7-(4-Alkylidenylpiperidinyl)-quinolone bacterial topoisomerase inhibitors.

Novel antibacterial fluoroquinolone agents bearing a 4-alkylidenylpiperidine 7-position substituent are active against quinolone-susceptible and quinolone-resistant gram-positive bacteria, including Streptococcus pneumoniae and MRSA. Analogs 22b, 23c, and 24 demonstrated superior in vitro and in vivo efficacy to ciprofloxacin against these cocci.

Toll-like receptor 3 is involved in airway epithelial cell response to nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is the etiological agent most frequently associated with bacterial exacerbations of chronic obstructive pulmonary disease (COPD). The present work shows that NTHi strains induced in primary normal human bronchial epithelial cells (NHBE) a cytokine/chemokine response in which CCL-5 and CXCL-10 were predominant. Production of both cytokines was inhibited by an anti-TLR3 monoclonal antibody (mAb) in a dose-dependent manner, but not by control human IgG4 antibodies, thus suggesting a TLR3-dependency of the NTHi stimulation. BEAS-2B, an immortalized human bronchial epithelial cell line, also showed a similar NTHi-induced response that was inhibited by the anti-TLR3 mAb. A BEAS-2B cell line stably expressing TLR3 siRNA showed significantly reduced cytokine/chemokine responses to NTHi stimulation, confirming the role of TLR3 in the response. These results indicate that TLR3 is a key component in the response of human bronchial epithelial cells to NTHi, and suggest that cognate neutralizing mAbs might be a useful therapeutic tool to regulate the inflammatory response.

Synthesis and antibacterial activity of 7-(1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-7-yl) quinolones.

A novel series of 7-(1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-7-yl) quinolones has been designed and synthesized in which the heterocyclic side chain is attached to the quinolone core through a carbon-carbon linkage. The antibacterial activity of the compounds was determined against a panel of Gram-positive and Gram-negative pathogens. Compounds 1b and 1e, bearing an 8-methoxy group as well as unsubstituted and (3S)-methyl substituted 1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-7-yl side chains, respectively, demonstrated notable activity against ciprofloxacin-resistant clinical isolates of Streptococcus pneumoniae.

In vivo activity of the pyrrolopyrazolyl-substituted oxazolidinone RWJ-416457.

RWJ-416457 is an investigational pyrrolopyrazolyl-substituted oxazolidinone with activity against antibiotic-susceptible and -resistant gram-positive pathogens. Efficacies of RWJ-416457, linezolid, and vancomycin against methicillin-susceptible Staphylococcus aureus (MSSA) and community-associated methicillin-resistant S. aureus (CA-MRSA) in murine skin and systemic infections were compared, as were efficacies against Streptococcus pneumoniae in a lower respiratory infection. In staphylococcal systemic infections, RWJ-416457 was equipotent with to twofold more potent than linezolid, with 50% effective dose values ranging from 1.5 to 5 mg/kg of body weight/day. RWJ-416457 was two- to fourfold less potent than vancomycin against MSSA but up to fourfold more potent than vancomycin against CA-MRSA. In MSSA and CA-MRSA skin infections, RWJ-416457 demonstrated an efficacy similar to that of linezolid, reducing CFU/g skin approximately 1.0 log(10) at all doses tested; vancomycin yielded greater reductions than the oxazolidinones, with decreases in CFU/g skin of 3 log(10) (MSSA) and 2 log(10) (CA-MRSA). In the pneumococcal model, RWJ-416457 was two- to fourfold more potent than linezolid. The free-drug area under the concentration-time curves at 24 h (fAUC(24)) were similar for RWJ-416457 and linezolid. The half-life of RWJ-416457 was up to threefold longer than that of linezolid for all routes of administration. The fAUC(24)/MIC ratio, the pharmacodynamic parameter considered predictive of oxazolidinone efficacy, was approximately twofold greater for RWJ-416457 than for linezolid. Since the fAUC values were similar for both compounds, the higher fAUC/MIC ratios of RWJ-416457 appear to result from its greater in vitro potency. These results demonstrate that RWJ-416457 is a promising new oxazolidinone with efficacy in S. aureus or S. pneumoniae mouse infection models.

Sporadic occurrences of fluoroquinolone-resistant Streptococcus pneumoniae in the United States: longitudinal analysis of institutions from the New England and West South Central regions during the TRUST 4-9 (2000-2005) Surveillance Studies.

Twenty-six institutions in New England and 24 institutions in West South Central regions participating in the Tracking Resistance in the United States Today (TRUST) 4-9 surveillance studies (2000-2005) were monitored for levofloxacin-resistant Streptococcus pneumoniae to determine if resistance was sporadic or persistent. Levofloxacin was used as a representative of the respiratory fluoroquinolones. Levofloxacin-resistant isolates were identified in 8 of the 26 New England institutions and in 11 of the 24 West South Central institutions during the surveillance period. Resistant isolates were recovered in consecutive years from 3 institutions: 1 each in Massachusetts, Oklahoma, and Texas. In total, 34 levofloxacin-resistant isolates (14 from New England, 20 from the West South Central region) were identified over the 6-year period. Two of these isolates from an institution in Connecticut and 2 from an institution in Oklahoma had the same serotype, pulsed-field gel electrophoresis pattern, and quinolone resistance-determining region (QRDR) mutations. States with elevated pneumococcal levofloxacin resistance rates, compared with the national average, did not maintain this status in consecutive years. Based on data from the same institutions over 6 years, levofloxacin resistance among US pneumococci has been sporadic, nonclonal, and rare.

Decline in the prevalence of pandemic clones Spain23F-1 and Spain9V-3 among US fluoroquinolone-resistant Streptococcus pneumoniae TRUST Surveillance isolates since 2001.

Fluoroquinolone-resistant variants of pandemic clones Spain(23F)-1, Spain(6B)-2, Spain(9V)-3, and Spain(14)-5 have been seen in various regions of the United States and the world, leading to the speculation that fluoroquinolone resistance among US Streptococcus pneumoniae may increase because of clonal spread. Using levofloxacin as a representative of the fluoroquinolone class, all 196 levofloxacin-resistant pneumococci from a total of 22 794 isolates in the Tracking Resistance in the United States Today (TRUST) 5-8 studies (2001-2004) were subjected to pulsed-field gel electrophoresis (PFGE), serotyping, and sequencing of parC/E and gyrA/B quinolone resistance-determining regions (QRDR) to measure the extent of clonality. In addition, susceptibility testing of these isolates to ciprofloxacin, gatifloxacin, levofloxacin, and moxifloxacin was performed. ATCC type strains of Spain(23F)-1, Spain(6B)-2, Spain(9V)-3, and Spain(14)-5 clones were included as comparators. Levofloxacin-resistant isolates with Spain(23F)-1-related PFGE patterns decreased from 28% of the resistant isolates in 2001 to 6% in 2004. These isolates, with serotypes 23F (n = 17), 19F (n = 17), or 19A (n = 1), had 15 different QRDR profiles and were all ciprofloxacin- and gatifloxacin-resistant. Levofloxacin-resistant isolates with Spain(9V)-3-related PFGE patterns decreased from 13% of the resistant isolates in 2001 to 2% in 2004. The Spain(9V)-3-related isolates were serotype 9V (n = 9), 9A (n = 2), and 9N (n = 1) and exhibited 6 different QRDR profiles. All were resistant to all fluoroquinolones tested. None of the levofloxacin-resistant isolates had PFGE patterns related to Spain(6B)-2 or Spain(14)-5. Resistance to respiratory fluoroquinolones among pneumococci has remained constant at about 1% (0.8%-1.1%) since 2001, and there has been a decrease in the prevalence of levofloxacin-resistant isolates similar to Spain(23F)-1 or Spain(9V)-3. Considerable QRDR variability among these strains appears to be the result of sporadic independent mutational events as opposed to clonal expansion.

Effects of the 7-valent pneumococcal conjugate vaccine on U.S. levofloxacin-resistant Streptococcus pneumoniae.

BACKGROUND: Seven-valent pneumococcal conjugate vaccine (PCV7) provides protection against invasive pneumococcal disease that extends to unvaccinated populations, such as elderly or immunocompromised adults. PCV7 also reduces incidence of pneumococcal penicillin resistance. In this study, the potential impact of PCV7 on pneumococcal fluoroquinolone resistance was examined. METHODS: U.S. levofloxacin-resistant isolates (264) from TRUST surveillance studies (1999-2004) were serotyped and quinolone resistance-determining region of parC/E and gyrA/B sequenced. Changes in prevalence of vaccine/nonvaccine serotypes during 2000-2004 and 1999-2004 were analyzed by regression analyses and chi-square trend test. RESULTS: The introduction of PCV7 (2000-2004) did not affect fluoroquinolone resistance prevalence, but mutants with vaccine serotypes declined linearly at -6.6 +/- 0.8% per year (p = 0.003), with concomitant replacement by nonvaccine serotypes; vaccine-related serotypes (6A, 9N, 19A, and 23N) increased (p = 0.04). Differential selection between vaccine and nonvaccine serotypes occurred for mutants containing amino acid substitutions at either ParC Ser79 (p = 0.01) or both ParC Ser79 and GyrA Ser81 (p = 0.04). Among mutants with ParC Ser79 substitutions, vaccine serotypes declined linearly (p = 0.02), whereas nonvaccine serotypes increased (p = 0.04). Additionally, a vaccine-independent effect became apparent during 1999-2004, as the incidence of ParC Ser79 and Asp83 mutations declined in fluoroquinolone-resistant strains, suggesting that these substitutions conferred decreased fitness. CONCLUSIONS: PCV7 has led to extensive replacement of vaccine serotypes by nonvaccine serotypes among levofloxacin-resistant pneumococci.

Tetrahydroindazole inhibitors of bacterial type II topoisomerases. Part 2: SAR development and potency against multidrug-resistant strains.

We have previously reported a novel class of tetrahydroindazoles that display potency against a variety of Gram-positive and Gram-negative bacteria, potentially via interaction with type II bacterial topoisomerases. Herein are reported SAR investigations of this new series. Several compounds possessing broad-spectrum potency were prepared. Further, these compounds exhibit activity against multidrug-resistant Gram-positive microorganisms equivalent to that against susceptible strains.

In vitro antibacterial activity of the pyrrolopyrazolyl-substituted oxazolidinone RWJ-416457.

RWJ-416457, an investigational pyrrolopyrazolyl-substituted oxazolidinone, inhibited the growth of linezolid-susceptible staphylococci, enterococci, and streptococci at concentrations of < or =4 microg/ml, generally exhibiting two- to fourfold-greater potency than that of linezolid. Time-kill studies demonstrated bacteriostatic effects for both RWJ-416457 and linezolid.

Antibacterial activity of pyrrolopyridine-substituted oxazolidinones: synthesis and in vitro SAR of various C-5 acetamide replacements.

A series of pyrrolopyridine-substituted oxazolidinones containing various C-5 acetamide isosteres was synthesized and the structure-antibacterial activity relationships determined against a representative panel of susceptible and resistant Gram-positive bacteria.

Infrequent occurrence of single mutations in topoisomerase IV and DNA gyrase genes among US levofloxacin-susceptible clinical isolates of Streptococcus pneumoniae from nine institutions (1999-2003).

OBJECTIVES: Prevalence of single quinolone-resistance determining region (QRDR) mutations in Streptococcus pneumoniae was studied from nine institutions over 5 years to track the incidence of single QRDR mutations. METHODS: All 1106 levofloxacin-susceptible pneumococci (MICs < or = 2.0 mg/L) identified from 1112 total isolates (99.5% susceptibility) in TRUST 3 (1999), TRUST 5 (2001) and TRUST 7 (2003) surveillance studies from the same nine hospitals in nine states were screened for QRDR mutations. Using pyrosequencing, the strains were screened for mutations corresponding to hot spots Asp-78, Ser-79 and Asp-83 in ParC; Asp-80, Ser-81 and Glu-85 in GyrA; Asp-435 in ParE and Asp-435 in GyrB. DNA sequencing of QRDRs was performed to confirm mutations. RESULTS: No QRDR mutations were found in any of the isolates with levofloxacin MICs < or = 0.5 mg/L and no gyrA or gyrB QRDR mutations were found in any of the screened isolates (MICs < or = 2 mg/L). Four single-step QRDR mutants with the following amino acid substitutions were found: ParE Asp-435 to Asn (isolated in 1999 in Colorado); ParC Asp-83 to Asn (isolated in 2001 in Kentucky); ParC Ser-79 to Phe (isolated in 2003 in Indiana) and ParC Ser-79 to Tyr (isolated in 2003 in California). These non-clonal strains had levofloxacin MICs of 1 mg/L and were non-susceptible to ciprofloxacin (MIC 2-4 mg/L). CONCLUSIONS: Overall prevalence of single QRDR mutations in levofloxacin-susceptible S. pneumoniae with MICs of < or = 2 mg/L was 0.4% (4/1106) and has remained <1% within nine institutions over 5 years (1999-2003).

Utility of muropeptide ligase for identification of inhibitors of the cell wall biosynthesis enzyme MurF.

MurF is a key enzyme in the biosynthesis of the bacterial cell wall in both gram-positive and gram-negative bacteria. This enzyme has not been extensively exploited as a drug target, possibly due to the difficulty in obtaining one of the substrates, UDP-MurNAc-L-Ala-gamma-D-Glu-meso-diaminopimelate, which is usually purified from bacteria. We have identified putative inhibitors of Escherichia coli MurF by a binding assay, thus bypassing the need for substrate. Inhibition of enzymatic activity was demonstrated in a high-performance liquid chromatography-based secondary assay with UDP-MurNAc-L-Ala-gamma-D-Glu-diaminopimelate substrate prepared in a novel way by using muropeptide ligase enzyme to add UDP-MurNAc to synthetic L-Ala-gamma-D-Glu-diaminopimelate; the substrate specificity of muropeptide ligase for peptides containing L-Lys in place of diaminopimelate was also investigated. Using the muropeptide ligase-generated MurF substrate, a thiazolylaminopyrimidine series of MurF enzyme inhibitors with 50% inhibitory concentration values as low as 2.5 microM was identified.

Identification of a dithiazoline inhibitor of Escherichia coli L,D-carboxypeptidase A.

The enzyme L,D-carboxypeptidase A is involved in the recycling of bacterial peptidoglycan and is essential in Escherichia coli during stationary phase. By high-throughput screening, we have identified a dithiazoline inhibitor of the enzyme with a 50% inhibitory concentration of 3 microM. The inhibitor appeared to cause lysis of E. coli during stationary phase, behavior that is similar to a previously described deletion mutant of L,D-carboxypeptidase A (M. F. Templin, A. Ursinus, and J.-V. Holtje, EMBO J. 18:4108-4117, 1999). As much as a one-log drop in CFU in stationary phase was observed upon treatment of E. coli with the inhibitor, and the amount of intracellular tetrapeptide substrate increased by approximately 33%, consistent with inhibition of the enzyme within bacterial cells. Stationary-phase targets such as L,D-carboxypeptidase A are largely underrepresented as targets of the antibiotic armamentarium but provide potential opportunities to interfere with bacterial growth and persistence.

Novel Ser79Leu and Ser81Ile substitutions in the quinolone resistance-determining regions of ParC topoisomerase IV and GyrA DNA gyrase subunits from recent fluoroquinolone-resistant Streptococcus pneumoniae clinical isolates.

Resistance of Streptococcus pneumoniae to fluoroquinolones is caused predominantly by amino acid substitutions at positions Ser79 of ParC and Ser81 of GyrA to either Phe or Tyr encoded in the quinolone resistance-determining regions of the parC topoisomerase IV and gyrA DNA gyrase genes. Analysis of highly resistant clinical isolates identified novel second-step substitutions, Ser79Leu (ParC) and Ser81Ile (GyrA). To determine contributions of these new mutations to fluoroquinolone resistance either alone or in combination with other Ser79/81 alleles, the substitutions Ser79Leu/Phe/Tyr in ParC and Ser81Ile/Phe/Tyr in GyrA were introduced into the R6 background, resulting in 15 isogenic strains. Their level of fluoroquinolone resistance was determined by susceptibility testing for ciprofloxacin, levofloxacin, moxifloxacin, gatifloxacin, gemifloxacin, garenoxacin, and norfloxacin. Leu79 and Ile81 alone as well as 79/81Phe/Tyr substitutions did not contribute significantly to resistance, with fluoroquinolone MICs increasing two- to fourfold compared to wild type for all agents tested. Fluoroquinolone MICs for double transformants ParC Ser79Phe/Tyr/Leu-GyrA Ser81Phe/Tyr were uniformly increased by 8- to 64-fold regardless of pairs of amino acid substitutions. However, combinations including Ile81 conferred two- to fourfold-higher levels of resistance than did combinations including any other Ser81 GyrA substitution, thus demonstrating the differential effects of diverse amino acid substitutions at particular hotspots on fluoroquinolone MICs.

The synthesis and antimicrobial evaluation of a new series of isoxazolinyl oxazolidinones.

A series of oxazolidinone antibacterial agents containing a 5-substituted isoxazol-3-yl moiety were synthesized via a nitrile oxide [3+2] dipolar cycloaddition reaction. These compounds were screened against a panel of susceptible and resistant Gram-positive organisms. Several analogs from this series were comparable to or more potent than linezolid in vitro.

Synthesis and antibacterial activity of pyrroloaryl-substituted oxazolidinones.

A novel series of oxazolidinones containing a pyrroloaryl substituent was synthesized and screened against a representative panel of susceptible and resistant Gram-positive bacteria. Several members of this series were found to have antibacterial activity comparable to or better than linezolid.

Cross-resistance, relatedness and allele analysis of fluoroquinolone-resistant US clinical isolates of Streptococcus pneumoniae (1998-2000).

OBJECTIVES: To detect relatedness among 68 (0.5%) of 13 795 US clinical isolates of Streptococcus pneumoniae from the TRUST 3 (1998-1999) and TRUST 4 (1999-2000) surveillance studies that were resistant to levofloxacin (MIC > or = 8 mg/l). METHODS: All levofloxacin-resistant isolates were analysed by broth microdilution reference method for susceptibility to four fluoroquinolones, DNA sequencing of quinolone resistance determining region (QRDR) of topoisomerase IV and DNA gyrase genes, serotyping, and pulsed-field gel electrophoresis (PFGE). RESULTS: All levofloxacin-resistant isolates were ciprofloxacin resistant (MIC > or = 4 mg/l, FDA breakpoint) and non-susceptible to gatifloxacin (MIC > or = 2 mg/l); 62 were non-susceptible to moxifloxacin (MIC > or = 2 mg/l). Resistant isolates were in 48 (20%) of 238 institutions in 29 states. Three institutions had levofloxacin-resistant isolates in both surveillance studies. Among the resistant isolates were 17 serotypes and 48 different PFGE patterns. Fourteen isolates had PFGE patterns closely related to the Spain(23F)-1 clone; one strain had a PFGE pattern closely related to the French(9V)-3 clone. All levofloxacin-resistant isolates had two or more mutations within the QRDR of parC, parE, gyrA and gyrB. CONCLUSIONS: US levofloxacin-resistant S. pneumoniae isolates were rare and most were unrelated with minimal clonal spread, and were associated with multiple QRDR mutations with extensive cross-resistance noted among fluoroquinolones.

Screening of large numbers of Streptococcus pneumoniae isolates for mutations associated with fluoroquinolone resistance using an oligonucleotide probe assay.

Mutations at a relatively small number of sites in parC, parE and gyrA account for most of the fluoroquinolone resistance in Streptococcus pneumoniae clinical isolates. A high throughput oligonucleotide probe assay was developed to screen for mutations in the quinolone-resistance determining region (QRDR) of parC (Ser79), gyrA (Ser81) and parE (Asp435) of Streptococcus pneumoniae. Eight oligonucleotide probes (17mers) were used in the presence of tetramethyl ammonium chloride so that the melting temperature was dependent on length and not on base composition. Using this assay it was possible to accurately detect QRDR mutations from several hundred S. pneumoniae clinical isolates that were grown on nylon membranes.

Novel piperidinyloxy oxazolidinone antibacterial agents. Diversification of the N-Substituent.

Oxazolidinone antibacterial agents, where the morpholino group of linezolid was replaced with an N-substituted piperidinyloxy moiety, were synthesized and shown to be active against a variety of resistant and susceptible Gram-positive organisms. The functionality attached to the piperidine nitrogen was varied extensively to determine the SAR for this series. One of the most potent compounds, 11, showed in vivo efficacy upon subcutaneous administration in a Staphylococcus aureus Smith murine systemic infection.

Prevalence of single mutations in topoisomerase type II genes among levofloxacin-susceptible clinical strains of Streptococcus pneumoniae isolated in the United States in 1992 to 1996 and 1999 to 2000.

Levofloxacin resistance in Streptococcus pneumoniae is rare, requiring at least two mutations in the quinolone resistance-determining region (QRDR) of topoisomerase IV and DNA gyrase. The prevalence of single QRDR mutations in these genes is unknown. Of 9,438 levofloxacin-susceptible pneumococci from the TRUST 4 surveillance study (1999-2000), 528 strains (MICs of 0.5 to 2.0 microg/ml) were selected for analysis. For comparison, 214 levofloxacin-susceptible strains (MICs of 0.5 to 1 microg/ml) isolated between 1992 and 1996 were analyzed. Oligonucleotide probe assay and DNA sequencing were used to detect QRDR mutations leading to changes at Ser79 and Asp83 in ParC, Ser81 in GyrA, and Asp435 in ParE, the most frequently found substitutions among levofloxacin-resistant strains. Among the 1992 to 1996 isolates only one strain (levofloxacin MIC, 1 microg/ml) had a mutation (Ser79 to Phe in ParC). No single mutations were found among 270 TRUST 4 strains with levofloxacin MICs of 0.5 microg/ml. Among 244 strains for which levofloxacin MICs were 1 microg/ml, 15 strains (6.1%) had a parC mutation and 3 strains (1.2%) had a parE mutation. Of 14 strains for which levofloxacin MICs were 2 microg/ml, 10 strains (71%) had a parC mutation; no parE mutations were found. No gyrA mutations were detected. It was estimated that 4.5% of the 9,438 levofloxacin-susceptible TRUST 4 isolates (MICs, < or =0.06 to 2 microg/ml) had a single parC or parE QRDR mutation. Although there has been an increase in the prevalence of single-step mutants, the increase may have been overestimated due in part to differences in geographical distribution for the two sets of isolates.