Genome of the bacterium Streptococcus pneumoniae strain R6.

Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.

Gene disruption studies of penicillin-binding proteins 1a, 1b, and 2a in Streptococcus pneumoniae.

The effects of inactivation of the genes encoding penicillin-binding protein 1a (PBP1a), PBP1b, and PBP2a in Streptococcus pneumoniae were examined. Insertional mutants did not exhibit detectable changes in growth rate or morphology, although a pbp1a pbp1b double-disruption mutant grew more slowly than its parent did. Attempts to generate a pbp1a pbp2a double-disruption mutant failed. The pbp2a mutants, but not the other mutants, were more sensitive to moenomycin, a transglycosylase inhibitor. These observations suggest that individually the pbp1a, pbp1b, and pbp2a genes are dispensable but that either pbp1a or pbp2a is required for growth in vitro. These results also suggest that PBP2a is a functional transglycosylase in S. pneumoniae.

Novel glycopeptide antibiotics: N-alkylated derivatives active against vancomycin-resistant enterococci.

LY264826 (A82846B) is a naturally-occurring glycopeptide antibiotic, differing from vancomycin in the stereochemistry of the amino-sugar of the disaccharide function, and the presence of a third sugar attached at the benzylic position of amino acid residue 6. Despite these seemingly subtle differences, LY264826 is approximately 10 times more active than vancomycin against the enterococci. In the pursuit of new antibiotics active against multiresistant Gram-positive organisms, an extensive side chain SAR was developed focusing on the reductive alkylation of LY264826 at the amino function of the disaccharide moiety. A new series of derivatives having varying degrees of structural diversity in the side chain (e.g. varying lengths and degrees of rigidity) was found to have potent activity against vancomycin-resistant enterococci (MIC's < 1.0 microgram/ml) as well as activity against staphylococci and streptococci as good or better than vancomycin.

Enzymatic deacylation of teicoplanin followed by reductive alkylation: synthesis and antibacterial activity of new glycopeptides.

Novel glycopeptides derived from teicoplanin were prepared and evaluated for activity against antibiotic-resistant gram-positive pathogens. Removal of the fatty acid sidechains of teicoplanin was accomplished by enzymatic deacylation. The resulting deacylated teicoplanin was subjected to reductive alkylation resulting in mono- and di-alkylated compounds at the 2 possible primary amines. Deacylated teicoplanin was less active than teicoplanin against enterococci and staphylococci (MIC > or =32 microg/ml). All mono- and di-alkylated products regained some activity, and some had potent activity against both staphylococci and glycopeptide-resistant enterococci. MICs of the most potent di-alkylated compounds ranged from 0.25 approximately 2 microg/ml against glycopeptide-resistant enterococci.

Beyond vancomycin: new therapies to meet the challenge of glycopeptide resistance.

The incidence of infections caused by resistant Gram-positive pathogens is increasing, while emergence of vancomycin resistance is reducing the number of therapeutic options. New agents are being rapidly evaluated as candidates to replace vancomycin; some of the most promising include semisynthetic glycopeptides, quinupristin-dalfopristin, oxazolidinones and everninomycins. Alternative strategies, including immunization and therapeutic vaccines, may also have a role.

Induction of VanA vancomycin resistance genes in Enterococcus faecalis: use of a promoter fusion to evaluate glycopeptide and nonglycopeptide induction signals.

To characterize induction of VanA resistance a plasmid was constructed in which the gene for firefly luciferase lucA was placed under the control of the promoter for the VanA resistance genes, the vanH promoter. This system afforded convenient quantitative measurement of induction of the VanA genes. Glycopeptide antibiotics and antibiotics representing 19 different mechanisms of action were evaluated for their ability to induce. Antibiotics that acted as inducers were all inhibitors of late steps of peptidoglycan synthesis. These included moenomycin, bacitracin, tunicamycin, ramoplanin and glycopeptides, but not penicillin or other beta-lactam antibiotics. Glycopeptide antibiotics were the most potent inducers. Both glycopeptides with little or no antimicrobial activity and semisynthetic glycopeptides active against VanA resistant enterococci were inducers. Overall, results suggest that an induction response may involve both an internal signal, such as precursor accumulation, and the glycopeptide molecule itself as a signal. The system may be useful as a screen for new antimicrobial agents.

Inhibition of peptidoglycan biosynthesis in vancomycin-susceptible and -resistant bacteria by a semisynthetic glycopeptide antibiotic.

LY191145 is a p-chlorobenzyl derivative of LY264826 (A82846B) with activity against both vancomycin-susceptible and -resistant enterococci. Incorporation of L-[14C]lysine into peptidoglycan of intact vancomycin-susceptible and -resistant Enterococcus faecium was inhibited by LY191145 (50% inhibitory concentrations of 1 and 5 microgram/ml, respectively). Inhibition was accompanied by accumulation of UDP-muramyl-peptide precursors in the cytoplasm. This agent inhibited late-stage steps in peptidoglycan biosynthesis in permeabilized E. faecium when either the UDP-muramyl-pentapeptide precursor from vancomycin-susceptible E. faecium or the UDP-muramyl-pentadepsipeptide precursor from vancomycin-resistant E. faecium was used as a substrate. Inhibition of late-stage steps led to accumulation of an N-acetyl-[14C]glucosamine-labeled lipid intermediate indicative of inhibition of the transglycosylation step. Inhibition of peptidoglycan polymerization without affecting cross-linking in a particulate membrane-plus-wall-fragment assay from Aerococcus viridans was consistent with this explanation. The fact that inhibition of peptidoglycan biosynthesis by LY191145 was not readily antagonized by an excess of free acyl-D-alanyl-D-alanine or acyl-D-alanyl-D-lactate ligands indicates that the manner in which this compound inhibits transglycosylation may not be identical to that of vancomycin.

Semisynthetic glycopeptide antibiotics derived from LY264826 active against vancomycin-resistant enterococci.

Certain derivatives of the glycopeptide antibiotic LY264826 with N-alkyl-linked substitutions on the epivancosamine sugar are active against glycopeptide-resistant enterococci. Six compounds representing our most active series were evaluated for activity against antibiotic-resistant, gram-positive pathogens. For Enterococcus faecium and E. faecalis resistant to both vancomycin and teicoplanin, the MICs of the six semisynthetic compounds for 90% of the strains tested were 1 to 4 micrograms/ml, compared with 2,048 micrograms/ml for vancomycin and 256 micrograms/ml for LY264826. For E. faecium and E. faecalis resistant to vancomycin but not teicoplanin, the MICs were 0.016 to 1 micrograms/ml, compared with 64 to 1,024 micrograms/ml for vancomycin. The compounds were highly active against vancomycin-susceptible enterococci and against E. gallinarum and E. casseliflavus and showed some activity against isolates of highly vancomycin-resistant leuconostocs and pediococci. The MICs for 90% of the strains of methicillin-resistant Staphylococcus aureus tested were typically 0.25 to 1 micrograms/ml, compared with 1 microgram/ml for vancomycin. Against methicillin-resistant S. epidermidis MICs ranged from 0.25 to 2 micrograms/ml, compared with 1 to 4 micrograms/ml for vancomycin and 4 to 16 micrograms/ml for teicoplanin. The spectrum of these new compounds included activity against teicoplanin-resistant, coagulase-negative staphylococci. The compounds exhibited exceptional potency against pathogenic streptococci, with MICs of < or = 0.008 microgram/ml against Streptococcus pneumoniae, including penicillin-resistant isolates. In in vivo studies with a mouse infection model, the median effective doses against a challenge by S. aureus, S. pneumoniae, or S. pyogenes were typically 4 to 20 times lower than those of vancomycin. Overall, these new glycopeptides, such as LY307599 and LY333328, show promise for use as agents against resistant enterococci, methicillin-resistant S. aureus, and penicillin-resistant pneumococci.

Reductive alkylation of glycopeptide antibiotics: synthesis and antibacterial activity.

Reductive alkylation of the A82846 family of glycopeptide antibiotics has the potential of producing seven products. N-Alkylation of the disaccharide amino function can be accomplished selectively, and offers the greatest increase in antibacterial activity. Products resulting from N-alkylation of LY264826 (A82846B) provide the most potent derivatives as compared to other members of this class of antibiotics. Two of these derivatives, LY307599 and LY333328 are approximately 500 times more active than vancomycin against vancomycin-resistant enterococci.

Activities of the semisynthetic glycopeptide LY191145 against vancomycin-resistant enterococci and other gram-positive bacteria.

LY191145 is the prototype of a series of compounds with activities against vancomycin-resistant enterococci derived by modification of the glycopeptide antibiotic LY264826. LY191145 had MICs for vancomycin- and teicoplanin-resistant enterococci of < or = 4 micrograms/ml for 50% of isolates and < or = 16 micrograms/ml for 90% of isolates. Its MICs for vancomycin-resistant, teicoplanin-susceptible enterococci were 1 to 8 micrograms/ml. LY191145 retains the potent activities of its parent compound against staphylococci and streptococci. In vivo studies in a mouse infection model confirmed these activities. This compound indicates the potential of semisynthetic glycopeptides as agents against antibiotic-resistant gram-positive bacteria.

beta-Lactamase inhibitors derived from N-tosyloxy-beta-lactams.

Electrophilic N-tosyloxy-beta-lactams, N-tosyloxy-4-phenyl-2-azetidinone (2b) and N-tosyloxy-3-(S)-phthalimido-4-(S)-2-azetidinone (2c), are described. These agents are novel potent beta-lactamase inhibitors.

In-vitro synergy and mechanism of interaction between vancomycin and ciprofloxacin against enterococcal isolates.

In-vitro synergy between vancomycin and ciprofloxacin against 44 enterococcal isolates was studied. Synergy occurred in chequerboard MIC determinations with six Enterococcus faecium strains resistant to both vancomycin and ciprofloxacin. The combination was additive for strains susceptible to one or both antibiotics. Time-kill studies involving selected strains with different susceptibility patterns confirmed the chequerboard results. The effect of ciprofloxacin on the induction of vancomycin resistance was compared in two vancomycin-resistant strains of E. faecium. Sub-inhibitory concentrations of ciprofloxacin prevented induction of vancomycin resistance in a ciprofloxacin-resistant strain, but not in a ciprofloxacin-susceptible strain. Membranes isolated from vancomycin-resistant ciprofloxacin-resistant cultures grown with vancomycin and ciprofloxacin at < or = 8 mg/L (0.125 x MIC) expressed a 39.5-kDa membrane protein involved in the expression of vancomycin resistance, but the protein was not detected in membranes from cultures grown in ciprofloxacin 16 mg/L. These findings indicated that a vancomycin-ciprofloxacin combination can be synergic against enterococci resistant to both vancomycin and ciprofloxacin, but would be unlikely to offer any advantage in the treatment of enterococcal infections because of the high concentrations required.

Inhibitors of two-component signal transduction systems: inhibition of alginate gene activation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa strains infecting cystic fibrosis patients often produce copious amounts of the exopolysaccharide alginate. Expression of alginate genes in P. aeruginosa is regulated by several proteins including members of the two-component bacterial signal transduction systems. Two of these regulatory proteins are AlgR1, the DNA-binding response regulator that transcriptionally activates alginate gene expression, and AlgR2, the kinase that modifies AlgR1 via phosphorylation to enhance its activity. In this paper, we report the identification of compounds that inhibit alginate gene expression by inhibiting (i) the phosphorylation/dephosphorylation of AlgR2 and (ii) the DNA-binding activity of AlgR1. Compounds with these activities may have potential as components of therapy for eliminating P. aeruginosa infection from the cystic fibrosis lung. In addition, we describe the effect of these compounds on the autophosphorylation activity of other known two-component kinases and show the ability of one compound to significantly inhibit the kinase activities of CheA, NRII, and KinA.

Modes of action and inhibitory activities of new siderophore-beta-lactam conjugates that use specific iron uptake pathways for entry into bacteria.

We describe here the mechanism of inhibition of two new siderophore-beta-lactam conjugates against Escherichia coli X580. One conjugate is a spermidine-based catechol siderophore-carbacephalosporin (JAM-2-263), and the other is an N5-acetyl-N5-hydroxy-L-ornithine tripeptide hydroxamate siderophore-carbacephalosporin (EKD-3-88). In an agar diffusion test, both conjugates produced large inhibitory zones against strain X580. Resistant strains (i.e., JAMR and EKDR) could be isolated after exposure of X580 to the conjugates JAM-2-263 and EKD-3-88, respectively. No cross-resistance was observed in these individual isolates. JAMR and EKDR were studied further to elucidate the mechanism of inhibition of each conjugated drug. The affinities of JAM-2-263 and EKD-3-88 for penicillin-binding proteins (PBPs) of isolated inner membranes were determined by a competition assay with 125I-penicillin V. JAM-2-263 targeted primarily PBPs 1A/B and 5/6, while EKD-3-88 targeted PBPs 1A/B and 3. Strains X580, JAMR, and EKDR showed similar PBP affinities for the conjugates. However, marked changes were observed in the iron-regulated outer membrane proteins of resistant isolates grown on agar plates depleted of iron. EKDR lost the expression of FhuA (78 kDa) and its sensitivity to phages T1 and T5, whereas JAMR lost the expression of Cir (74 kDa) and its sensitivity to colicin Ia. These results revealed the requirement of FhuA and Cir for the inhibitory activities of EKD-3-88 and JAM-2-263, respectively. In an antibiotic diffusion assay, ferrichrome (1 microM) strongly antagonized the activities of both conjugates against X580 and JAMR, including the residual activity of JAM-2-263 against JAMR. However, the susceptibility of strain EKDR lacking the ferrichrome receptor (FhuA-) to the two conjugates remained the same in the presence of ferrichrome. The antagonistic effect of ferrichrome on the activity of JAM-2-263 may also indicate a role for FhuA in the activity of this beta-lactam conjugate. A FhuA- Cir- double mutant confirmed this hypothesis, since it showed a higher level of resistance to JAM-2-263. To reproduce iron-restricted in vivo growth conditions, we grew X580 and EKDR cells in diffusion chambers implanted in the peritoneal cavities of rats. Strain EKDR showed impaired growth in such a cultivation system. This is the first report of beta-lactam drug transport into E. coli cells that involves the FhuA outer membrane protein.

Comparison of the outer membrane protein and lipopolysaccharide profiles of mucoid and nonmucoid Pseudomonas aeruginosa.

Laboratory-derived mucoid variants of Pseudomonas aeruginosa were selected by plating the standard PAO1 laboratory strain with bacteriophage. These mucoid variants formed two distinct groups of strains on the basis of phage typing. The first group had the same phage-typing pattern as the parent PAO1 strain, while the second group had a distinctly different phage-typing pattern. One strain from each group was assessed along with the parent PAO1 strain for its outer membrane protein (OMP) and lipopolysaccharide (LPS) profiles by sodium dodecyl sulfate-gel electrophoresis followed by appropriate staining. The mucoid derivatives were found to differ from the parent PAO1 nonmucoid strain in having lost a high-molecular-weight LPS species. Furthermore, the reversion of the mucoid strains to the nonmucoid phenotype was accompanied by a return of the missing high-molecular-weight LPS species. No observable difference between the mucoid derivatives and the parent nonmucoid strain was noted in the OMP profiles. The opposite was found in the case of four isolates of mucoid P. aeruginosa from patients with cystic fibrosis. Two OMP bands (of approximately 55 and 25 kilodaltons) were present in the mucoid isolates but missing in their sister nonmucoid strains. In the case of the cystic fibrosis isolates, no difference in the LPS profiles within mucoid-nonmucoid pairs was noted.

Activity of glycopeptides against vancomycin-resistant gram-positive bacteria.

Gram-positive bacteria resistant to vancomycin are rare; but they include members of the genera Leuconostoc, Lactobacillus, and Pediococcus, as well as recently emerging vancomycin-resistant strains of Enterococcus faecium and Enterococcus faecalis. Vancomycin, teicoplanin, and several vancomycin derivatives were tested for their activities against vancomycin-resistant gram-positive bacteria. Vancomycin-resistant E. faecium and E. faecalis were generally cross-resistant to other glycopeptides, but some N-substituted vancomycin derivatives were active against the resistant strains, with MICs of 2 to 32 micrograms/ml. These vancomycin derivatives also had significant levels of activity against intrinsically vancomycin-resistant organisms such as Leuconostoc sp. While vancomycin resistance in E. faecium and E. faecalis was inducible, resistance in members of the genera Leuconostoc, Lactobacillus, and Pediococcus appeared to be expressed constitutively. Antibody to a vancomycin-induced membrane protein found in membranes of resistant enterococci did not detect a cross-reacting protein in other vancomycin-resistant species.