intestinal flora and support the structural and functional recovery of the gut mucosa in antiretroviral therapy treated PLWH.

The MOX lineage of β-lactamases includes a group of molecular class C enzymes (AmpCs) encoded by genes mobilized from the chromosomes of Aeromonas spp. to plasmids. MOX-9, previously identified as a plasmid-encoded enzyme from a Citrobacter freundii isolate, belongs to a novel sublineage of MOX enzymes, derived from the resident Aeromonas media AmpC. The bla(MOX-9) gene was found to be carried on a transposon, named Tn7469, likely responsible for its mobilization to plasmidic context. MOX-9 was overexpressed in Escherichia coli, purified, and subjected to biochemical characterization. Kinetic analysis showed a relatively narrow-spectrum profile with strong preference for cephalosporin substrates, with some differences compared with MOX-1 and MOX-2. MOX-9 was not inhibited by clavulanate and sulbactam, while both tazobactam and avibactam acted as inhibitors in the micromolar range.

KPC-53 enzyme is a natural KPC variant which showed a duplication of L167E168 residues in the Ω-loop structure. The bla(KPC-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 k(cat) and k(cat)/K(m) versus several β-lactams, in particular carbapenems. Indeed, a 2,000-fold reduction was observed in the k(cat) 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.

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.

Purpose: The mediator (MED) multisubunit-complex modulates the activity of the transcriptional machinery, and genetic defects in different MED subunits (17, 20, 27) have been implicated in neurologic diseases. In this study, we identified a recurrent homozygous variant in MED11 (c.325C>T; p.Arg109Ter) in 7 affected individuals from 5 unrelated families. Methods: To investigate the genetic cause of the disease, exome or genome sequencing were performed in 5 unrelated families identified via different research networks and Matchmaker Exchange. Deep clinical and brain imaging evaluations were performed by clinical pediatric neurologists and neuroradiologists. The functional effect of the candidate variant on both MED11 RNA and protein was assessed using reverse transcriptase polymerase chain reaction and western blotting using fibroblast cell lines derived from 1 affected individual and controls and through computational approaches. Knockouts in zebrafish were generated using clustered regularly interspaced short palindromic repeats/Cas9. Results: The disease was characterized by microcephaly, profound neurodevelopmental impairment, exaggerated startle response, myoclonic seizures, progressive widespread neurodegeneration, and premature death. Functional studies on patient-derived fibroblasts did not show a loss of protein function but rather disruption of the C-terminal of MED11, likely impairing binding to other MED subunits. A zebrafish knockout model recapitulates key clinical phenotypes. Conclusion: Loss of the C-terminal ofMED subunit 11 may affect its binding efficiency to otherMED subunits, thus implicating the MED-complex stability in brain development and neurodegeneration.

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.

The ability to form biofilms is a recognized trait of Stenotrophomonas maltophilia, but the extent of its clinical relevance is still unclear. The present multicenter prospective study (ANSELM) aims at investigating the association between biofilm formation and clinical outcomes of S. maltophilia infections. One hundred and nine isolates were collected from various geographical origins and stratified according to their clinical relevance. Biofilm formation was evaluated by the microtiter plate assay and correlated with microbiological and clinical data from the associated strains. Antibiotic susceptibility of the planktonic cells was tested by the disk diffusion technique, while antibiotic activity against mature biofilms was spectrophotometrically assessed. Most strains (91.7%) were able to form biofilm, although bloodborne strains produced biofilm amounts significantly higher than strains causing hospital- rather than community-acquired infections, and those recognized as &ldquo, definite&rdquo, pathogens. Biofilm formation efficiency was positively correlated with mechanical ventilation (p = 0.032), whereas a negative relationship was found with antibiotic resistance (r2 = 0.107, p &lt, 0.001), specifically in the case of the pathogenic strains. Mature S. maltophilia biofilms were markedly more resistant (up to 128 times) to cotrimoxazole and levofloxacin compared with their planktonic counterparts, especially in the case of bloodborne strains. Our findings indicate that biofilm formation by S. maltophilia is obviously a contributing factor in the pathogenesis of infections, especially in deep ones, thus warranting additional studies with larger cohort of patients and isolates.

Background: We previously reported that severe COVID-19 patients had higher chances of survival and a reduced risk of developing respiratory failure when administered with the probiotic formulation SLAB51. This study aimed to investigate further bacteriotherapy mechanisms and how early they are activated. Methods: We performed an analysis on the blood oxygenation parameters collected in sixty-nine severe COVID-19 patients requiring non-invasive oxygen therapy and presenting a CT lung involvement ≥50%. Twenty-nine patients received low-molecular-weight heparin, azithromycin and Remdesivir. In addition, forty subjects received SLAB51. Blood gas analyses were performed before the beginning of treatments and at 24 h. Results: The patients receiving only standard therapy needed significantly increased oxygen amounts during the 24 h observation period. Furthermore, they presented lower blood levels of pO2, O2Hb and SaO2 than the group also supplemented with oral bacteriotherapy. In vitro data suggest that SLAB51 can reduce nitric oxide synthesis in intestinal cells. Conclusions: SARS-CoV-2 infected patients may present lesions in the lungs compromising their gas exchange capability. The functionality of the organs essential for these patients’ survival depends mainly on the levels of pO2, O2Hb and SaO2. SLAB51 contains enzymes that could reduce oxygen consumption in the intestine, making it available for the other organs.

The Guiana extended-spectrum (GES) β-lactamase GES G170H , GES G170L , and GES G170K mutants showed k cat , K m , and k cat / K m 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.

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., 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. ​

We report a 64-years old man with B-cell CLL infected by SARS-CoV-2 during his hospitalization for a spondylodiscitis. Because of his health conditions the duration of the antiviral therapy was restricted to one week where we observed a transient disappearance of CD19+/CD5+ B-lymphocytes in peripheral blood.

The ability to form biofilms is a recognized trait of Stenotrophomonas maltophilia, but the extent of its clinical relevance is still unclear. The present multicenter prospective study (ANSELM) aims at investigating the association between biofilm formation and clinical outcomes of S. maltophilia infections. One hundred and nine isolates were collected from various geographical origins and stratified according to their clinical relevance. Biofilm formation was evaluated by the microtiter plate assay and correlated with microbiological and clinical data from the associated strains. Antibiotic susceptibility of the planktonic cells was tested by the disk diffusion technique, while antibiotic activity against mature biofilms was spectrophotometrically assessed. Most strains (91.7%) were able to form biofilm, although bloodborne strains produced biofilm amounts significantly higher than strains causing hospital- rather than community-acquired infections, and those recognized as “definite” pathogens. Biofilm formation efficiency was positively correlated with mechanical ventilation (p = 0.032), whereas a negative relationship was found with antibiotic resistance (r2 = 0.107; p < 0.001), specifically in the case of the pathogenic strains. Mature S. maltophilia biofilms were markedly more resistant (up to 128 times) to cotrimoxazole and levofloxacin compared with their planktonic counterparts, especially in the case of bloodborne strains. Our findings indicate that biofilm formation by S. maltophilia is obviously a contributing factor in the pathogenesis of infections, especially in deep ones, thus warranting additional studies with larger cohort of patients and isolates.

The Guiana extended-spectrum (GES) β-lactamase GES(G170H), GES(G170L), and GES(G170K) mutants showed k(cat), K(m), and k(cat)/K(m) 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.

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.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).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 MICThe inhibition level of NDM enzymes provided by taniborbactam protects the antibacterial activity of cefepime from this important metallo-β-lactamase.

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

New Delhi Metallo-&beta, lactamase-1 (NDM-1) is the most prevalent type of metallo-&beta, lactamase, able to hydrolyze almost all antibiotics of the &beta, 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-&beta, lactamase (VIM-1) (subclass B1) and L1 metallo-&beta, lactamase (L1) (subclass B3) with inhibitory concentration (IC50) values ranging from 100 to 0.04 &mu, M. 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 &beta, 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.

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.

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.

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

The New Delhi metallo-β-lactamase-1 (NDM-1) enzyme is the most common metallo-β-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 K(m), k(cat), and k(cat)/K(m) 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 k(cat) 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 k(cat)/K(m) 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 β-lactams. NDM numbering is used in order to facilitate the comparison with other NDM-1 studies.

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 MIC50/MIC90 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.

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 blaKPC-3 was found in Tn4401a transposon with the tnpR, tnpA, ISKpn6, and ISKpn7 mobile elements whereas blaCTX-M-15 was detected downstream ISEcp1 genetic element. A class 1 integron with a gene cassette of 780 bp corresponding to aadA2 gene was identified in 33 K. pneumoniae ST512 isolates.

GES-type β-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-1 P174E and GES-5 P174E mutants, selected by site saturation mutagenesis, were purified and kinetically characterized. In comparison with GES-1 and GES-5 wild-type enzymes, GES-1 P174E and GES-5 P174E mutants exhibited lower k cat and k cat / K m values for cephalosporins and penicillins. Concerning carbapenems, GES-1 P174E shared higher k cat values but lower K m values than those calculated for GES-1. The GES-1 P174E and GES-5 P174E mutants showed high hydrolytic efficiency for imipenem, with k cat / K m values 100- and 660-fold higher, respectively, than those of GES-1. Clavulanic acid and tazobactam are good inhibitors for both GES-1 P174E and GES-5 P174E . Molecular dynamic (MD) simulations carried out for GES-1, GES-5, GES-1 P174E , and GES-5 P174E 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.

TEM-184, a novel TEM-derived extended-spectrum β-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.

Objectives The aim of this study was to evaluate the role of residue 238 in CTX-M-15 and CTX-M-15G238C mutant with respect to carbapenems and various β-lactamase inhibitors. Methods A CTX-M-15G238C laboratory mutant was generated by site-directed mutagenesis from CTX-M-15 enzyme by replacing glycine 238 with cysteine. Thiol titration and p-chloromercuribenzoate (PCMB) inactivation assays were used to ascertain the presence of a disulfide bridge in the active site of CTX-M-15G238C. Kinetic parameters were determined both for CTX-M-15 and CTX-M-15G238C enzymes by analysing either the complete hydrolysis time courses or under initial rate conditions. Results In CTX-M-15G238C mutant, the two cysteines (C69 and C238) located in the enzyme active site were unable to form a disulfide bridge. CTX-M-15 and thermostable CTX-M-15G238C were used to study the kinetic interaction with carbapenems, which behaved as poor substrates for both enzymes. Meropenem and ertapenem acted as transient inactivators for CTX-M-15 and CTX-M-15G238C, and for these compounds the variation of kobs versus the inactivator concentration was linear. Imipenem behaved as a transient inactivator for CTX-M-15 and as an inactivator (with k+3 = 0) for CTX-M-15G238C. In any case, the k+2/K values for CTX-M-15G238C were higher than those for CTX-M-15. Conclusions Compared with CTX-M-15, CTX-M-15G238C mutant appears to have a more favourable conformation for carbapenem acylation and higher activity against cefotaxime, which could be due to the presence of free –SH groups in the enzyme active site.

The New Delhi metallo-&beta, 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 DH5&alpha, /NDM-24 showed a MIC value 4-fold higher than that of E. coli DH5&alpha, /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 &beta, lactams tested, except penicillins. The thermostability testing revealed that V88L generated a destabilized effect on NDM-24. The V88L substitution occurred in the &beta, strand and low &beta, 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.

The aim of this study was to evaluate the role of residue 238 in CTX-M-15 and CTX-M-15A CTX-M-15In CTX-M-15Compared with CTX-M-15, CTX-M-15

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-β-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.