Rudolf L. Then

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.

Thymidine concentrations in serum and urine of different animal species and man.

Abstract Thymidine concentrations were determined in the serum and urine of different animal species and man with a sensitive microbiological method, using Lactobacillus arabinosus ATCC 8014. In contrast to man, dog and other higher animals, where little or no thymidine could be found, considerable amounts (0.1-1 μg/ml) were present in serum and urine from rat and mouse. In mice thymidine concentrations were seen to be enhanced by infections. Some tissue values of rats were determined and the influence of the sample preparation on the amount of thymidine found was investigated.

The Targets of Currently Used Antibacterial Agents: Lessons for Drug Discovery

Based on the mode of action of antibacterial drugs currently used, targets can be defined as distinct cellular constituents such as enzymes, enzyme substrates, RNA, DNA, and membranes which exhibit very specific binding sites at the surface of these components or at the interface of macromolecular complexes assembled in the cell. Intriguingly, growth inhibition or even complete loss of bacterial viability is often the result of a cascade of events elicited upon treatment with an antibacterial agent. In addition, their mode of action frequently involves more than one single target. A comprehensive description of the targets exploited so far by commercialized antibacterial agents, including anti-mycobacterial agents, is given. The number of targets exploited so far by commercial antibacterial agents is estimated to be about 40. The most important biosynthetic pathways and cellular structures affected by antibacterial drugs are the cell wall biosynthesis, protein biosynthesis, DNA per se, replication, RNA per se, transcription and the folate biosynthetic pathway. The disillusionment with the genomics driven antibacterial drug discovery is a result of the restrictive definition of targets as products of essential and conserved genes. Emphasis is made to not only focus on proteins as potential drug targets, but increase efforts and devise screening technologies to discover new agents interacting with different RNA species, DNA, new protein families or macromolecular complexes of these constituents.

Cloning and characterization of a novel, plasmid-encoded trimethoprim-resistant dihydrofolate reductase from Staphylococcus haemolyticus MUR313.

In recent years resistance to the antibacterial agent trimethoprim (Tmp) has become more widespread, and several trimethoprim-resistant (Tmpr) dihydrofolate reductases (DHFRs) have been described from gram-negative bacteria. In staphylococci, only one Tmpr DHFR has been described, the type S1 DHFR, which is encoded by the dfrA gene found on transposon Tn4003. In order to investigate the coincidence of high-level Tmp resistance and the presence of dfrA, we analyzed the DNAs from various Tmpr staphylococci for the presence of dfrA sequences by PCR with primers specific for the thyE-dfrA genes from Tn4003. We found that 30 or 33 isolates highly resistant to Tmp (MICs, > or = 512 micrograms/ml) contained dfrA sequences, whereas among the Tmpr (MICs, < or = 256 micrograms/ml) and Tmps isolates only the Staphylococcus epidermidis isolates (both Tmpr and Tmps) seemed to contain the dfrA gene. Furthermore, we have cloned and characterized a novel, plasmid-encoded Tmpr DHFR from Staphylococcus haemolyticus MUR313. The dfrD gene of plasmid pABU17 is preceded by two putative Shine-Dalgarno sequences potentially allowing for the start of translation at two triplets separated by nine nucleotides. The predicted protein of 166 amino acids, designated S2DHFR, encoded by the longer open reading frame was overproduced in Escherichia coli, purified, and characterized. The molecular size of the recombinant S2DHFR was determined by ion spray mass spectrometry to be 19,821.2 +/- 2 Da, which is in agreement with the theoretical value of 19,822 Da. In addition, the recombinant S2DHFR was shown to exhibit DHFR activity and to be highly resistant to Tmp.

Low Trimethoprim Susceptibility of Anaerobic Bacteria Due to Insensitive Dihydrofolate Reductases

All the 28 Bacteroides fragilis strains investigated were susceptible to sulfamethoxazole (minimal inhibitory concentration < 16 μg/ml) and resistant to trimethoprim (TMP; minimal inhibitory concentration > 4 μg/ml). Synergism between sulfamethoxazole and TMP was present in all strains at a ratio of 1:1. The few clostridia investigated proved more resistant to both compounds. Dihydrofolate reductases from B. fragilis, C. perfringens, and some other anaerobic species were isolated. Inhibition profiles with six structurally different inhibitors revealed major differences in all enzymes. For 50% inhibition, the enzyme from B. fragilis and all clostridia required concentrations of TMP which were between several hundredfold and 1,000-fold higher than those required for the enzyme of Escherichia coli, whereas the enzyme from Propionibacterium acnes only needed a threefold higher concentration. In vitro activities of TMP were seen to correspond to the activity at the enzymatic level in B. fragilis and P. acnes, but correspond to a much lesser extent to the activity at the enzymatic level in clostridia, where a poor penetration is assumed to be involved. Dihydrofolate reductase inhibitors other than TMP were found to be as active as TMP both at the enzyme and in vitro. In B. fragilis, higher concentrations of exogenous thymidine were required for increasing the minimal inhibitory concentration of TMP than in E. coli and probably also in C. perfringens.

Characterization of the gene for chromosomal trimethoprim-sensitive dihydrofolate reductase of Staphylococcus aureus ATCC 25923.

The gene for the trimethoprim-sensitive (Tmps) chromosomal dihydrofolate reductase (DHFR) of Staphylococcus aureus ATCC 25923 was cloned and characterized. The structural gene encodes a polypeptide of 159 amino acid residues and has a calculated molecular weight of 18,251. The amino acid sequences of this Tmps DHFR and those of the trimethoprim-resistant type S1 DHFR encoded by transposon Tn4003 are 80% identical. In contrast to the trimethoprim-resistant enzyme, the Tmps DHFR can be highly overexpressed in Escherichia coli, with most of the recombinant protein occurring in a soluble and an active form. Images

Identical genes for trimethoprim-resistant dihydrofolate reductase from Staphylococcus aureus in Australia and Central Europe

The nucleotide sequence of a 1.25 kb BglII/EcoRI fragment from the 34 kb trimethoprim (Tp)‐resistant plasmid pABU1 of Staphylococcus aureus 157/4696, isolated in Zürich, was determined. It contained the entire Tp‐resistant dihydrofolate reductase gene, 197 bp of the thymidylate synthetase, 395 bp of a truncated gene and 111 bp of IS257R1. With the exception of one single base pair at position 862 the sequence of the whole fragment was identical to nucleotides 1633 to 2885 of the Tp‐resistant transposon Tn4OO3 in plasmid pSK 1 from an Australian S. aureus isolate. This suggests the worldwide dissemination of Tn4OO3 in multiresistant Staphylococci.

In vitro antibacterial properties of cefetamet and in vivo activity of its orally absorbable ester derivative, cefetamet pivoxil

The in vitro activity of cefetamet, the microbiologically active metabolite of the orally administered prodrug cefetamet pivoxil, was compared with that of cephalexin, cefaclor, cefuroxime and amoxicillin. Cefetamet was highly active againstEnterobacteriaceae, Neisseria spp.,Vibrio spp.,Haemophilus influenzae and streptococci other than enterococci. Cefetamet inhibited cefaclor-resistant species such asProteus vulgaris, Providencia stuartii, Providencia rettgeri andSerratia marcescens. Staphylococci,Pseudomonas aeruginosa and cephalosporinase-overproducing strains ofEnterobacter cloacae were resistant to cefetamet. The superior activity of cefetamet compared with older oral beta-lactam antibiotics against a large number of gram-negative pathogens correlated with enhanced stability towards betalactamases. In accordance with the in vitro findings, cefetamet pivoxil showed good activity in experimental infections in the mouse and rat, suggesting satisfactory bioavailability in these animals after oral administration.

Cephalosporin resistance in strains of Klebsiella oxytoca isolated during antibiotic therapy

In the course of multiple antibiotic treatment of a seriously ill patient, a high level of resistance to cefamandole, cefuroxime and ceftriaxone was found among consecutive isolates of Klebsiella oxytoca, other species isolated remaining susceptible. Susceptibility to cefotaxime was less affected, and the activity of cefoxitin and moxalactam was unchanged. Resistant K. oxytoca strains synthesized a large amount of a broad-spectrum beta-lactamase, able to hydrolyze ceftriaxone, cefuroxime and cefotaxime. The enzyme level as well as the ability to hydrolyze different cephalosporins with different rates explained the in vitro susceptibilities observed reasonably well. The initially susceptible K. oxytoca strain easily converted in vitro to variants producing a high level of beta-lactamase after exposure to ceftriaxone, without concomitant acquisition of high-level resistance to this antibiotic, suggesting that additional and at present unknown factors also contributed to resistance of the clinical isolates.

Selective Inhibition of Dihydrofolate Reductase from Problem Human Pathogens

Considerable changes in the prevalence and epidemiology of human pathogenic microorganisms have occurred over the last decade. Two main factors are responsible: 1 Resistance development, which is a particular concern among grampositive cocci, e.g. staphylococci1 2 The increased number of patients with impaired immuneresponse, highlighted by the spread of HIV, who suffer infections with opportunistic pathogens such as Pneumocystis carinii, Toxoplasma gondii, Mycobacterium avium and others.