Camel-Derived Nanobodies as Potent Inhibitors of New Delhi Metallo-ß-Lactamase-1 Enzyme.

The injudicious usage of antibiotics during infections caused by Gram-negative bacteria leads to the emergence of ß-lactamases. Among them, the NDM-1 enzyme poses a serious threat to human health. Developing new antibiotics or inhibiting ß-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.

Molecular and Kinetic Characterization of MOX-9, a Plasmid-Mediated Enzyme Representative of a Novel Sublineage of MOX-Type Class C ß-Lactamases.

The MOX lineage of ß-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 blaMOX-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.

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.

Mutational Effects on Carbapenem Hydrolysis of YEM-1, a New Subclass B2 Metallo-ß-Lactamase from Yersinia mollaretii.

Analysis of the genome sequence of Yersinia mollaretii ATCC 43969 identified the blaYEM gene, encoding YEM-1, a putative subclass B2 metallo-ß-lactamase. The objectives of our work were to produce and purify YEM-1 and to complete its kinetic characterization. YEM-1 displayed the narrowest substrate range among known subclass B2 metallo-ß-lactamases, since it can hydrolyze imipenem, but not other carbapenems, such as biapenem, meropenem, doripenem, and ertapenem, with high catalytic efficiency. A possible explanation of this activity profile is the presence of tyrosine at residue 67 (loop L1), threonine at residue 156 (loop L2), and serine at residue 236 (loop L3). We showed that replacement of Y67 broadened the activity profile of the enzyme for all carbapenems but still resulted in poor activity toward the other ß-lactam classes.

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.

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.

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.

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.

Coexistence of blaNDM-5, blaCTX-M-15, blaOXA-232, blaSHV-182 genes in multidrug-resistant K. pneumoniae ST437-carrying OmpK36 and OmpK37 porin mutations: First report in Italy.

OBJECTIVES: K. pneumoniae is a common cause of severe hospital-acquired infections. In the present study, we have characterised the whole-genome of two K. pneumoniae ST437 belonging to the clonal complex CC258. METHODS: The whole-genome sequencing was performed by MiSeq Illumina, with a 2 × 300bp paired-end run. ResFinder 4.4.2 was used to detect acquired antimicrobial resistance genes (ARGs) and chromosomal mutations. Mobile genetic elements (plasmids and ISs) were identified by MobileElementFinder v1.0.3. The genome was also assigned to ST using MLST 2.0.9. Virulence factors were detected using the Virulence Factor Database (VFDB). RESULTS: K. pneumoniae KPNAQ_1/23 and KPNAQ_2/23 strains, isolated from urine samples of hospitalised patients, showed resistance to most antibiotics, including ceftazidime-avibactam, ceftolozane-tazobactam, and meropenem-vaborbactam combinations. Both strains were susceptible only to cefiderocol. Multiple mechanisms of resistance were identified. Resistance to ß-lactams was due to the presence of NDM-5, OXA-232, CTX-M-15, SHV-182 ß-lactamases, and OmpK36 and OmpK37 porin mutations. Resistance to fluoroquinolones was mediated by chromosomal mutations in acrR, oqxAB efflux pumps, and the bifunctional gene aac(6')-Ib-cr. CONCLUSION: The presence of different virulence genes makes these KPNAQ_1/23 and KPNAQ_2/23 high-risk clones.

Synergistic Activity of Temocillin and Fosfomycin Combination against KPC-Producing Klebsiella pneumoniae Clinical Isolates.

Infections caused by KPC-producing K. pneumoniae continue to pose a significant clinical challenge due to their emerging resistance to new antimicrobials. We investigated the association between two drugs whose roles have been repurposed against multidrug-resistant bacteria: fosfomycin and temocillin. Temocillin exhibits unusual stability against KPC enzymes, while fosfomycin acts as a potent "synergizer". We conducted in vitro antimicrobial activity studies on 100 clinical isolates of KPC-producing K. pneumoniae using a combination of fosfomycin and temocillin. The results demonstrated synergistic activity in 91% of the isolates. Subsequently, we assessed the effect on Galleria mellonella larvae using five genetically different KPC-Kp isolates. The addition of fosfomycin to temocillin increased larvae survival from 73 to 97% (+Δ 32%; isolate 1), from 93 to 100% (+Δ 7%; isolate 2), from 63 to 86% (+Δ 36%; isolate 3), from 63 to 90% (+Δ 42%; isolate 4), and from 93 to 97% (+Δ 4%; isolate 10). Among the temocillin-resistant KPC-producing K. pneumoniae isolates (24 isolates), the addition of fosfomycin reduced temocillin MIC values below the resistance breakpoint in all isolates except one. Temocillin combined with fosfomycin emerges as a promising combination against KPC-producing K. pneumoniae, warranting further clinical evaluation.

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.