Essentiality, expression, and characterization of the class II 3-hydroxy-3-methylglutaryl coenzyme A reductase of Staphylococcus aureus.

Sequence comparisons have implied the presence of genes encoding enzymes of the mevalonate pathway for isopentenyl diphosphate biosynthesis in the gram-positive pathogen Staphylococcus aureus. In this study we showed through genetic disruption experiments that mvaA, which encodes a putative class II 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is essential for in vitro growth of S. aureus. Supplementation of media with mevalonate permitted isolation of an auxotrophic mvaA null mutant that was attenuated for virulence in a murine hematogenous pyelonephritis infection model. The mvaA gene was cloned from S. aureus DNA and expressed with an N-terminal His tag in Escherichia coli. The encoded protein was affinity purified to apparent homogeneity and was shown to be a class II HMG-CoA reductase, the first class II eubacterial biosynthetic enzyme isolated. Unlike most other HMG-CoA reductases, the S. aureus enzyme exhibits dual coenzyme specificity for NADP(H) and NAD(H), but NADP(H) was the preferred coenzyme. Kinetic parameters were determined for all substrates for all four catalyzed reactions using either NADP(H) or NAD(H). In all instances optimal activity using NAD(H) occurred at a pH one to two units more acidic than that using NADP(H). pH profiles suggested that His378 and Lys263, the apparent cognates of the active-site histidine and lysine of Pseudomonas mevalonii HMG-CoA reductase, function in catalysis and that the general catalytic mechanism is valid for the S. aureus enzyme. Fluvastatin inhibited competitively with HMG-CoA, with a K(i) of 320 microM, over 10(4) higher than that for a class I HMG-CoA reductase. Bacterial class II HMG-CoA reductases thus are potential targets for antibacterial agents directed against multidrug-resistant gram-positive cocci.

Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in gram-positive cocci.

The mevalonate pathway and the glyceraldehyde 3-phosphate (GAP)-pyruvate pathway are alternative routes for the biosynthesis of the central isoprenoid precursor, isopentenyl diphosphate. Genomic analysis revealed that the staphylococci, streptococci, and enterococci possess genes predicted to encode all of the enzymes of the mevalonate pathway and not the GAP-pyruvate pathway, unlike Bacillus subtilis and most gram-negative bacteria studied, which possess only components of the latter pathway. Phylogenetic and comparative genome analyses suggest that the genes for mevalonate biosynthesis in gram-positive cocci, which are highly divergent from those of mammals, were horizontally transferred from a primitive eukaryotic cell. Enterococci uniquely encode a bifunctional protein predicted to possess both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and acetyl-CoA acetyltransferase activities. Genetic disruption experiments have shown that five genes encoding proteins involved in this pathway (HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase) are essential for the in vitro growth of Streptococcus pneumoniae under standard conditions. Allelic replacement of the HMG-CoA synthase gene rendered the organism auxotrophic for mevalonate and severely attenuated in a murine respiratory tract infection model. The mevalonate pathway thus represents a potential antibacterial target in the low-G+C gram-positive cocci.

Efflux and target mutations as quinolone resistance mechanisms in clinical isolates of Streptococcus pneumoniae.

The aim of this study was to characterize quinolone resistance mechanisms in strains of Streptococcus pneumoniae with increased MICs of ofloxacin. These strains were also tested for their susceptibility to a battery of quinolone antimicrobial agents, including gemifloxacin. Of the S. pneumoniae isolates used, 27 were susceptible to ofloxacin, 18 intermediate and 48 resistant (ofloxacin MIC <4, 4 and >4 mg/L, respectively). In general, the ofloxacin-susceptible strains had no amino acid substitutions in GyrA, GyrB, ParC or ParE. Moderate increases in MIC were associated with substitutions in the quinolone resistance-determining region (QRDR) of ParC, while the highest MICs were found for strains that also had substitutions in the QRDR of GyrA. The most common substitutions were Ser79-->Phe in ParC and Ser81-->Phe in GyrA. Other substitutions were identified within the QRDR of ParC and outside the QRDR of ParC and ParE; these did not appear to affect susceptibility. The effects of antimicrobial efflux pumps were studied by determining MICs of a range of quinolones in the presence and absence of reserpine, an inhibitor of Gram-positive efflux pumps. Our results indicated that high-level resistance, caused entirely by efflux, was seen in a minority of ofloxacin-resistant S. pneumoniae strains. Testing the susceptibility of quinolone-resistant strains to gemifloxacin, ciprofloxacin, norfloxacin, ofloxacin and trovafloxacin revealed that gemifloxacin was least affected by this large variety of resistance mechanisms and was the only quinolone with MICs of < or =0.5 mg/L for all strains in this study. These results suggest that gemifloxacin is highly potent against S. pneumoniae and may also be effective against strains resistant to other quinolones.

Dimerization and membrane anchors in extracellular targeting of vancomycin group antibiotics.

Antibiotics of the vancomycin group are shown to enhance their affinities for the bacterial cell wall by the devices of either dimerization (vancomycin and other glycopeptides which dimerize even more strongly) or use of a membrane anchor (teicoplanin); a chelate mechanism is suggested in both cases, as supported by antagonism experiments with the cell wall analog di-N-acetyl-L-Lys-D-Ala-D-Ala. These results may have implications for other binding processes which occur near membrane surfaces.

SB-202742, a novel beta-lactamase inhibitor isolated from Spondias mombin.

SB-202742 [1], an anacardic acid derivative possessing beta-lactamase inhibitory activity, has been isolated from a hexane extract of the plant, Spondias mombin. Its isolation, structure determination, and biological activity are reported herein.

MM 45289, a potent glycopeptide antibiotic which interacts weakly with diacetyl-L-lysyl-D-alanyl-D-alanine.

MM 45289 (A82846A, eremomycin), a glycopeptide antibiotic of the vancomycin type, was confirmed to have improved antibacterial activity over vancomycin. However its affinity (Ka) for the target site peptide mimetic diacetyl-L-lysyl-D-alanyl-D-alanine (DALAA) was 23-fold lower. Concentrations of DALAA required to reverse the antibacterial activity of MM 45289 were in the order of 10 to 50-fold higher than for vancomycin. These results have implications for both mode of action studies and mechanism-based screening strategies for this class of antibiotic.

MM 42842, a new member of the monobactam family produced by Pseudomonas cocovenenans. I. Identification of the producing organism.

A bacterial soil isolate designated 326-32B produces a new member of the monobactam series of antibiotics, MM 42842, and the bulgecins. Identification studies show isolate 326-32B to be a strain of Pseudomonas cocoveneans which is a species previously noted for the production of toxoflavin. A description of P. cocovenenans does not appear to have been previously published and the identify of strain 326-32B was established by means of a direct comparison with the deposited organism P. cocovenenans NCIB 9450. The properties of strain 326-32B, and P. cocovenenans NCIB 9450 were compared with those of the monobactam and bulgecin producing organisms Pseudomonas acidophila ATCC 31363 and Pseudomonas mesoacidophila ATCC 31433. The four organisms were found to share certain properties, including the ability to grow at pH 4.0.

MM 42842, a new member of the monobactam family produced by Pseudomonas cocovenenans. II. Production, isolation and properties of MM 42842.

A new member of the monobactam family of beta-lactam antibiotics, designated MM 42842, has been detected in a culture of Pseudomonas cocovenenans. The production, isolation and some properties of the antibiotic are described. Structural studies show MM 42842 to be closely related to the previously described antibiotic sulfazecin.

Selection of variants of Gram-negative bacteria with elevated production of type 1 beta-lactamase.

Variants of Pseudomonas aeruginosa, Citrobacter freundii and Enterobacter cloacae were isolated that produced elevated levels of type 1 beta-lactamase. The variants were readily isolated on antibiotic-containing agar and showed resistance to a wide range of beta-lactam antibiotics including some regarded as being relatively stable to the beta-lactamase produced.

Regrowth of Pseudomonas aeruginosa and other bacteria after the bactericidal action of carbenicillin and other beta-lactam antibiotics.

Exposure of Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus to bactericidal concentrations of beta-lactam antibiotics in broth culture resulted in a decrease in viability over the first 6--8 hr, followed by regrowth which was not due to the selection of resistant variants or loss of antibiotic potency. During incubation, bacteria adhered to the surface of the culture vessel and multiplied despite the presence of bactericidal concentrations of antibiotic in the medium. It is concluded that the phenomenon of "regrowth" results from such adhesion and the subsequent dispersal of some of these cells into the culture medium. The significance of these findings is discussed in relation to the treatment of infection, the determination of minimal bactericidal concentrations, and the phenomena of tolerance and persisters.

Selection of variants of Pseudomonas aeruginosa resistant of Beta-lactam antibiotics.

In broth cultures of Pseudomonas aeruginosa, containing carbenicillin or azlocillin, regrowth occurred after a period of bactericidal action, to reach visible proportions overnight. Regrowth in the presence of relatively high concentrations of carbenicillin or azlocillin could not be accounted for on the basis of growth of resistant variants nor as a result of drug inactivation. On the other hand, resistant variants could be selected from the regrowth which occurred at concentrations of carbenicillin or azlocillin only slight in excess of the minimum inhibitory concentrations (MIC). Antibiotic resistant variants could also be isolated from individual colonies growing on agar plates containing carbenicillin, ticarcillin, azlocillin or piperaccillin at concentrations above the MIC for the majority of the population. Two types of resistant variant were isolated. The first showed a 2-5 fold increase in resistance to carbenicillin, ticarcillin, azlocillin and piperacillin while Beta-lactamase production in these variants appeared to be unchanged. The second type of resistant variant showed unchanged sensitivity to carbenicillin and ticarcillin, or only a slight increase in resistance, whereas resistance to azlocillin and piperacillin was increased as much as 40-fold or more. These variants showed increased constitutive Beta-lactamase production and may be derepressed mutants of the parent culture. Variants of this type were readily selected by culture in the presence of azlocillin or piperacillin but only infrequently as a result of culture in the presence of carbenicillin or ticarcillin. The existence in cultures of P. aeruginosa of variants showing elevated Beta-lactamase production may account at least in part for the effect of inoculum size on the activity of azlocillin and piperacillin against P. aeruginosa and the marked discrepancy between MIC and minimum bactericidal concentration (MBC) which is characteristic of the ureido penicillins.

Comparative activities of ampicillin, epicillin and amoxycillin in vitro and in vivo.

The antibacterial activities of three aminopenicillins ampicillin, epicillin and amoxycillin were compared in vitro and in vivo. The minimum inhibitory concentrations (MIC) of the three penicillins were very similar and the compounds were active against non-beta-lactamase-producing strains of Escherichia coli, Salmonella and Shigella species, Proteus mirabilis, Haemophilus influenzae and Neisseria gonorrhoeae. Streptococci including Streptococcus faecalis, and non-beta-lactamase-producing staphylococci were also sensitive to the compounds but Pseudomonas aeruginosa, Klebsiella aerogenes, Enterobacter and indole-positive Proteus species were resistant. At concentrations close to MIC value epicillin and ampicillin showed similar bactericidal activity against E. coli and against S. typhi and both compounds caused a slower rate of kill than was seen with amoxycillin. Microscopical observation of the cells exposed to ampicillin and epicillin for 1 h showed the presence of filamentous forms which lysed slowly, whereas cells exposed to amoxycillin for the same period rapidly. Epicillin was similar to or slightly less active than ampicillin against experimental mouse infections, and against the majority of infections both compounds were significantly less effective than amoxycillin by the oral and subcutaneous routes of administration.