Structural Investigations of the Inhibition of Escherichia coli AmpC ß-Lactamase by Diazabicyclooctanes.

ß-Lactam antibiotics are presently the most important treatments for infections by pathogenic Escherichia coli, but their use is increasingly compromised by ß-lactamases, including the chromosomally encoded class C AmpC serine-ß-lactamases (SBLs). The diazabicyclooctane (DBO) avibactam is a potent AmpC inhibitor; the clinical success of avibactam combined with ceftazidime has stimulated efforts to optimize the DBO core. We report kinetic and structural studies, including four high-resolution crystal structures, concerning inhibition of the AmpC serine-ß-lactamase from E. coli (AmpC EC ) by clinically relevant DBO-based inhibitors: avibactam, relebactam, nacubactam, and zidebactam. Kinetic analyses and mass spectrometry-based assays were used to study their mechanisms of AmpC EC inhibition. The results reveal that, under our assay conditions, zidebactam manifests increased potency (apparent inhibition constant [Kiapp], 0.69 µM) against AmpC EC compared to that of the other DBOs (Kiapp = 5.0 to 7.4 µM) due to an ∼10-fold accelerated carbamoylation rate. However, zidebactam also has an accelerated off-rate, and with sufficient preincubation time, all the DBOs manifest similar potencies. Crystallographic analyses indicate a greater conformational freedom of the AmpC EC -zidebactam carbamoyl complex compared to those for the other DBOs. The results suggest the carbamoyl complex lifetime should be a consideration in development of DBO-based SBL inhibitors for the clinically important class C SBLs.

A Unified Numbering Scheme for Class C ß-Lactamases.

A standard numbering scheme has been proposed for class C ß-lactamases. This will significantly enhance comparison of biochemical and biophysical studies performed on different members of this class of enzymes and facilitate communication in the field.

Complexes formed by the siderophore-based monosulfactam antibiotic BAL30072 and their interaction with the outer membrane receptor PiuA of P. aeruginosa.

Nuclear magnetic resonance and infrared spectroscopy have been used to investigate the formation of complexes of BAL30072 with Fe3+ and Ga3+ in solution and to collect geometrical parameters supporting reliable 3D structure models. Structural models for the ligand-metal complexes with different stoichiometries have been characterized using density functional theory calculations. Blind ensemble docking to the PiuA receptor from P. aeruginosa was performed for the different complexes to compare binding affinities and statistics of the residues most frequently contacted. When compared to analogues, BAL30072 was found to have an intrinsic propensity to form complexes with low ligand-to-metal stoichiometry. By using one of the sulfate oxygen atoms as a third donor in addition to the bidentate pyridinone moiety, BAL30072 can form a L2M complex, which was predicted to be the one with the best binding affinity to PiuA. The example of BAL30072 strongly suggests that a lower stoichiometry might be the one recognized by the receptor, so that to focus only on the highest stoichiometry might be misleading for siderophores with less than six donors.

Molecular Basis of Filtering Carbapenems by Porins from ß-Lactam-resistant Clinical Strains of Escherichia coli.

Integral membrane proteins known as porins are the major pathway by which hydrophilic antibiotics cross the outer membrane of Gram-negative bacteria. Single point mutations in porins can decrease the permeability of an antibiotic, either by reduction of channel size or modification of electrostatics in the channel, and thereby confer clinical resistance. Here, we investigate four mutant OmpC proteins from four different clinical isolates of Escherichia coli obtained sequentially from a single patient during a course of antimicrobial chemotherapy. OmpC porin from the first isolate (OmpC20) undergoes three consecutive and additive substitutions giving rise to OmpC26, OmpC28, and finally OmpC33. The permeability of two zwitterionic carbapenems, imipenem and meropenem, measured using liposome permeation assays and single channel electrophysiology differs significantly between OmpC20 and OmpC33. Molecular dynamic simulations show that the antibiotics must pass through the constriction zone of porins with a specific orientation, where the antibiotic dipole is aligned along the electric field inside the porin. We identify that changes in the vector of the electric field in the mutated porin, OmpC33, create an additional barrier by "trapping" the antibiotic in an unfavorable orientation in the constriction zone that suffers steric hindrance for the reorientation needed for its onward translocation. Identification and understanding the underlying molecular details of such a barrier to translocation will aid in the design of new antibiotics with improved permeation properties in Gram-negative bacteria.

Structure and Function of the PiuA and PirA Siderophore-Drug Receptors from Pseudomonas aeruginosa and Acinetobacter baumannii.

The outer membrane of Gram-negative bacteria presents an efficient barrier to the permeation of antimicrobial molecules. One strategy pursued to circumvent this obstacle is to hijack transport systems for essential nutrients, such as iron. BAL30072 and MC-1 are two monobactams conjugated to a dihydroxypyridone siderophore that are active against Pseudomonas aeruginosa and Acinetobacter baumannii Here, we investigated the mechanism of action of these molecules in A. baumannii We identified two novel TonB-dependent receptors, termed Ab-PiuA and Ab-PirA, that are required for the antimicrobial activity of both agents. Deletion of either piuA or pirA in A. baumannii resulted in 4- to 8-fold-decreased susceptibility, while their overexpression in the heterologous host P. aeruginosa increased susceptibility to the two siderophore-drug conjugates by 4- to 32-fold. The crystal structures of PiuA and PirA from A. baumannii and their orthologues from P. aeruginosa were determined. The structures revealed similar architectures; however, structural differences between PirA and PiuA point to potential differences between their cognate siderophore ligands. Spontaneous mutants, selected upon exposure to BAL30072, harbored frameshift mutations in either the ExbD3 or the TonB3 protein of A. baumannii, forming the cytoplasmic-membrane complex providing the energy for the siderophore translocation process. The results of this study provide insight for the rational design of novel siderophore-drug conjugates against problematic Gram-negative pathogens.

What we may expect from novel antibacterial agents in the pipeline with respect to resistance and pharmacodynamic principles.

There are some 43 small molecules in the antibiotic development pipeline from late preclinical stage (7 compounds) through Phase 1 (11 molecules), Phase 2 (13 molecules) to Phase 3 (12 molecules). The majority of these are representatives of established antibiotic classes that have been modified to address problems of resistance. In addition, there is considerable activity around the discovery of novel classes of ß-lactamase inhibitors with 10 combinations representing 4 inhibitor classes, at different stages of development. The combination of such inhibitors, which have broad activity against serine ß-lactamases and may even inhibit some penicillin binding proteins, with carbapenems, cephalosporins or aztreonam, provides enhanced activity against multi-drug resistant Gram-negative bacteria. There are 6 molecules representing novel classes of antibiotics but only one of these, murepavadin, is expected to have activity against a Gram-negative pathogenic bacterium (Pseudomonas aeruginosa). Although the new analogues of existing classes, and novel combinations, have been designed to address specific resistance problems, it is by no means certain than they will not be affected by the general mechanisms of resistance, particularly decreased net flux across the Gram-negative outer membrane. The potential impact of resistance mechanisms on the new agents is assessed and the ways in which PK/PD studies are used to design dosing regimens for the new agents, especially combinations, as well as to improve dosing of existing antibiotics are discussed.

The Pseudomonas aeruginosa PA14 ABC Transporter NppA1A2BCD Is Required for Uptake of Peptidyl Nucleoside Antibiotics.

UNLABELLED: Analysis of the genome sequence of Pseudomonas aeruginosa PA14 revealed the presence of an operon encoding an ABC-type transporter (NppA1A2BCD) showing homology to the Yej transporter of Escherichia coli. The Yej transporter is involved in the uptake of the peptide-nucleotide antibiotic microcin C, a translation inhibitor that targets the enzyme aspartyl-tRNA synthetase. Furthermore, it was recently shown that the Opp transporter from P. aeruginosa PAO1, which is identical to Npp, is required for uptake of the uridyl peptide antibiotic pacidamycin, which targets the enzyme translocase I (MraY), which is involved in peptidoglycan synthesis. We used several approaches to further explore the substrate specificity of the Npp transporter. Assays of growth in defined minimal medium containing peptides of various lengths and amino acid compositions as sole nitrogen sources, as well as Biolog Phenotype MicroArrays, showed that the Npp transporter is not required for di-, tri-, and oligopeptide uptake. Overexpression of the npp operon increased susceptibility not just to pacidamycin but also to nickel chloride and the peptidyl nucleoside antibiotic blasticidin S. Furthermore, heterologous expression of the npp operon in a yej-deficient mutant of E. coli resulted in increased susceptibility to albomycin, a naturally occurring sideromycin with a peptidyl nucleoside antibiotic. Additionally, heterologous expression showed that microcin C is recognized by the P. aeruginosa Npp system. Overall, these results suggest that the NppA1A2BCD transporter is involved in the uptake of peptidyl nucleoside antibiotics by P. aeruginosa PA14. IMPORTANCE: One of the world's most serious health problems is the rise of antibiotic-resistant bacteria. There is a desperate need to find novel antibiotic therapeutics that either act on new biological targets or are able to bypass known resistance mechanisms. Bacterial ABC transporters play an important role in nutrient uptake from the environment. These uptake systems could also be exploited by a Trojan horse strategy to facilitate the transport of antibiotics into bacterial cells. Several natural antibiotics mimic substrates of peptide uptake routes. In this study, we analyzed an ABC transporter involved in the uptake of nucleoside peptidyl antibiotics. Our data might help to design drug conjugates that may hijack this uptake system to gain access to cells.

A kinetic analysis of the inhibition of FOX-4 ß-lactamase, a plasmid-mediated AmpC cephalosporinase, by monocyclic ß-lactams and carbapenems.

OBJECTIVES: Class C ß-lactamases are prevalent among Enterobacteriaceae; however, these enzymes are resistant to inactivation by commercially available ß-lactamase inhibitors. In order to find novel scaffolds to inhibit class C ß-lactamases, the comparative efficacy of monocyclic ß-lactam antibiotics (aztreonam and the siderophore monosulfactam BAL30072), the bridged monobactam ß-lactamase inhibitor BAL29880, and carbapenems (imipenem, meropenem, doripenem and ertapenem) were tested in kinetic assays against FOX-4, a plasmid-mediated class C ß-lactamase (pmAmpC). METHODS: The FOX-4 ß-lactamase was purified. Steady-state kinetics, electrospray ionization mass spectrometry (ESI-MS) and ultraviolet difference (UVD) spectroscopy were conducted using the ß-lactam scaffolds described. RESULTS: The K(i) values for the monocyclic ß-lactams against FOX-4 ß-lactamase were 0.04 ± 0.01 µM (aztreonam) and 0.66 ± 0.03 µM (BAL30072), and the Ki value for the bridged monobactam BAL29880 was 8.9 ± 0.5 µM. For carbapenems, the Ki values ranged from 0.27 ± 0.05 µM (ertapenem) to 2.3 ± 0.3 µM (imipenem). ESI-MS demonstrated the formation of stable covalent adducts when the monocyclic ß-lactams and carbapenems were reacted with FOX-4 ß-lactamase. UVD spectroscopy suggested the appearance of different chromophoric intermediates. CONCLUSIONS: Monocyclic ß-lactam and carbapenem antibiotics are effective mechanism-based inhibitors of FOX-4 ß-lactamase, a clinically important pmAmpC, and provide stimulus for the development of new inhibitors to inactivate plasmidic and chromosomal class C ß-lactamases.

Structural insights into the anti-methicillin-resistant Staphylococcus aureus (MRSA) activity of ceftobiprole.

Methicillin-resistant Staphylococcus aureus (MRSA) is an antibiotic-resistant strain of S. aureus afflicting hospitals and communities worldwide. Of greatest concern is its development of resistance to current last-line-of-defense antibiotics; new therapeutics are urgently needed to combat this pathogen. Ceftobiprole is a recently developed, latest generation cephalosporin and has been the first to show activity against MRSA by inhibiting essential peptidoglycan transpeptidases, including the ß-lactam resistance determinant PBP2a, from MRSA. Here we present the structure of the complex of ceftobiprole bound to PBP2a. This structure provides the first look at the molecular details of an effective ß-lactam-resistant PBP interaction, leading to new insights into the mechanism of ceftobiprole efficacy against MRSA.

Involvement of Fe uptake systems and AmpC ß-lactamase in susceptibility to the siderophore monosulfactam BAL30072 in Pseudomonas aeruginosa.

BAL30072 is a monosulfactam conjugated with an iron-chelating dihydroxypyridone moiety. It is active against Gram-negative bacteria, including multidrug-resistant Pseudomonas aeruginosa. We selected mutants with decreased susceptibilities to BAL30072 in P. aeruginosa PAO1 under a variety of conditions. Under iron-deficient conditions, mutants with overexpression of AmpC ß-lactamase predominated. These mutants were cross-resistant to aztreonam and ceftazidime. Similar mutants were obtained after selection at >16× the MIC in iron-sufficient conditions. At 4× to 8× the MIC, mutants with elevated MIC for BAL30072 but unchanged MICs for aztreonam or ciprofloxacin were selected. The expression of ampC and the major efflux pump genes were also unchanged. These BAL30072-specific mutants were characterized by transcriptome analysis, which revealed upregulation of the Fe-dicitrate operon, FecIRA. Whole-genome sequencing showed that this resulted from a single nucleotide change in the Fur-box of the fecI promoter. Overexpression of either the FecI ECF sigma factor or the FecA receptor increased BAL30072 MICs 8- to 16-fold. A fecI mutant and a fecA mutant of PAO1 were hypersusceptible to BAL30072 (MICs < 0.06 µg/ml). The most downregulated gene belonged to the pyochelin synthesis operon, although mutants in pyochelin receptor or synthesis genes had unchanged MICs. The piuC gene, coding for a Fe(II)-dependent dioxygenase located next to the piuA iron receptor gene, was also downregulated. The MICs of BAL30072 for piuC and piuA transposon mutants were increased 8- and 16-fold, respectively. We conclude that the upregulation of the Fe-dicitrate system impacts the expression of other TonB-dependent iron transporters and that PiuA and PiuC contribute to the susceptibility of P. aeruginosa PAO1 to BAL30072.

Combined effects of the siderophore monosulfactam BAL30072 and carbapenems on multidrug-resistant Gram-negative bacilli.

OBJECTIVES: Carbapenem resistance in Gram-negative bacteria, mediated by restricted net influx and carbapenem-hydrolysing ß-lactamases, is a growing problem. The monosulfactam antibiotic BAL30072 is stable to most carbapenemases, suggesting that it could be complementary to carbapenems. We have investigated the antimicrobial activity of BAL30072 combined with imipenem, meropenem and doripenem. METHODS: The in vitro activities of the combinations were evaluated using broth microdilution susceptibility and agar disc diffusion tests, broth dilution chequerboard titration and time-kill studies, using strains of Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter with carbapenem MICs ≥ 2 mg/L. RESULTS: The combinations were effective against 70%-80% of the isolates tested in the presence of 1 mg/L of each antibiotic, whereas the carbapenems were ineffective and BAL30072 alone was effective against 20%-40% of the strains. Synergistic effects were observed with many Enterobacteriaceae and P. aeruginosa, but were less common among the Acinetobacter, although additive effects, where the activity of one partner compensated for lack of activity of the other, were common. None of the combinations exhibited an antagonistic effect in all tests, in contrast to other ß-lactams where negative interactions were frequently observed. Animal models of septicaemia demonstrated that the synergy observed in vitro with BAL30072 and meropenem can translate into greater in vivo efficacy. CONCLUSIONS: BAL30072/carbapenem combinations were effective against a broader range of multidrug-resistant Gram-negative bacteria than either of the single agents. Additive and synergistic effects were observed in Enterobacteriaceae and P. aeruginosa, and this enhanced activity was frequently associated with suppression of resistance development. The in vitro activity translated into improved in vivo efficacy.

In vitro activity of the siderophore monosulfactam BAL30072 against meropenem-non-susceptible Acinetobacter baumannii.

OBJECTIVES: The activity of BAL30072 was compared with that of anti-Acinetobacter reference drugs against meropenem-non-susceptible Acinetobacter baumannii isolates associated with up-regulation of the intrinsic OXA-51-like enzyme or an acquired OXA. METHODS: Antimicrobial susceptibility testing was investigated by broth microdilution of 310 non-duplicate, meropenem-non-susceptible A. baumannii isolates to BAL30072, amikacin ampicillin/sulbactam, aztreonam, cefepime, colistin, imipenem, levofloxacin, meropenem, rifampicin, tigecycline and tobramycin. RESULTS: BAL30072 showed greater activity than the ß-lactam comparators, levofloxacin, amikacin, tobramycin and rifampicin. The activity of BAL30072 was comparable to that of tigecycline, with an MIC(50) of 2 mg/L. Elevated BAL30072 MICs were found, but there was no correlation with elevated MICs of the other antimicrobials. CONCLUSIONS: BAL30072 is a promising new agent with good activity against carbapenem-non-susceptible A. baumannii.

The role of the outer membrane of Gram-negative bacteria in antibiotic resistance: Ajax' shield or Achilles' heel?

There has been an enormous increase in our knowledge of the fundamental steps in the biosynthesis and assembly of the outer membrane of Gram-negative bacteria. Lipopolysaccharide is a major component of the outer membrane of Gram-negative bacteria as is peptidoglycan. Porins, efflux pumps and other transport proteins of the outer membrane are also present. It is clear that there are numerous essential proteins that have the potential to be targets for novel antimicrobial agents. Progress, however, has been slow. Much of the emphasis has been on cytoplasmic processes that were better understood earlier on, but have the drawback that two penetration barriers, with different permeability properties, have to be crossed. With the increased understanding of the late-stage events occurring in the periplasm, it may be possible to shift focus to these more accessible targets. Nevertheless, getting drugs across the outer membrane will remain a challenge to the ingenuity of the medicinal chemist.

Imitation of ß-lactam binding enables broad-spectrum metallo-ß-lactamase inhibitors.

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-ß-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential ß-lactamase stable ß-lactam mimics. Subsequent structure-activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL-carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.

Synthesis and characterization of the arylomycin lipoglycopeptide antibiotics and the crystallographic analysis of their complex with signal peptidase.

Glycosylation of natural products, including antibiotics, often plays an important role in determining their physical properties and their biological activity, and thus their potential as drug candidates. The arylomycin class of antibiotics inhibits bacterial type I signal peptidase and is comprised of three related series of natural products with a lipopeptide tail attached to a core macrocycle. Previously, we reported the total synthesis of several A series derivatives, which have unmodified core macrocycles, as well as B series derivatives, which have a nitrated macrocycle. We now report the synthesis and biological evaluation of lipoglycopeptide arylomycin variants whose macrocycles are glycosylated with a deoxy-α-mannose substituent, and also in some cases hydroxylated. The synthesis of the derivatives bearing each possible deoxy-α-mannose enantiomer allowed us to assign the absolute stereochemistry of the sugar in the natural product and also to show that while glycosylation does not alter antibacterial activity, it does appear to improve solubility. Crystallographic structural studies of a lipoglycopeptide arylomycin bound to its signal peptidase target reveal the molecular interactions that underlie inhibition and also that the mannose is directed away from the binding site into solvent which suggests that other modifications may be made at the same position to further increase solubility and thus reduce protein binding and possibly optimize the pharmacokinetics of the scaffold.

In vitro and in vivo properties of BAL30376, a ß-lactam and dual beta-lactamase inhibitor combination with enhanced activity against Gram-negative Bacilli that express multiple ß-lactamases.

BAL30376 is a triple combination comprising a siderophore monobactam, BAL19764; a novel bridged monobactam, BAL29880, which specifically inhibits class C ß-lactamases; and clavulanic acid, which inhibits many class A and some class D ß-lactamases. The MIC(90) was ≤ 4 µg/ml (expressed as the concentration of BAL19764) for most species of the Enterobacteriaceae family, including strains that produced metallo-ß-lactamases and were resistant to all of the other ß-lactams tested. The MIC(90) for Stenotrophomonas maltophilia was 2 µg/ml, for multidrug-resistant (MDR) Pseudomonas aeruginosa it was 8 µg/ml, and for MDR Acinetobacter and Burkholderia spp. it was 16 µg/ml. The presence of the class C ß-lactamase inhibitor BAL29880 contributed significantly to the activity of BAL30376 against strains of Citrobacter freundii, Enterobacter species, Serratia marcescens, and P. aeruginosa. The presence of clavulanic acid contributed significantly to the activity against many strains of Escherichia coli and Klebsiella pneumoniae that produced class A extended-spectrum ß-lactamases. The activity of BAL30376 against strains with metallo-ß-lactamases was largely attributable to the intrinsic stability of the monobactam BAL19764 toward these enzymes. Considering its three components, BAL30376 was unexpectedly refractory toward the development of stable resistance.

In vivo and in vitro activity of the siderophore monosulfactam BAL30072 against Acinetobacter baumannii.

OBJECTIVES: New antibiotics that are active against multidrug-resistant (MDR) Acinetobacter baumannii are urgently needed. BAL30072, a siderophore monosulfactam antibiotic that rapidly penetrates the outer membrane of A. baumannii and has potent activity against most isolates, including those harbouring AmpC ß-lactamases and metallo- (class B) or OXA- (class D) carbapenemases, is being developed to meet that need. METHODS: We assessed the in vitro activity of BAL30072, meropenem and the combination of BAL30072 and meropenem (2:1 and 1:1 ratios) by MIC and time-kill studies. Proof-of-principle in vivo efficacy was determined using a rat soft-tissue infection model. Five diverse strains with defined phenotypic and genetic profiles were tested (AB307-0294, AB8407, AB1697, AB3340 and AB0057). RESULTS: In microdilution assays, combining BAL30072 with meropenem lowered meropenem MICs 2-8-fold. In time-kill studies, the BAL30072 and meropenem combinations resulted in bactericidal concentrations 2-8-fold lower than those of meropenem or BAL30072 alone. In the rat model, BAL30072 was active against four of five strains (AB307-0294, AB8407, AB1697 and AB3340), including meropenem-susceptible and -non-susceptible strains. AB0057 was the only strain resistant to BAL30072 in vivo and in vitro (MIC >64 mg/L). Meropenem was active in vivo against two of the five strains tested (AB307-0294 and AB3340). Both BAL30072 and BAL30072 with meropenem were equally effective in vivo. CONCLUSIONS: These data support the continued evaluation of BAL30072 for use in the treatment of infections caused by MDR A. baumannii.

Enhancing resistance to cephalosporins in class C beta-lactamases: impact of Gly214Glu in CMY-2.

The biochemical properties of CMY-32, a class C enzyme possessing a single-amino acid substitution in the Omega loop (Gly214Glu), were compared to those of the parent enzyme, CMY-2, a widespread class C beta-lactamase. In parallel with our microbiological characterization, the Gly214Glu substitution in CMY-32 reduced catalytic efficiency (k(cat)/K(m)) by 50-70% against "good" substrates (i.e., cephalothin) while increasing k(cat)/K(m) against "poor" substrates (i.e., cefotaxime). Additionally, CMY-32 was more susceptible to inactivation by sulfone beta-lactamase inhibitors (i.e., sulbactam and tazobactam) than CMY-2. Timed electrospray ionization mass spectrometry (ESI-MS) analysis of the reaction of CMY-2 and CMY-32 with different substrates and inhibitors suggested that both beta-lactamases formed similar intermediates during catalysis and inactivation. We next showed that the carbapenems (imipenem, meropenem, and doripenem) form long-lived acyl-enzyme intermediates and present evidence that there is beta-lactamase-catalyzed elimination of the C(6) hydroxyethyl substituent. Furthermore, we discovered that the monobactam aztreonam and BAL29880, a new beta-lactamase inhibitor of the monobactam class, inactivate CMY-2 and CMY-32 by forming an acyl-enzyme intermediate that undergoes elimination of SO(3)(2-). Molecular modeling and dynamics simulations suggest that the Omega loop is more constrained in CMY-32 than CMY-2. Our model also proposes that Gln120 adopts a novel conformation in the active site while new interactions form between Glu214 and Tyr221, thus explaining the increased level of cefotaxime hydrolysis. When it is docked in the active site, we observe that BAL29880 exploits contacts with highly conserved residues Lys67 and Asn152 in CMY-2 and CMY-32. These findings highlight (i) the impact of single-amino acid substitutions on protein evolution in clinically important AmpC enzymes and (ii) the novel insights into the mechanisms by which carbapenems and monobactams interact with CMY-2 and CMY-32 beta-lactamases.

In vitro properties of BAL30072, a novel siderophore sulfactam with activity against multiresistant gram-negative bacilli.

BAL30072 is a new monocyclic beta-lactam antibiotic belonging to the sulfactams. Its spectrum of activity against significant Gram-negative pathogens with beta-lactam-resistant phenotypes was evaluated and was compared with the activities of reference drugs, including aztreonam, ceftazidime, cefepime, meropenem, imipenem, and piperacillin-tazobactam. BAL30072 showed potent activity against multidrug-resistant (MDR) Pseudomonas aeruginosa and Acinetobacter sp. isolates, including many carbapenem-resistant strains. The MIC(90)s were 4 microg/ml for MDR Acinetobacter spp. and 8 microg/ml for MDR P. aeruginosa, whereas the MIC(90) of meropenem for the same sets of isolates was >32 microg/ml. BAL30072 was bactericidal against both Acinetobacter spp. and P. aeruginosa, even against strains that produced metallo-beta-lactamases that conferred resistance to all other beta-lactams tested, including aztreonam. It was also active against many species of MDR isolates of the Enterobacteriaceae family, including isolates that had a class A carbapenemase or a metallo-beta-lactamase. Unlike other monocyclic beta-lactams, BAL30072 was found to trigger the spheroplasting and lysis of Escherichia coli rather than the formation of extensive filaments. The basis for this unusual property is its inhibition of the bifunctional penicillin-binding proteins PBP 1a and PBP 1b, in addition to its high affinity for PBP 3, which is the target of monobactams, such as aztreonam.

Propenylamide and propenylsulfonamide cephalosporins as a novel class of anti-MRSA beta-lactams.

Novel C(3) propenylamide and propenylsulfonamide cephalosporins have been synthesized and tested for their ability to inhibit the penicillin-binding protein 2' (PBP2') from Staphylococcus epidermidis and the growth of a panel of clinically relevant bacterial species, including methicillin-resistant Staphylococcus aureus (MRSA). The most potent compounds inhibited the growth of MRSA strains with minimum inhibitory concentrations (MIC) as low as 1 microg/mL. The structure-activity relationship revealed the potential for further optimization of this new cephalosporin class.