Fastidious Gram-negative Pathogens Research Articles

In Vitro and In Vivo Properties of CUO246, a Novel Bacterial DNA Gyrase/Topoisomerase IV Inhibitor.

CUO246, a novel DNA gyrase/topoisomerase IV inhibitor, is active in vitro against a broad range of Gram-positive, fastidious Gram-negative, and atypical bacterial pathogens and retains activity against quinolone-resistant strains in circulation. The frequency of selection for single step mutants of wild-type S. aureus with reduced susceptibility to CUO246 was <4.64 × 10-9 at 4× and 8× MIC and remained low when using an isogenic QRDR mutant (<5.24 × 10-9 at 4× and 8× MIC). Biochemical assays indicated that CUO246 had potent inhibitory activity against both DNA gyrase (GyrAB) and topoisomerase IV (ParCE). Furthermore, CUO246 showed rapid bactericidal activity in time-kill assays and potent in vivo efficacy against S. aureus in a neutropenic murine thigh infection model. These results suggest that CUO246 may be useful in treating infections by various causative agents of acute skin and skin structure infections, respiratory tract infections, and sexually transmitted infections.

Expanding the potential of NAI-107 for treating serious ESKAPE pathogens: synergistic combinations against Gram-negatives and bactericidal activity against non-dividing cells

ObjectivesTo characterize NAI-107 and related lantibiotics for their in vitro activity against Gram-negative pathogens, alone or in combination with polymyxin, and against non-dividing cells or biofilms of Staphylococcus aureus. NAI-107 was also evaluated for its propensity to select or induce self-resistance in Gram-positive bacteria.MethodsWe used MIC determinations and chequerboard experiments to establish the antibacterial activity of the examined compounds against target microorganisms. Time–kill assays were used to evaluate killing of exponential and stationary-phase cells. The effects on biofilms (growth inhibition and biofilm eradication) were evaluated using biofilm-coated pegs. The frequency of spontaneous resistant mutants was evaluated by either direct plating or by continuous sub-culturing at 0.5 × MIC levels, followed by population analysis profiles.ResultsThe results showed that NAI-107 and its brominated variant are highly active against Neisseria gonorrhoeae and some other fastidious Gram-negative pathogens. Furthermore, all compounds strongly synergized with polymyxin against Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, and showed bactericidal activity. Surprisingly, NAI-107 alone was bactericidal against non-dividing A. baumannii cells. Against S. aureus, NAI-107 and related lantibiotics showed strong bactericidal activity against dividing and non-dividing cells. Activity was also observed against S. aureus biofilms. As expected for a lipid II binder, no significant resistance to NAI-107 was observed by direct plating or serial passages.ConclusionsOverall, the results of the current work, along with previously published results on the efficacy of NAI-107 in experimental models of infection, indicate that this lantibiotic represents a promising option in addressing the serious threat of antibiotic resistance.

Pleuromutilins: Potent Drugs for Resistant Bugs—Mode of Action and Resistance

Pleuromutilins are antibiotics that selectively inhibit bacterial translation and are semisynthetic derivatives of the naturally occurring tricyclic diterpenoid pleuromutilin, which received its name from the pleuromutilin-producing fungus Pleurotus mutilus Tiamulin and valnemulin are two established derivatives in veterinary medicine for oral and intramuscular administration. As these early pleuromutilin drugs were developed at a time when companies focused on major antibacterial classes, such as the β-lactams, and resistance was not regarded as an issue, interest in antibiotic research including pleuromutilins was limited. Over the last decade or so, there has been a resurgence in interest to develop this class for human use. This has resulted in a topical derivative, retapamulin, and additional derivatives in clinical development. The most advanced compound is lefamulin, which is in late-stage development for the intravenous and oral treatment of community-acquired bacterial pneumonia and acute bacterial skin infections. Overall, pleuromutilins and, in particular, lefamulin are characterized by potent activity against Gram-positive and fastidious Gram-negative pathogens as well as against mycoplasmas and intracellular organisms, such as Chlamydia spp. and Legionella pneumophila Pleuromutilins are unaffected by resistance to other major antibiotic classes, such as macrolides, fluoroquinolones, tetracyclines, β-lactam antibiotics, and others. Furthermore, pleuromutilins display very low spontaneous mutation frequencies and slow, stepwise resistance development at sub-MIC in vitro. The potential for resistance development in clinic is predicted to be slow as confirmed by extremely low resistance rates to this class despite the use of pleuromutilins in veterinary medicine for >30 years. Although rare, resistant strains have been identified in human- and livestock-associated environments and as with any antibiotic class, require close monitoring as well as prudent use in veterinary medicine. This review focuses on the structural characteristics, mode of action, antibacterial activity, and resistance development of this potent and novel antibacterial class for systemic use in humans.

Biological profiling of novel tricyclic inhibitors of bacterial DNA gyrase and topoisomerase IV.

The objective of this study was to characterize the in vitro and in vivo biological properties of a novel series of small-molecule bacterial type IIA topoisomerase inhibitors. Bacterial susceptibility testing was performed by broth microdilution. Resistance frequencies were determined by plating bacteria onto agar containing test compound and enumerating mutants. Bacteria were passaged using subinhibitory concentrations of antibacterials to generate resistance. Target enzyme inhibition was determined by exposure to antibacterials and DNA; topoisomers were visualized by gel electrophoresis. Oral and intravenous pharmacokinetic profiles were determined in mice. In vivo efficacy was determined using a mouse model of septicaemia and thigh infection with MSSA and MRSA, respectively. Representative compounds REDX04139, REDX05604 and REDX05931 demonstrated in vitro potency against a range of Gram-positive and fastidious Gram-negative pathogens. Clinical isolate testing revealed REDX04139 and REDX05931 had MIC90 values of 0.25 and 0.5 mg/L, respectively, for MRSA and MIC90 values of 2 mg/L for streptococci. REDX04139 was bactericidal in vitro against Staphylococcus aureus at 8× MIC over 6 h. Pharmacokinetic profiling of REDX04139 and REDX05604 in mice revealed low clearance and excellent bioavailability (≥71%). REDX04139 provided 100% survival against S. aureus in a mouse septicaemia model, while REDX05604 reduced bacterial load by up to 3.7 log units in the MRSA mouse thigh infection model. Redx Pharma has discovered a novel series of topoisomerase inhibitors that are being further developed for drug-resistant bacteria.

In Vitro Activity of AZD0914, a Novel Bacterial DNA Gyrase/Topoisomerase IV Inhibitor, against Clinically Relevant Gram-Positive and Fastidious Gram-Negative Pathogens.

AZD0914, a new spiropyrimidinetrione bacterial DNA gyrase inhibitor with a novel mode of inhibition, has activity against bacterial species commonly cultured from patient infection specimens, including fluoroquinolone-resistant isolates. This study assessed the in vitro activity of AZD0914 against key Gram-positive and fastidious Gram-negative clinical isolates collected globally in 2013. AZD0914 demonstrated potent activity, with MIC90s for AZD0914 of 0.25 mg/liter against Staphylococcus aureus (n = 11,680), coagulase-negative staphylococci (n = 1,923), streptococci (n = 4,380), and Moraxella catarrhalis (n = 145), 0.5 mg/liter against Staphylococcus lugdunensis (n = 120) and Haemophilus influenzae (n = 352), 1 mg/liter against Enterococcus faecalis (n = 1,241), and 2 mg/liter against Haemophilus parainfluenzae (n = 70). The activity against Enterococcus faecium was more limited (MIC90, 8 mg/liter). The spectrum and potency of AZD0914 included fluoroquinolone-resistant isolates in each species group, including methicillin-resistant staphylococci, penicillin-resistant streptococci, vancomycin-resistant enterococci, β-lactamase-producing Haemophilus spp., and M. catarrhalis. Based on these in vitro findings, AZD0914 warrants further investigation for its utility against a variety of Gram-positive and fastidious Gram-negative bacterial species.

Discovery of 4''-ether linked azithromycin-quinolone hybrid series: influence of the central linker on the antibacterial activity.

A series of novel C-4''-substituted azithromycins was synthesized and evaluated for in vitro antibacterial activity against a panel of representative erythromycin-susceptible and macrolide-lincosamide-streptogramin (MLS) resistant pathogens. In summary, azithromycin and quinolone substructures merged in a mutually SAR-compatible design gave rise to a new class of antimicrobials with an improved spectrum and potency over azithromycin. Prototypical analogues 7f and 8f display an improved potency versus azithromycin against Gram-positive and fastidious Gram-negative pathogens. In particular, these new leads maintain activity against MLS-resistant strains of Streptococcus pneumoniae and Streptococcus pyogenes. In addition, they represent an improvement over telithromycin (1) and cethromycin (2) against the fastidious Gram-negative pathogen Haemophilus influenzae.

Novel hybrids of 15-membered 8a- and 9a-azahomoerythromycin A ketolides and quinolones as potent antibacterials

A series of novel 6-O-substituted and 6,12-di-O-substituted 8a-aza-8a-homoerythromycin A and 9a-aza-9a-homoerythromycin A ketolides were synthesized and evaluated for in vitro antibacterial activity against a panel of representative erythromycin-susceptible and erythromycin-resistant test strains. Another series of ketolides based on 14-membered erythromycin oxime scaffold was also synthesized and their antibacterial activity compared to those of 15-membered azahomoerythromycin analogues. In general, structure–activity studies have shown that 14-membered ketolides displayed favorable antibacterial activity in comparison to their corresponding 15-membered analogues within 9a-azahomoerythromycin series. However, within 8a-azahomoerythromycin series, some compounds incorporating a ketolide combined with either quinoline or quinolone pharmacophore substructures showed significantly potent activity against a variety of erythromycin-susceptible and macrolide-lincosamide-streptogramin B (MLS B)-resistant Gram-positive pathogens as well as fastidious Gram-negative pathogens. The best compounds in this series overcome all types of resistance in relevant clinical Gram-positive pathogens and display hitherto unprecedented in vitro activity against the constitutively MLS B-resistant strain of Staphylococcus aureus. In addition, they also represent an improvement over telithromycin ( 2) and cethromycin ( 3) against fastidious Gram-negative pathogens Haemophilus influenzae and Moraxella catarrhalis.

Synthesis and Structure−Activity Relationships of Novel 8a-Aza-8a-homoerythromycin A Ketolides

A series of novel 6-O-substituted 8a-aza-8a-homoerythromycin A ketolides was synthesized and evaluated for in vitro antibacterial activity. Key strategic elements of the synthesis include the base-induced E-Z isomerization of 3-O-descladinosyl-6-O-allylerythromycin A 9(E)-oxime followed by ring-expanding reaction of the resulting 9(Z)-oxime via Beckmann rearrangement. The ketolides showed potent activity against a variety of erythromycin-susceptible and macrolide-lincosamide-streptogramin B (MLS(B)) resistant Gram-positive and fastidious Gram-negative pathogens. The best compounds in this series overcome all types of resistance in relevant clinical Gram-positive pathogens and display in vitro activity comparable to telithromycin and cethromycin.

A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore

As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.

Conformational Constraint in Oxazolidinone Antibacterials. Synthesis and Structure−Activity Studies of (Azabicyclo[3.1.0]hexylphenyl)oxazolidinones

The oxazolidinones are a new class of synthetic antibacterials effective against a broad range of pathogenic Gram-positive bacteria, including multi-drug-resistant strains. Linezolid is the first drug from this class to reach the market and has become an important new option for the treatment of serious infections, particularly those caused by methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enteroccocus faecium (VRE). In the search for novel oxazolidinones with improved potency and spectrum, we have prepared and evaluated the antibacterial properties of conformationally constrained analogues in which the morpholine ring of linezolid is replaced with various substituted azabicyclo[3.1.0]hexyl ring systems. Several classes of azabicyclic analogues were identified with activity comparable or superior to that of linezolid. These include analogues bearing hydroxyl, amino, amido, or carboxyl groups on the azabicyclic ring. The azabicyclic acid analogue 50 was 4 times more potent than linezolid against key Gram-positive and fastidious Gram-negative pathogens (S. aureus, Streptococcus pneumoniae, and E. faecalis MICs < or = 1 microg/mL; Haemophilus influenzae MIC = 4 microg/mL).

Novel oxazolidinone–quinolone hybrid antimicrobials

Antimicrobial compounds incorporating oxazolidinone and quinolone pharmacophore substructures have been synthesized and evaluated. Representative analogues 2, 5, and 6 display an improved potency versus linezolid against gram-positive and fastidious gram-negative pathogens. The compounds are also active against linezolid- and ciprofloxacin-resistant Staphylococcus aureus and Enterococcus faecium strains. The MOA for these new antimicrobials is consistent with a combination of protein synthesis and gyrase A/topoisomerase IV inhibition, with a structure-dependent degree of the contribution from each inhibitory mechanism.

Carbon–carbon-linked (pyrazolylphenyl)oxazolidinones with antibacterial activity against multiple drug resistant gram-positive and fastidious gram-negative bacteria

In an effort to expand the spectrum of activity of the oxazolidinone class of antibacterial agents to include Gram-negative bacteria, a series of new carbon–carbon linked pyrazolylphenyl analogues has been prepared. The α- N-substituted methyl pyrazole ( 10α) in the C3-linked series exhibited very good Gram-positive activity with MICs ≤0.5–1 μg/mL and moderate Gram-negative activity with MICs=2–8 μg/mL against Haemophilus influenzae and Moraxella catarrhalis. This analogue was also found to have potent in vivo activity with an ED 50=1.9 mg/kg. β-Substitution at the C3-linked pyrazole generally results in a loss of activity. The C4-linked pyrazoles are slightly more potent than their counterparts in the C3-linked series. Most of the analogues in the C4-linked series exhibited similar levels of activity in vitro, but lower levels of activity in vivo than 10α. In addition, incorporation of a thioamide moiety in selected C4-linked pyrazole analogues results in an enhancement of in vitro activity leading to compounds several times more potent than eperezolid, linezolid and vancomycin. The thioamide of the N-cyanomethyl pyrazole analogue ( 34) exhibited an exceptional in vitro activity with MICs of ≤ 0.06–0.25 μg/mL against Gram-positive pathogens and with MICs of 1 μg/mL against fastidious Gram-negative pathogens.

A Critical Review of Oxazolidinones: An Alternative or Replacement for Glycopeptides and Streptogramins?

To review the available data on the oxazolidinones linezolid and eperezolid. Published reports were obtained by searching MEDLINE for articles published between 1992 and 2000, inclusive. References of published papers were also obtained and reviewed. Abstracts from scientific proceedings were reviewed. Due to the limited data available regarding these agents, the criteria for study inclusion were not restrictive. The oxazolidinones (eg, linezolid) are a new antimicrobial class with a unique mechanism of action. They are active against resistant Gram-positive cocci including methicillin-susceptible and -resistant Staphylococcus aureus (MRSA), methicillin-susceptible and -resistant Staphylococccus epidermidis, vancomycin-resistant enterococci (VRE) and penicillin-resistant Streptococcus pneumoniae (PRSP). Linezolid is active against anaerobes and displays modest activity against fastidious Gram-negative pathogens such as Haemophilus influenzae, but is not active against Enterobacteriaceae. Linezolid is available both orally and parenterally, and has a bioavailability of 100%. Clinical trials comparing linezolid with standard therapy have demonstrated similar bacteriological and clinical cures rates to standard therapy in community- and hospital-acquired pneumonia, uncomplicated and complicated skin and soft tissue infections, and infections caused by MRSA and VRE. Adverse effects have been minor and infrequent; however, platelets should be monitored in patients who have received more than two weeks of linezolid therapy. It is expected that these agents will have a bright future due to their excellent spectrum of activity against antibiotic-resistant Gram-positive organisms, such as MRSA, VRE and PRSP, and their excellent bioavailability. The oxazolidinones represent a new class of antimicrobials with a unique mechanism of action. They have excellent activity against susceptible and resistant Gram-positive organisms such as MRSA, methicillin-susceptible S epidermidis, VRE and PRSP, and a good adverse effect profile; they can be administered both intravenously and orally. Their potential use in Canada may be as an intravenous and oral alternative to glycopeptides and streptogramins.