Although β-lactams have been the most effective class of antibacterial agents used in clinical practice for the past half century, their effectiveness on Gram-negative bacteria has been eroded due to the emergence and spread of β-lactamase enzymes that are not affected by currently marketed β-lactam/β-lactamase inhibitor combinations. Avibactam is a novel, covalent, non-β-lactam β-lactamase inhibitor presently in clinical development in combination with either ceftaroline or ceftazidime. In vitro studies show that avibactam may restore the broad-spectrum activity of cephalosporins against class A, class C, and some class D β-lactamases. Here we describe the structures of two clinically important β-lactamase enzymes bound to avibactam, the class A CTX-M-15 extended-spectrum β-lactamase and the class C Pseudomonas aeruginosa AmpC β-lactamase, which together provide insight into the binding modes for the respective enzyme classes. The structures reveal similar binding modes in both enzymes and thus provide a rationale for the broad-spectrum inhibitory activity of avibactam. Identification of the key residues surrounding the binding pocket allows for a better understanding of the potency of this scaffold. Finally, avibactam has recently been shown to be a reversible inhibitor, and the structures provide insights into the mechanism of avibactam recyclization. Analysis of the ultra-high-resolution CTX-M-15 structure suggests how the deacylation mechanism favors recyclization over hydrolysis.
Acquired metallo-β-lactamases (MBLs) are resistance determinants of increasing clinical importance in Gram-negative bacterial pathogens, which confer a broad-spectrum β-lactam resistance, including carbapenems. Several such enzymes have been described since the 1990s. In the present study, a novel acquired MBL, named FIM-1, was identified and characterized. The blaFIM-1 gene was cloned from a multidrug-resistant Pseudomonas aeruginosa clinical isolate (FI-14/157) cultured from a patient with a vascular graft infection in Florence, Italy. The isolate belonged in the sequence type 235 epidemic clonal lineage. The FIM-1 enzyme is a member of subclass B1 and, among acquired MBLs, exhibited the highest similarity (ca. 40% amino acid identity) with NDM-type enzymes. In P. aeruginosa FI-14/157, the blaFIM-1 gene was apparently inserted into the chromosome and associated with ISCR19-like elements that were likely involved in the capture and mobilization of this MBL gene. Transfer experiments of the blaFIM-1 gene to an Escherichia coli strain or another P. aeruginosa strain by conjugation or electrotransformation were not successful. The FIM-1 protein was produced in E. coli and purified by two chromatography steps. Analysis of the kinetic parameters, carried out with the purified enzyme, revealed that FIM-1 has a broad substrate specificity, with a preference for penicillins (except the 6α-methoxy derivative temocillin) and carbapenems. Aztreonam was not hydrolyzed. Detection of this novel type of acquired MBL in a P. aeruginosa clinical isolate underscores the increasing diversity of such enzymes that can be encountered in the clinical setting.
TEM-72 is a quadruple mutant of TEM-1 and shows extended-spectrum β-lactamase properties. The present structure shows the presence of a citrate anion bound to the TEM-72 active site and supports the use of polycarboxylates as a scaffold for the design of broad-spectrum inhibitors of serine β-lactamases.
TEM-72, a class A β-lactamase identified in isolates of Enterobacteriaceae, is a quadruple mutant of TEM-1 (Q39K, M182T, G238S and E240K) and shows extended-spectrum β-lactamase (ESBL) properties arising from the G238S and E240K substitutions. Although many structures of TEM variants have been published, they do not include an enzyme with the simultaneous presence of both of the ESBL-conferring G238S and E240K substitutions. Furthermore, the structure shows the presence of a citrate anion bound to the TEM-72 active site, where it interacts with all of the conserved residues of class A β-lactamases. The present structure supports the use of polycarboxylates as a scaffold for the design of broad-spectrum inhibitors of serine β-lactamases.
class A β-lactamases; extended-spectrum β-lactamases; inhibitors; citrate; polycarboxylate
Streptococcus pneumoniae expresses three distinct sialidases, NanA, NanB, and NanC, that are believed to be key virulence factors and thus, potential important drug targets. We previously reported that the three enzymes release different products from sialosides, but could share a common catalytic mechanism before the final step of product formation. However, the kinetic investigations of the three sialidases have not been systematically done thus far, due to the lack of an easy and steady measurement of sialidase reaction rate.
In this work, we present further kinetic characterization of pneumococcal sialidases by using a direct spectrophotometric method with the chromogenic substrate p-nitrophenyl-N-acetylneuraminic acid (p-NP-Neu5Ac). Using our assay, the measured kinetic parameters of the three purified pneumococcal sialidase, NanA, NanB and NanC, were obtained and were in perfect agreement with the previously published data. The major advantage of this alternative method resides in the direct measurement of the released product, allowing to readily determine of initial reaction rates and record complete hydrolysis time courses.
We developed an accurate, fast and sensitive spectrophotometric method to investigate the kinetics of sialidase-catalyzed reactions. This fast, sensitive, inexpensive and accurate method could benefit the study of the kinetics and inhibition of sialidases in general.
Sialidase; Neuraminidase; Chromogenic sialic acids; Kinetic assay; Streptococcus pneumoniae
Sialic acid (N-acetylneuraminic acid; NeuNAc) is one of the most important carbohydrates for Streptococcus pneumoniae due of its role as a carbon and energy source, receptor for adhesion and invasion and molecular signal for promotion of biofilm formation, nasopharyngeal carriage and invasion of the lung.
In this work, NeuNAc and its metabolic derivative N-acetyl mannosamine (ManNAc) were used to analyze regulatory mechanisms of the neuraminidase locus expression. Genomic and metabolic comparison to Streptococcus mitis, Streptococcus oralis, Streptococcus gordonii and Streptococcus sanguinis elucidates the metabolic association of the two amino sugars to different parts of the locus coding for the two main pneumococcal neuraminidases and confirms the substrate specificity of the respective ABC transporters. Quantitative gene expression analysis shows repression of the locus by glucose and induction of all predicted transcriptional units by ManNAc and NeuNAc, each inducing with higher efficiency the operon encoding for the transporter with higher specificity for the respective amino sugar. Cytofluorimetric analysis demonstrated enhanced surface exposure of NanA on pneumococci grown in NeuNAc and ManNAc and an activity assay allowed to quantify approximately twelve times as much neuraminidase activity on induced cells as opposed to glucose grown cells.
The present data increase the understanding of metabolic regulation of the nanAB locus and indicate that experiments aimed at the elucidation of the relevance of neuraminidases in pneumococcal virulence should possibly not be carried out on bacteria grown in glucose containing media.
Sialic acid; Metabolic regulation; Carbon catabolite repression
The TEM-107 extended-spectrum β-lactamase detected in a Klebsiella pneumoniae clinical isolate had a Gly238Ser substitution compared to the TEM-43 β-lactamase. The MIC of ceftazidime was higher (64 μg/ml) than that of cefotaxime (2 μg/ml) for the isolate. Clavulanic acid reduced the MIC of ceftazidime 64-fold.
The metallo-β-lactamase VIM-4, mainly found in Pseudomonas aeruginosa or Acinetobacter baumannii, was produced in Escherichia coli and characterized by biochemical and X-ray techniques. A detailed kinetic study performed in the presence of Zn2+ at concentrations ranging from 0.4 to 100 μM showed that VIM-4 exhibits a kinetic profile similar to the profiles of VIM-2 and VIM-1. However, VIM-4 is more active than VIM-1 against benzylpenicillin, cephalothin, nitrocefin, and imipenem and is less active than VIM-2 against ampicillin and meropenem. The crystal structure of the dizinc form of VIM-4 was solved at 1.9 Å. The sole difference between VIM-4 and VIM-1 is found at residue 228, which is Ser in VIM-1 and Arg in VIM-4. This substitution has a major impact on the VIM-4 catalytic efficiency compared to that of VIM-1. In contrast, the differences between VIM-2 and VIM-4 seem to be due to a different position of the flapping loop and two substitutions in loop 2. Study of the thermal stability and the activity of the holo- and apo-VIM-4 enzymes revealed that Zn2+ ions have a pronounced stabilizing effect on the enzyme and are necessary for preserving the structure.
The IMP-13 metallo-β-lactamase was overproduced in Escherichia coli BL21(DE3) and purified by chromatography. Analysis of kinetic parameters revealed some notable differences with other IMP-type enzymes, noteworthily a higher catalytic efficiency toward ticarcillin and piperacillin and a marked preference for imipenem over meropenem.
The production of metallo-β-lactamase (MBL) is an important mechanism of resistance to β-lactam antibiotics, including carbapenems. Despite the discovery and emergence of many acquired metallo-β-lactamases, IMP-type determinants (now counting at least 27 variants) remain the most prevalent in some geographical areas. In Asian countries, and notably Japan, IMP-1 and its closely related variants are most widespread. Some other variants have been detected in other countries and show either an endemic (e.g., IMP-13 in Italy) or sporadic (e.g., IMP-12 in Italy or IMP-18 in the United States) occurrence. The IMP-18-producing Pseudomonas aeruginosa strain PS 297 from the southwestern United States carried at least two class 1 integrons. One was identical to In51, while the other, named In133 and carrying the blaIMP-18 gene cassette in the third position, showed an original array of five gene cassettes, including aacA7, qacF, aadA1, and an unknown open reading frame (ORF). Interestingly. In133 differed significantly from In96, the blaIMP-18-carrying integron identified in a P. aeruginosa isolate from Mexico. The meropenem and ertapenem MIC values were much lower for Escherichia coli strains producing IMP-18 (0.06 and 0.12 μg/ml, respectively) than for strains producing IMP-1 (2 μg/ml for each). Kinetic data obtained with the purified enzyme revealed lower turnover rates of IMP-18 than of other IMP-type enzymes with most substrates.
Metallo-β-lactamases (MBLs) are important enzymatic factors in resistance to β-lactam antibiotics that show important structural and functional heterogeneity. BJP-1 is a subclass B3 MBL determinant produced by Bradyrhizobium japonicum that exhibits interesting properties. BJP-1, like CAU-1 of Caulobacter vibrioides, overall poorly recognizes β-lactam substrates and shows an unusual substrate profile compared to other MBLs. In order to understand the structural basis of these properties, the crystal structure of BJP-1 was obtained at 1.4-Å resolution. This revealed significant differences in the conformation and locations of the active-site loops, determining a rather narrow active site and the presence of a unique N-terminal helix bearing Phe-31, whose side chain binds in the active site and represents an obstacle for β-lactam substrate binding. In order to probe the potential of sulfonamides (known to inhibit various zinc-dependent enzymes) to bind in the active sites of MBLs, the structure of BJP-1 in complex with 4-nitrobenzenesulfonamide was also obtained (at 1.33-Å resolution), thereby revealing the mode of interaction of these molecules in MBLs. Interestingly, sulfonamide binding resulted in the displacement of the side chain of Phe-31 from its hydrophobic binding pocket, where the benzene ring of the molecule is now found. These data further highlight the structural diversity shown by MBLs but also provide interesting insights in the structure-function relationships of these enzymes. More importantly, we provided the first structural observation of MBL interaction with sulfonamides, which might represent an interesting scaffold for the design of MBL inhibitors.
Metallo-β-lactamase (MBL)-producing bacteria are emerging worldwide and represent a formidable threat to the efficacy of relevant β-lactams, including carbapenems, expanded-spectrum cephalosporins, and β-lactamase inactivator/β-lactam combinations. VIM-2 is currently the most widespread MBL and represents a primary target for MBL inhibitor research, the clinical need for which is expected to further increase in the future. Using a saturation mutagenesis approach, we probed the importance of four residues (Phe-61, Ala-64, Tyr-67, and Trp-87) located close to the VIM-2 active site and putatively relevant to the enzyme activity based on structural knowledge of the enzyme and on structure-activity relationships of the subclass B1 MBLs. The ampicillin MIC values shown by the various mutants were affected very differently depending on the randomized amino acid position. Position 64 appeared to be rather tolerant to substitution, and kinetic studies showed that the A64W mutation did not significantly affect substrate hydrolysis or binding, representing an important difference from IMP-type enzymes. Phe-61 and Tyr-67 could be replaced with several amino acids without the ampicillin MIC being significantly affected, but in contrast, Trp-87 was found to be critical for ampicillin resistance. Further kinetic and biochemical analyses of W87A and W87F variants showed that this residue is apparently important for the structure and proper folding of the enzyme but, surprisingly, not for its catalytic activity. These data support the critical role of residue 87 in the stability and folding of VIM-2 and might have strong implications for MBL inhibitor design, as this residue would represent an ideal target for interaction with small molecules.
Class D β-lactamases represent a heterogeneous group of active-site serine β-lactamases that show an extraordinary panel of functional features and substrate profiles, thus representing relevant models for biochemical and structural studies. OXA-46 is a narrow-spectrum enzyme belonging to the OXA-2 subgroup which was found in a Pseudomonas aeruginosa clinical isolate from northern Italy. In this work, we obtained the three-dimensional structure of OXA-46, which shows the overall fold of active serine β-lactamases and a dimeric quaternary structure. Significant differences with currently available structures of class D β-lactamases were found in the loops located close to the active site, which differ in length and conformation. Interestingly, the three subunits present in the asymmetric unit showed some structural heterogeneity, only one of which presented a carbamylated lysine recognized as an important functional feature of class D enzymes. The carbamylation state of residue Lys75 appeared to be associated with different shapes and dimensions of the active site. Moreover, a tartrate molecule from the crystallization buffer was found in the active site of the noncarbamylated subunits, which interacts with catalytically relevant residues. The OXA-46 crystal asymmetric units thus interestingly present the structures of the free carbamylated active site and of the ligand-bound uncarbamylated active site, offering the structural basis for investigating the potential of new scaffolds of β-lactamase inhibitors.
Aeromonas enteropelogenes (formerly A. tructi) was described to be an ampicillin-susceptible and cephalothin-resistant Aeromonas species, which suggests the production of a cephalosporinase. Strain ATCC 49803 was susceptible to amoxicillin, cefotaxime, and imipenem but resistant to cefazolin (MICs of 2, 0.032, 0.125, and >256 μg/ml, respectively) and produced an inducible β-lactamase. Cefotaxime-resistant mutants (MIC, 32 μg/ml) that showed constitutive β-lactamase production could be selected in vitro. The gene coding for the cephalosporinase of A. enteropelogenes ATCC 49803 was cloned, and its biochemical properties were investigated. Escherichia coli transformants showing resistance to various β-lactams carried a 3.5-kb plasmid insert whose sequence revealed a 1,146-bp open reading frame (ORF) encoding a class C β-lactamase, named TRU-1, showing the highest identity scores with A. punctata CAV-1 (75%), A. salmonicida AmpC (75%), and A. hydrophila CepH (71%). The blaTRU-1 locus includes open reading frames (ORFs) showing significant homology with genes found in the genomes of other Aeromonas species, although it exhibits a different organization, as reflected by the presence of additional ORFs located downstream of the β-lactamase gene in the A. hydrophila and A. salmonicida genomes. Specific PCR assays were negative for cphA-like and blaOXA-12-like genes in three A. enteropelogenes ATCC strains. Purified TRU-1 showed a broad substrate profile, efficiently hydrolyzing benzylpenicillin, cephalothin, cefoxitin, and, although with significantly lower turnover rates, oxyiminocephalosporins. Cephaloridine and cefepime were poorly recognized by the enzyme, as reflected by the high Km values observed with these substrates. Thus far, A. enteropelogenes represents the only known example of an Aeromonas species that produces only one β-lactamase belonging to molecular class C.
A Pseudomonas aeruginosa isolate recovered in Belgium produced a novel extended-spectrum ß-lactamase, BEL-2, differing from BEL-1 by a single Leu162Phe substitution. That modification significantly altered the kinetic properties of the enzyme, increasing its affinity for expanded-spectrum cephalosporins. The blaBEL-2 gene was identified from a P. aeruginosa isolate clonally related to another blaBEL-1-positive isolate.
The genus Chryseobacterium and other genera belonging to the family Flavobacteriaceae include organisms that can behave as human pathogens and are known to cause different kinds of infections. Several species of Flavobacteriaceae, including Chryseobacterium indologenes, are naturally resistant to β-lactam antibiotics (including carbapenems), due to the production of a resident metallo-β-lactamase. Although C. indologenes presently constitutes a limited clinical threat, the incidence of infections caused by this organism is increasing in some settings, where isolates that exhibit multidrug resistance phenotypes (including resistance to aminoglycosides and quinolones) have been detected. Here, we report the identification and characterization of a new IND-type variant from a C. indologenes isolate from Burkina Faso that is resistant to β-lactams and aminoglycosides. The levels of sequence identity of the new variant to other IND-type metallo-β-lactamases range between 72 and 90% (for IND-4 and IND-5, respectively). The purified enzyme exhibited N-terminal heterogeneity and a posttranslational modification consisting of the presence of a pyroglutamate residue at the N terminus. IND-6 shows a broad substrate profile, with overall higher turnover rates than IND-5 and higher activities than IND-2 and IND-5 against ceftazidime and cefepime.
Metallo-β-lactamases (MBLs) are zinc-dependent bacterial enzymes characterized by an efficient hydrolysis of carbapenems and a lack of sensitivity to commercially available β-lactamase inactivators. Apart from the acquired subclass B1 enzymes, which exhibit increasing clinical importance and whose evolutionary origin remains unclear, most MBLs are encoded by resident genes found in the genomes of organisms belonging to at least three distinct phyla. Using genome database mining, we identified an open reading frame (ORF) (ECA2849) encoding an MBL-like protein in the sequenced genome of Erwinia carotovora, an important plant pathogen. Although no detectable β-lactamase activity could be found in E. carotovora, a recombinant Escherichia coli strain in which the ECA2849 ORF was cloned showed decreased susceptibility to several β-lactams, while carbapenem MICs were surprisingly poorly affected. The enzyme, named CAR-1, was purified by means of ion-exchange chromatography steps, and its characterization revealed unique structural and functional features. This new MBL was able to efficiently hydrolyze cephalothin, cefuroxime, and cefotaxime and, to a lesser extent, penicillins and the other cephalosporins but only poorly hydrolyzed meropenem, while imipenem was not recognized. CAR-1 is the first example of a functional naturally occurring MBL in the family Enterobacteriaceae (order Enterobacteriales) and highlights the extraordinary structural and functional diversity exhibited by MBLs.
A new natural IND-type metallo-β-lactamase variant, IND-5, was identified in a clinical isolate of Chryseobacterium indologenes. IND-5 shared 92.8% and 92.4% amino acid homology with IND-1 and IND-3, respectively. Purified enzyme (pI = 8.8, Mr = 25,000) was able to hydrolyze penicillins, some narrow- and expanded-spectrum cephalosporins, and carbapenems but not monobactams.
Previous studies have reported β-lactamase-mediated penicillin resistance in Fusobacterium nucleatum, but no β-lactamase gene has yet been identified in this species. An F. nucleatum subsp. polymorphum strain resistant to penicillin and amoxicillin was isolated from a human periodontitis sample. DNA cloning and sequencing revealed a 765-bp open reading frame encoding a new class D β-lactamase named FUS-1 (OXA-85). A recombinant Escherichia coli strain carrying the blaFUS-1 gene exhibited resistance to amoxicillin with a moderate decrease in the MICs with clavulanic acid. The blaFUS-1 gene was found in two additional clonally unrelated F. nucleatum subsp. polymorphum isolates. It was located on the chromosome in a peculiar genetic environment where a gene encoding a putative transposase-like protein is found, suggesting a possible acquisition of this class D β-lactamase gene. The FUS-1 enzyme showed the closest ancestral relationship with OXA-63 from Brachyspira pilosicoli (53% identity) and with putative chromosomal β-lactamases of Campylobacter spp. (40 to 42% identity). FUS-1 presents all of the conserved structural motifs of class D β-lactamases. Kinetic analysis revealed that FUS-1 exhibits a narrow substrate profile, efficiently hydrolyzing benzylpenicillin and oxacillin. FUS-1 was poorly inactivated by clavulanate and NaCl. FUS-1 is the first example of a class D β-lactamase produced by a gram-negative, anaerobic, rod-shaped bacterium to be characterized.
The diffusion of metallo-β-lactamases (MBLs) among clinically important human pathogens represents a therapeutic issue of increasing importance. However, the origin of these resistance determinants is largely unknown, although an important number of proteins belonging to the MBL superfamily have been identified in microbial genomes. In this work, we analyzed the distribution and function of genes encoding MBL-like proteins in the class Rhizobiales. Among 12 released complete genomes of members of the class Rhizobiales, a total of 57 open reading frames (ORFs) were found to have the MBL conserved motif and identity scores with MBLs ranging from 8 to 40%. On the basis of the best identity scores with known MBLs, four ORFs were cloned into Escherichia coli for heterologous expression. Among their products, one (blr6230) encoded by the Bradyrhizobium japonicum USDA110 genome, named BJP-1, hydrolyzed β-lactams when expressed in E. coli. BJP-1 enzyme is most closely related to the CAU-1 enzyme from Caulobacter vibrioides (40% amino acid sequence identity), a member of subclass B3 MBLs. A kinetic analysis revealed that BJP-1 efficiently hydrolyzed most β-lactam substrates, except aztreonam, ticarcillin, and temocillin, with the highest catalytic efficiency measured with meropenem. Compared to other MBLs, BJP-1 was less sensitive to inactivation by chelating agents.
An outbreak of Pseudomonas aeruginosa showing a multidrug-resistant (MDR) phenotype (including carbapenems, ceftazidime, cefepime, gentamicin, tobramycin, and fluoroquinolones) was observed, during a 5-month period, in a general intensive care unit of a large tertiary care and clinical research hospital in southern Italy. The outbreak involved 15 patients, with a total of 87 isolates, mostly from lower respiratory tract specimens. Analysis of isolates involved in the outbreak revealed production of metallo-β-lactamase (MBL) activity, and genotyping by pulsed-field gel electrophoresis of genomic DNA digested by SpeI revealed clonal relatedness among isolates. Molecular analysis of the MBL determinant showed the presence of a blaIMP-13 gene carried on a gene cassette inserted in a class 1 integron which also contained an aacA4 aminoglycoside resistance cassette encoding an AAC(6′)-Ib enzyme. The blaIMP-13-containing integron and its genetic environment appeared to be similar to those found in P. aeruginosa isolates producing IMP-13 from a hospital in Rome. The blaIMP-13 gene was not transferable by conjugation and was apparently carried on the chromosome. The outbreak was coincidental with a shortage of nursing personnel, and resolution was apparently associated with reinstatement of nursing personnel and reinforcement of general infection control practices within the intensive care unit. To our best knowledge this is the first description of a nosocomial outbreak of relatively large size caused by an IMP-producing gram-negative pathogen in Europe.
Carbapenem resistance mediated by acquired carbapenemase genes has been increasingly reported, particularly for clinical isolates of Pseudomonas aeruginosa and Acinetobacter spp. Of 1,234 nonduplicate isolates of carbapenem-resistant Pseudomonas spp. and Acinetobacter spp. isolated at a tertiary-care hospital in Seoul, Korea, 211 (17%) were positive for metallo-β-lactamase (MBL). Of these, 204 (96%) had either the blaIMP-1 or blaVIM-2 allele. In addition, seven Acinetobacter baumannii isolates were found to have a novel MBL gene, which was designated blaSIM-1. The SIM-1 protein has a pI of 7.2, is a new member of subclass B1, and exhibits 64 to 69% identity with the IMP-type MBLs, which are its closest relatives. All SIM-1-producing isolates exhibited relatively low imipenem and meropenem MICs (8 to 16 μg/ml) and had a multidrug resistance phenotype. Expression of the cloned blaSIM-1 gene in Escherichia coli revealed that the encoded enzyme is capable of hydrolyzing a broad array of β-lactams, including penicillins, narrow- to expanded-spectrum cephalosporins, and carbapenems. The blaSIM-1 gene was carried on a gene cassette inserted into a class 1 integron, which included three additional cassettes (arr-3, catB3, and aadA1). The strains were isolated from sputum and urine specimens from patients with pneumonia and urinary tract infections, respectively. All patients had various underlying diseases. Pulsed-field gel electrophoresis of SmaI-digested genomic DNAs showed that the strains belonged to two different clonal lineages, indicating that horizontal transfer of this gene had occurred and suggesting the possibility of further spread of resistance in the future.
A novel OXA-type enzyme, named OXA-46, was found to be encoded by a gene cassette inserted into a class 1 integron from a multidrug-resistant Pseudomonas aeruginosa clinical isolate. The variable region of the integron also contained a blaVIM-1 metallo-β-lactamase cassette and a duplicated aacA4 aminoglycoside acetyltransferase cassette. OXA-46 belongs to the OXA-2 lineage of class D β-lactamases. It exhibits 78% sequence identity with OXA-2 and the highest similarity (around 92% identity) with another OXA-type enzyme detected in clinical isolates of Burkholderia cepacia and in unidentified bacteria from a wastewater plant. Expression of blaOXA-46 in Escherichia coli decreased susceptibility to penicillins and narrow-spectrum cephalosporins but not to extended-spectrum cephalosporins, cefsulodin, aztreonam, or carbapenems. The enzyme was overproduced in E. coli and purified by two anion-exchange chromatography steps (approximate yield, 6 mg/liter). OXA-46 was made of a 28.5-kDa polypeptide and exhibited an alkaline pI (7.8). In its native form OXA-46 appeared to be dimeric, and the oligomerization state was not affected by EDTA. Kinetic analysis of OXA-46 revealed a specificity for narrow-spectrum substrates, including oxacillin, other penicillins (but not temocillin), and narrow-spectrum cephalosporins. The enzyme apparently did not interact with temocillin, oxyimino-cephalosporins, or aztreonam. OXA-46 was inactivated by tazobactam and carbapenems and, although less efficiently, also by clavulanic acid. Enzyme activity was not affected either by EDTA or by divalent cations and exhibited low susceptibility to NaCl. These findings underscore the functional and structural diversity that can be encountered among class D β-lactamases.
Three epidemiologically independent Pseudomonas aeruginosa isolates, representative of the first VIM-1 metallo-β-lactamase producers detected at three different hospitals in northern Italy, were investigated to determine their genomic relatedness and to compare the structures of the genetic supports for the VIM-1 determinants. The three isolates, all of serotype O11, appeared to be clonally related according to the results of genotyping by macrorestriction analysis of genomic DNA by pulsed-field gel electrophoresis and random amplification of polymorphic DNA. Investigation of the genetic support for the blaVIM-1 determinant revealed that it was carried on identical or almost identical integrons (named In70.2 and In70.3) located within a conserved genomic context. The integrons were structurally related to In70 and In110, two plasmid-borne blaVIM-1-containing integrons from Achromobacter xylosoxidans and Pseudomonas putida isolates, respectively, from the same geographic area (northern Italy) and were found to be inserted close to the res site of a Tn5051-like transposon, different from any of those described previously, that was apparently carried on the bacterial chromosome. The present findings suggest that the three VIM-1-producing isolates are members of the same clonal complex which have been spreading in hospitals in northern Italy since the late 1990s and point to a common ancestry of their blaVIM-1-containing integrons.
The THIN-B metallo-β-lactamase, a subclass B3 enzyme produced by the environmental species Janthinobacterium lividum, was overproduced in Escherichia coli by means of a T7-based expression system. The enzyme was purified (>95%) by two ion-exchange chromatography steps and subjected to biochemical analysis. The native THIN-B enzyme is a monomeric protein of 31 kDa. It exhibits the highest catalytic efficiencies with carbapenem substrates and cephalosporins, except for cephaloridine, which acts as a poor inactivator. Individual rate constants for inactivation by chelators were measured, suggesting that inactivation occurred by a mechanism involving formation of a ternary complex.