Cong-Ran Li

In vivo antibacterial activity of nemonoxacin, a novel non-fluorinated quinolone

OBJECTIVES To evaluate the in vivo antibacterial efficacy of nemonoxacin, a novel C8-methoxy non-fluorinated quinolone in murine systemic and local infection models. METHODS The efficacy of nemonoxacin in systemic infections was evaluated in mouse peritonitis models using isolates of methicillin-susceptible Staphylococcus aureus (MSSA, n=1), methicillin-resistant S. aureus (MRSA, n=1), methicillin- and levofloxacin-resistant Staphylococcus capitis (levofloxacin-resistant MRSC, n=1), penicillin-intermediate Streptococcus pneumoniae (PISP, n=1), penicillin-resistant S. pneumoniae (PRSP, n=2), Enterococcus faecalis (n=2, including 1 vancomycin-resistant Enterococcus, VRE) and Escherichia coli (n=3). The local infections included mouse pulmonary infections caused by PRSP (n=1), Klebsiella pneumoniae (n=1) and mouse ascending urinary tract infection caused by E. coli (n=1). RESULTS In the mouse systemic infection model, nemonoxacin demonstrated potent activity against MSSA (ED(50) =2.08 mg/kg), MRSA (ED(50) =2.59 mg/kg), levofloxacin-resistant MRSC (ED(50) =2.52 mg/kg), PISP (ED(50) =5.47 mg/kg), PRSP (ED(50) =3.68-5.28 mg/kg) and E. coli (ED(50) =3.13-5.28 mg/kg), and moderate activity towards E. faecalis infection (ED(50) =8.48-15.16 mg/kg). The therapeutic efficacy of nemonoxacin was significantly higher (P<0.01) than that of levofloxacin in infections caused by Gram-positive isolates (MSSA, MRSA, levofloxacin-resistant MRSC, PISP, PRSP and E. faecalis), but less potent than that of levofloxacin against E. coli infection (P<0.01). Nemonoxacin in vivo efficacy results with Gram-positive isolates (2- to 5-fold ED(50) advantage over levofloxacin) are consistent with the MIC data (4- to 16-fold MIC advantage of nemonoxacin over levofloxacin). In the mouse pulmonary infection model, nemonoxacin showed potent activity towards PRSP (higher than levofloxacin) and K. pneumoniae (lower than levofloxacin) infections. In the mouse ascending urinary tract infection model, nemonoxacin exhibited potent activity against E. coli infection (lower than levofloxacin). CONCLUSIONS The results validated the potent efficacy of nemonoxacin in vivo. The higher efficacy of nemonoxacin than of levofloxacin towards infections caused by Gram-positive cocci (especially MRSA, levofloxacin-resistant MRSC, PRSP and VRE) warrants investigation of its clinical use.

Toxicokinetic study of melamine in the presence and absence of cyanuric acid in rats

Several lines of evidence show that the nephrotoxic effect of melamine (MEL) in animals is consistent with combined ingestion of MEL and cyanuric acid (CYA). The aim of the present study was to compare the toxicokinetics of MEL in the presence and absence of CYA, and to elucidate the correlation between toxicity and kinetic properties of MEL. Sprague–Dawley rats were administered a single oral dose of MEL (100 mg kg−1) with or without CYA (100 mg kg−1). Plasma and tissue samples were analyzed by liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay. Significant changes in toxicokinetic parameters of MEL such as lower maximum concentration (7.4 ± 3.5 vs 78.0 ± 11.0 µg ml−1) and area under curve (94.9 ± 53.5 vs 295.1 ± 93.7 µg h ml−1), higher plasma elimination half‐life (7.0 ± 3.3 vs 2.5 ± 0.3 h) and volume of distribution (11 505.5 ± 5030.3 vs 1312.7 ± 337.7 ml kg−1), as well as significantly higher concentration of MEL in rat kidney (2.96–274.15 vs < 1 µg g−1) were detected in the CYA co‐administration group when compared with MEL alone group (P < 0.05). The differences in kinetic parameters between the two groups meant that CYA co‐administration could lower absorption, slow excretion and induce tissue accumulation of MEL, which correlated well with the generation and development of renal toxicity. In conclusion, co‐administration with CYA leads to the alteration of the kinetic characteristics of MEL, which provides an additional explanation for renal toxicity. Copyright

In Vivo Antibacterial Activity of MRX-I, a New Oxazolidinone

ABSTRACT MRX-I is a potent oxazolidinone antibiotic against Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae (PRSP), penicillin-intermediate S. pneumoniae (PISP), and vancomycin-resistant enterococci (VRE). In this study, the in vivo efficacy of orally administered MRX-I was evaluated using linezolid as a comparator. MRX-I showed the same or better efficacy than linezolid in both systemic and local infection models against the tested strains.

Genetic basis of high level aminoglycoside resistance in Acinetobacter baumannii from Beijing, China

The objective of this study was to investigate the genetic basis of high level aminoglycoside resistance in Acinetobacter baumannii clinical isolates from Beijing, China. 173 A. baumannii clinical isolates from hospitals in Beijing from 2006 to 2009 were first subjected to high level aminoglycoside resistance (HLAR, MIC to gentamicin and amikacin>512 µg/mL) phenotype selection by broth microdilution method. The strains were then subjected to genetic basis analysis by PCR detection of the aminoglycoside modifying enzyme genes (aac(3)-I, aac(3)-IIc, aac(6′)-Ib, aac(6′)-II, aph(4)-Ia, aph(3′)-I, aph(3′)-IIb, aph(3′)-IIIa, aph(3′)-VIa, aph(2″)-Ib, aph(2″)-Ic, aph(2″)-Id, ant(2″)-Ia, ant(3″)-I and ant(4′)-Ia) and the 16S rRNA methylase genes (armA, rmtB and rmtC). Correlation analysis between the presence of aminoglycoside resistance gene and HLAR phenotype were performed by SPSS. Totally 102 (58.96%) HLAR isolates were selected. The HLAR rates for year 2006, 2007, 2008 and 2009 were 52.63%, 65.22%, 51.11% and 70.83%, respectively. Five modifying enzyme genes (aac(3)-I, detection rate of 65.69%; aac(6′)-Ib, detection rate of 45.10%; aph(3′)-I, detection rate of 47.06%; aph(3′)-IIb, detection rate of 0.98%; ant(3″)-I, detection rate of 95.10%) and one methylase gene (armA, detection rate of 98.04%) were detected in the 102 A. baumannii with aac(3)-I+aac(6′)-Ib+ant(3″)-I+armA (detection rate of 25.49%), aac(3)-I+aph(3′)-I+ant(3″)-I+armA (detection rate of 21.57%) and ant(3″)-I+armA (detection rate of 12.75%) being the most prevalent gene profiles. The values of chi-square tests showed correlation of armA, ant(3″)-I, aac(3)-I, aph(3′)-I and aac(6′)-Ib with HLAR. armA had significant correlation (contingency coefficient 0.685) and good contingency with HLAR (kappa 0.940). The high rates of HLAR may cause a serious problem for combination therapy of aminoglycoside with β-lactams against A. baumannii infections. As armA was reported to be able to cause high level aminoglycoside resistance to most of the clinical important aminoglycosides (gentamicin, amikacin, tobramycin, etc), the function of aminoglycoside modifying enzyme gene(s) in A. baumannii carrying armA deserves further investigation.

In Vivo Antibacterial Activity of Vertilmicin, a New Aminoglycoside Antibiotic

ABSTRACT Vertilmicin is a novel aminoglycoside antibiotic with potent activity against gram-negative and -positive bacteria in vitro. In this study, we further evaluated the efficacy of vertilmicin in vivo in systemic and local infection animal models. We demonstrated that vertilmicin had relatively high and broad-spectrum activities against mouse systemic infections caused by Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis. The 50% effective doses of subcutaneously administered vertilmicin were 0.63 to 0.82 mg/kg, 0.18 to 0.29 mg/kg, 0.25 to 0.99 mg/kg, and 4.35 to 7.11 mg/kg against E. coli, K. pneumoniae, S. aureus, and E. faecalis infections, respectively. The therapeutic efficacy of vertilmicin was generally similar to that of netimicin, better than that of gentamicin in all the isolates tested, and better than that of verdamicin against E. coli 9612 and E. faecalis HH22 infections. The therapeutic efficacy of vertilmicin was further confirmed in local infection models of rabbit skin burn infection and mouse ascending urinary tract infection.

In Vitro Antibacterial Activity of Vertilmicin and Its Susceptibility to Modifications by the Recombinant AAC(6′)-APH(2″) Enzyme

ABSTRACT Vertilmicin is a new semisynthetic aminoglycoside with a structure similar to that of netilmicin except for a methyl group at the C-6′ position. In the present study, the in vitro antibacterial activity of vertilmicin was studied, and its susceptibility to modifications by the recombinant aminoglycoside bifunctional modifying enzyme AAC(6′)-APH(2″) was compared with those of verdamicin and netilmicin. A total of 1,185 clinical isolates collected from hospitals in Beijing between 2000 and 2001 were subjected to the in vitro antibacterial activity evaluations, including MIC, minimum bactericidal concentration (MBC), and time-kill curve tests. The MICs were evaluated in non-gentamicin-resistant (gentamicin-susceptible and gentamicin-intermediate) strains and gentamicin-resistant strains, respectively. For most of the non-gentamicin-resistant bacteria (except for the isolates of Pseudomonas spp.), the MIC90s of vertilmicin were in the range of 0.5 to 8 μg/ml, comparable to those of the reference aminoglycosides. For the gentamicin-resistant isolates, the three semisynthetic aminoglycosides (vertilmicin, netilmicin, and amikacin) demonstrated low MIC50s and/or MIC90s, as well as high percent susceptibility values. Among the study drugs, vertilmicin showed the lowest MIC90s, 16 μg/ml, for the gram-positive gentamicin-resistant isolates of Staphylococcus aureus and Staphylococcus epidermidis. Meanwhile, vertilmicin was a potent bactericidal agent, with MBC/MIC ratios in the range of 1 to 2 for Escherichia coli, Klebsiella pneumoniae, and S. aureus and 1 to 4 for S. epidermidis. The time-kill curve determination further demonstrated that this effect was rapid and concentration dependent. In evaluations of susceptibility to modifications by the recombinant AAC(6′)-APH(2″) with maximum rate of metabolism/Km measurements, vertilmicin exhibited susceptibilities to both acetylation and phosphorylation lower than those of netilmicin and verdamicin.

Recombinant expression and biochemical characterization of Mycobacterium tuberculosis 3Fe-4S ferredoxin Rv1786

Ferredoxins are iron-sulfur protein that mediate electron transfer in cytochrome P450 mono-oxygenase (CYP)-related catalytic reactions in a wide variety of organisms. Rv1786 is a putative ferredoxin, encoded by a gene located downstream of the gene encoding CYP143A1 in the Mycobacterium tuberculosis genome. However, the structure and function of Rv1786 have remained unclear. Here, the recombinant Mtb Rv1786 was expressed, purified as a His-tagged form and characterized with [3Fe-4S] clusters as its cofactors using a series of measurements including SDS-PAGE, western blot, UV/Visible, MALDI-TOF/TOF-MS, and electron paramagnetic resonance spectroscopic analysis. Based on the assessments of surface plasmon resonance (SPR) and steady state kinetic assays, Rv1786 was found to be able to couple with both ferredoxin reductase A (FdrA) and flavoprotein reductase A (FprA) as redox partner, but with a stronger binding to FprA and a better coupling activity to FdrA. Preliminary structural and biochemical characterization of Mtb Rv1786 as a redox partner is presented here.

Validated LC–MS/MS method for determination of YH-8, a novel PKnB inhibitor, in rat plasma and its application to pharmacokinetic study

(E)-Methyl-4-aryl-4-oxabut-2-enoate (YH-8) is a novel PKnB protein kinase inhibitor with good anti-tuberculosis activity. To evaluate its pharmacokinetics in rats, a sensitive and selective high performance liquid chromatography–tandem mass spectrometric (LC--MS/MS) method has been developed and validated for the quantification of YH-8 in rat plasma for the first time. Samples were pre-treated using a liquid--liquid extraction with ethyl acetate and the chromatographic separation was performed on a C18 column by gradient elution with methanol--water as the mobile phase. YH-8 was detected using a tandem mass spectrometer in positive selected reaction monitoring (SRM) mode. Method validation revealed good linearity over the range of 1–500 ng/mL for YH-8 with a lower limit of quantification (LLOQ) of 1 ng/mL. Intra- and inter-day precision of YH-8 assay in rat plasma samples were 2.0%–6.8%, with accuracy of the method being 100.69%–106.18%. Stability test showed that when spiked into rat plasma, YH-8 was stable for 12 h at room temperature, for up to 15 days at −70 °C, and after three freeze-thaw cycles. Extracted samples were found to be stable over 12 h in an auto-sampler. The method was successfully applied to the pharmacokinetic study of YH-8 in rats after oral administration at 100 mg/kg and 200 mg/kg.

In vivo antibacterial activity of chinfloxacin, a new fluoroquinolone antibiotic

OBJECTIVES To evaluate the in vivo antibacterial efficacy of chinfloxacin, a novel fluoroquinolone, in murine systemic and local infection models. METHODS The efficacy of chinfloxacin in systemic infection was evaluated in a mouse peritonitis model using isolates of methicillin-susceptible Staphylococcus aureus (MSSA, n = 3), methicillin-resistant Staphylococcus aureus (MRSA; n = 1), penicillin-intermediate Streptococcus pneumoniae (PISP; n = 1), penicillin-resistant S. pneumoniae (PRSP; n = 2), vancomycin-susceptible Enterococcus faecalis (VSE; n = 1), vancomycin-resistant E. faecalis (VRE; n = 2), Escherichia coli (n = 3) and Klebsiella pneumoniae (n = 2). The local infections included mouse pulmonary infections caused by penicillin-susceptible S. pneumoniae (PSSP; n = 1), PRSP (n = 1) and K. pneumoniae (n = 2). RESULTS In the mouse systemic infection model, chinfloxacin demonstrated potent activity against MSSA [50% effective dose (ED(50)) 2.28-4.15 mg/kg], MRSA (ED(50) 14.75 mg/kg), PISP (ED(50) 6.20 mg/kg), PRSP (ED(50) 3.51-5.03 mg/kg), VSE (ED(50) 25.02 mg/kg), VRE (ED(50) 5.18-15.39 mg/kg), E. coli (ED(50) 1.25-1.90 mg/kg) and K. pneumoniae (ED(50) 2.92-8.28 mg/kg). The therapeutic efficacy of chinfloxacin was generally similar to (P > 0.05) that of moxifloxacin, significantly higher (P < 0.01 or P < 0.05) than that of levofloxacin in Gram-positive isolate infections (MSSA, MRSA, PISP, PRSP, VSE and VRE), and less than that of levofloxacin against E. coli and K. pneumoniae infections (P < 0.01). In the mouse pulmonary infection model, chinfloxacin showed potent activity towards S. pneumoniae (higher than levofloxacin and ciprofloxacin) and K. pneumoniae (lower than levofloxacin and similar to or higher than ciprofloxacin) infections. CONCLUSIONS The results validated the potent efficacy of chinfloxacin in vivo. The high efficacy of chinfloxacin in murine systemic and local infections warrants investigation of its clinical use.

γ-Glutamyl Spermine Synthetase PauA2 as a Potential Target of Antibiotic Development against Pseudomonas aeruginosa

ABSTRACT Polyamines are absolute requirements for cell growth. When in excess, Pseudomonas aeruginosa possesses six γ-glutamylpolyamine synthetases (GPSs) encoded by the pauA1-pauA7 genes to initiate polyamine catabolism. Recently, the pauA2 mutant was reported to lose the capability to grow on spermine (Spm) and spermidine (Spd) as sole carbon and nitrogen sources. Although this mutant grew normally in defined minimal medium and LB broth, growth was completely abolished by the addition of Spm or Spd. These two compounds exert a bactericidal effect (Spm > Spd) on the mutants as demonstrated by MIC measurements (over 500-fold reduction) and time-killing curves. Spm toxicity in the pauA2 mutant was attenuated when the major uptake system was further deleted from the strain, suggesting cytoplasmic targets of toxicity. In addition, the synergistic effect of Spm and carbenicillin in the wild-type strain PAO1 was diminished in mutants without functional PauA2. Furthermore, Spm MIC was reduced by 8-fold when the Spm uptake system was deleted from the wild-type strain, suggesting a second target of Spm toxicity in the periplasm. Experiments were also conducted to test the hypothesis that native Spm and Spd in human serum may be sufficient to kill the pauA2 mutant. Growth of the mutant was completely inhibited by 40% (vol/vol) human serum, whereas the parental strain required 80%. Colony counts indicated that the mutant but not the parent was in fact killed by human plasma. In addition, carbenicillin MIC against the mutant was reduced by 16-fold in the presence of 20% human serum while that of the parental strain remained unchanged. Taking PauA2 as the template, sequence comparison indicates that putative PauA2 homologues are widespread in a variety of Gram-negative bacteria. In summary, this study reveals the importance of GPS in alleviation of polyamine toxicity when in excess, and it provides strong support to the feasibility of GPS as a molecular target for new antibiotic development.