Malcolm G. P. Page

Prospects for the next anti-Pseudomonas drug.

Pseudomonas aeruginosa is one of the most dreaded Gram-negative bacterial pathogens in hospitals. Not only it is among the most frequently isolated Gram-negative organisms in bloodstream and wound infections, pneumonia, intra-abdominal-sepsis and urogenital-sepsis, but also it is frequently found in patients with comorbid illnesses and compromised by in-dwelling catheters, tubes and surgery where mortality rates of more than 60% have been reported. Besides its intrinsic resistance to a number of widely used antibiotics, Pseudomonas also managed to acquire resistance via additional mechanisms, including target mutations, increased expression of efflux pumps and of antibiotic-degrading enzymes. Taken together, the increased incidence in certain types of infections, the increased use of invasive devices in the hospital as well as the increased frequency of multi-resistant Pseudomonas strains, have clearly led to a shortage of treatment options for nosocomial Pseudomonas infections. Even the recommended combination therapy of an antibiotic of the beta-lactam class together with an aminoglycoside or a fluoroquinolone, is no longer always successful and sometimes a polymyxin has to be given as last resort. Despite growing concerns of clinicians and medical societies about the very limited number of novel drugs in the pipeline to fight multi-resistant Pseudomonas strains, only a very small number of novel anti-Pseudomonas drugs are currently in late stage of pre-clinical or clinical development. However, and possibly as a reflection of the magnitude of the problem, quite a variety of approaches are being pursued. Among these are next-generation analogues of successful antibiotic classes (e.g. novel beta-lactams and combinations of novel beta-lactamase inhibitors with known penicillins or cephalosporins), antibodies, phages and selective peptides. It is to be hoped that a number of these novel drugs will show clinical utility and reach the market over the next 6-10 years.

Membrane transport proteins: implications of sequence comparisons☆

Analyses of the sequences and structures of many transport proteins that differ in substrate specificity, direction of transport and mechanism of transport suggest that they form a family of related proteins. Their sequence similarities imply a common mechanism of action. This hypothesis provides an objective basis for examining their mechanisms of action and relationships to other transporters.

In Vitro and In Vivo Properties of Ro 63-9141, a Novel Broad-Spectrum Cephalosporin with Activity against Methicillin-Resistant Staphylococci

ABSTRACT Ro 63-9141 is a new member of the pyrrolidinone-3-ylidenemethyl cephem series of cephalosporins. Its antibacterial spectrum was evaluated against significant gram-positive and gram-negative pathogens in comparison with those of reference drugs, including cefotaxime, cefepime, meropenem, and ciprofloxacin. Ro 63-9141 showed high antibacterial in vitro activity against gram-positive bacteria except ampicillin-resistant enterococci, particularly vancomycin-resistant strains of Enterococcus faecium. Its MIC at which 90% of the isolates tested were inhibited (MIC90) for methicillin-resistant Staphylococcus aureus (MRSA) was 4 μg/ml. Ro 63-9141 was bactericidal against MRSA. Development of resistance to the new compound in MRSA was not observed. Ro 63-9141 was more potent than cefotaxime against penicillin-resistant Streptococcus pneumoniae(MIC90 = 2 μg/ml). It was active against ceftazidime-susceptible strains of Pseudomonas aeruginosaand against Enterobacteriaceae except Proteus vulgaris and some isolates producing extended-spectrum β-lactamases. The basis for the antibacterial spectrum of Ro 63-9141 lies in its affinity to essential penicillin-binding proteins, including PBP 2′ of MRSA, and its stability towards β-lactamases. The in vivo findings were in accordance with the in vitro susceptibilities of the pathogens. These data suggest the potential utility of Ro 63-9141 for the therapy of infections caused by susceptible pathogens, including MRSA. Since insufficient solubility of Ro 63-9141 itself precludes parenteral administration in humans, a water-soluble prodrug, Ro 65-5788, is considered for development.

In Vitro Properties of BAL30072, a Novel Siderophore Sulfactam with Activity against Multiresistant Gram-Negative Bacilli

ABSTRACT BAL30072 is a new monocyclic β-lactam antibiotic belonging to the sulfactams. Its spectrum of activity against significant Gram-negative pathogens with β-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 MIC90s were 4 μg/ml for MDR Acinetobacter spp. and 8 μg/ml for MDR P. aeruginosa, whereas the MIC90 of meropenem for the same sets of isolates was >32 μg/ml. BAL30072 was bactericidal against both Acinetobacter spp. and P. aeruginosa, even against strains that produced metallo-β-lactamases that conferred resistance to all other β-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-β-lactamase. Unlike other monocyclic β-lactams, BAL30072 was found to trigger the spheroplasting and lysis of Escherichiacoli 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.

Peptide Deformylase as an Antibacterial Drug Target: Target Validation and Resistance Development

ABSTRACT New inhibitors of peptide deformylase (PDF) which are very potent against the isolated enzyme and show a certain degree of antibacterial activity have recently been synthesized by our group. Several lines of experimental evidence indicate that these inhibitors indeed interfere with the target enzyme in the bacterial cell. (i) The inhibition ofEscherichia coli growth could be counteracted by overexpression of PDF from different organisms, including E. coli, Streptococcus pneumoniae, and Haemophilus influenzae. Conversely, reduced expression of PDF in S. pneumoniae resulted in an increased susceptibility to the inhibitors. (ii) Proteome analysis on two-dimensional gels revealed a shift for many proteins towards lower pI in the presence of PDF inhibitors, as would be expected if the proteins still carry theirN-formyl-Met terminus. (iii) PDF inhibitors show no antimicrobial activity against E. coli under conditions that make growth independent of formylation and deformylation. The antibacterial activity in E. coli was characterized as bacteriostatic. Furthermore, the development of resistance in E. coli was observed to occur with high frequency (10−7). Resistant mutants show a reduced growth rate, and DNA sequence analysis revealed mutations in their formyl transferase gene. Taking all these aspects into account, we conclude that PDF may not be an optimal target for broad-spectrum antibacterial agents.

Crystal Structure of the Class D Beta-Lactamase OXA-10.

We report the crystal structure of a class D β-lactamase, the broad spectrum enzyme OXA-10 from Pseudomonas aeruginosa at 2.0 Å resolution. There are significant differences between the overall fold observed in this structure and those of the evolutionarily related class A and class C β-lactamases. Furthermore, the structure suggests the unique, cation mediated formation of a homodimer. Kinetic and hydrodynamic data shows that the dimer is a relevant species in solution and is the more active form of the enzyme. Comparison of the molecular details of the active sites of the class A and class C enzymes with the OXA-10 structure reveals that there is no counterpart in OXA-10 to the residues proposed to act as general bases in either of these enzymes (Glu 166 and Tyr 150, respectively). Our structures of the native and chloride inhibited forms of OXA-10 suggest that the class D enzymes have evolved a distinct catalytic mechanism for β-lactam hydrolysis. Clinical variants of OXA-10 are also discussed in light of the structure.

Genetic Analysis and Functional Characterization of the Streptococcus pneumoniae vic Operon

ABSTRACT The vic two-component signal transduction system of Streptococcus pneumoniae is essential for growth. The vic operon comprises three genes encoding the following: VicR, a response regulator of the OmpR family; VicK, its cognate histidine kinase; and VicX, a putative protein sharing 55% identity to the predicted product (YycJ) of an open reading frame in the Bacillus subtilis genome. We show that not only is vic essential for viability but it also influences virulence and competence. A putative transcriptional start site for the vic operon was mapped 16 bp upstream of the ATG codon of vicR. Only one transcript of 2.9 kb, encoding all three genes, was detected by Northern blot analysis. VicK, an atypical PAS domain-containing histidine kinase, can be autophosphorylated in vitro, and VicR functions in vitro as a phospho-acceptor protein. (PAS is an acronym formed from the names of the proteins in which the domains were first recognized: the Drosophila period clock protein [PER], vertebrate aryl hydrocarbon receptor nuclear translocator [ARNT], and Drosophila single-minded protein [SIM].) PAS domains are commonly involved in sensing intracellular signals such as redox potential, which suggests that the signal for vic might also originate in the cytoplasm. Growth rate, competence, and virulence were monitored in strains with mutations in the vic operon. Overexpression of the histidine kinase, VicK, resulted in decreased virulence, whereas the transformability of a null mutant decreased by 3 orders of magnitude.

Impact of Specific pbp5 Mutations on Expression of β-Lactam Resistance in Enterococcus faecium

ABSTRACT We tested the impact of individual PBP 5 mutations on expression of ampicillin resistance in Enterococcus faecium using a shuttle plasmid designed to facilitate expression of cloned pbp5 in ampicillin-susceptible E. faecium D344SRF. Substitutions that had been implicated in contributing to the resistance of clinical strains conferred only modest levels of resistance when they were present as single point mutations. The levels of resistance were amplified when some mutations were present in combination. In particular, a methionine-to-alanine change at position 485 (in close proximity to the active site) combined with the insertion of a serine at position 466 (located in a loop that forms the outer edge of the active site) was associated with the highest levels of resistance to all β-lactams. Affinity for penicillin generally correlated with β-lactam MICs for the mutants, but these associations were not strictly proportional.

Involvement of the Putative ATP-Dependent Efflux Proteins PatA and PatB in Fluoroquinolone Resistance of a Multidrug-Resistant Mutant of Streptococcus pneumoniae

ABSTRACT The multidrug-resistant mutant Streptococcus pneumoniae M22 constitutively overexpresses two genes (patA and patB) that encode proteins homologous to known efflux proteins belonging to the ABC transporter family. It is shown here that PatA and PatB were strongly induced by quinolone antibiotics and distamycin in fluoroquinolone-sensitive strains. PatA was very important for growth of S. pneumoniae, and it could not be disrupted in strain M22. PatB appeared to control metabolic activity, particularly in amino acid biosynthesis, and it may have a pivotal role in coordination of the response to quinolone antibiotics. The induction of PatA and PatB by antibiotics showed a pattern similar to that exhibited by SP1861, a homologue of ABC-type transporters of choline and other osmoprotectants. A second group of quinolone-induced transporter genes comprising SP1587 and SP0287, which are homologues of, respectively, oxalate/formate antiporters and xanthine or uracil permeases belonging to the major facilitator family, showed a different pattern of induction by other antibiotics. There was no evidence for the involvement of PmrA, the putative proton-dependent multidrug transporter that has been implicated in norfloxacin resistance, in the response to quinolone antibiotics in either the resistant mutant or the fluoroquinolone-sensitive strains.

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 β-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.