Antimicrobial activity of Mimosa caesalpiniifolia Benth and its interaction with antibiotics against Staphylococcus aureus strains overexpressing efflux pump genes.

This study aimed to evaluate the antimicrobial activity of the dichloromethane fraction (DCMF) from the stem bark of Mimosa caesalpiniifolia and its effect on the activity of conventional antibiotics against Staphylococcus aureus strains overexpressing specific efflux pump genes. DCMF showed activity against S. aureus, Staphylococcus epidermidis and Candida albicans. Addition of DCMF at subinhibitory concentrations to the growth media enhanced the activity of norfloxacin, ciprofloxacin and ethidium bromide against S. aureus strains overexpressing norA suggesting the presence of efflux pump inhibitors in its composition. Similar results were verified for tetracycline against S. aureus overexpressing tetK, as well as, for ethidium bromide against S. aureus overexpressing qacC. These results indicate that M. caesalpiniifolia is a source of molecules able to modulate the fluoroquinolone- and tetracycline-resistance in S. aureus probably by inhibition of NorA, TetK and QacC respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Drug resistance is a common problem in patients with infectious diseases. Dichloromethane fraction from the stem bark of Mimosa caesalpiniifolia showed antimicrobial activity against Gram-positive bacterium Staphylococcus aureus and against Candida albicans, but did not show activity against Gram-negative specie Escherichia coli. Moreover, this fraction was able to potentiate the action of norfloxacin, ciprofloxacin and tetracycline against S. aureus strains overexpressing different efflux pump genes. Thus, Mimosa caesalpiniifolia is a source of efflux pump inhibitors which could be used in combination with fluoroquinolones or tetracycline in the treatment of infectious diseases caused by S. aureus strains overexpressing efflux pump genes.

Inhibition of the NorA efflux pump of Staphylococcus aureus by synthetic riparins.

AIM: The goal of this study was to increase knowledge about the antimicrobial activity of some synthetic Riparin-derived compounds, alone or in combination with fluoroquinolone antibiotics, against a strain of Staphylococcus aureus resistant to fluoroquinolone by way of overexpression of the NorA efflux pump. METHODS AND RESULTS: Microdilution tests showed that Riparins A and B did not show any significant antibacterial activity against Staph. aureus strains. On the other hand, the intrinsic antibacterial activity increased with increasing lipophilicity of the compounds, in the following order: Riparin-D (MIC 256 µg ml-1 ; Log P 2·95) < Riparin-C (MIC 102 µg ml-1 ; Log P 3·22) < Riparin-E (MIC 16 µg ml-1 ; Log P 3·57). The addition of all riparins to growth media at subinhibitory concentrations caused an increase in the antibacterial activity of antibiotics against the NorA-overexpressing test strain. Riparin-B, which has two methoxyl groups at the phenethyl moiety, showed the best modulatory effect. CONCLUSIONS: Riparin-E is a good anti-staphylococci agent, while Riparin-B functions as a NorA efflux pump inhibitor. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest the possibility of using Riparin-B in combination with norfloxacin or ciprofloxacin for therapy of infections caused by multi-drug resistant Staph. aureus.

The multidrug efflux pump NorA is not required for salicylate-induced reduction in drug accumulation by Staphylococcus aureus.

Growth of Staphylococcus aureus in the presence of salicylate leads to reduced ciprofloxacin and ethidium accumulation and increased resistance to ethidium. Salicylate induced reduction in ciprofloxacin accumulation is energy-independent while salicylate induced alterations in ethidium accumulation and efflux is proton motive force-dependent. NorA is an intrinsic multidrug efflux pump that contributes to intrinsic levels of fluoroquinolone and ethidium resistance in S. aureus. The NorA inhibitor reserpine did not dramatically affect the ability of salicylate to induce increased ciprofloxacin and ethidium resistance. Inactivation of norA did not alter the ability of salicylate to induce increased ciprofloxacin and ethidium resistance levels and a reduction in ciprofloxacin accumulation. These data demonstrate that NorA is not absolutely required for the salicylate-inducible multidrug resistance mechanism of S. aureus.

Activities of newer fluoroquinolones against ciprofloxacin-resistant Streptococcus pneumoniae.

The incidence of ciprofloxacin resistance in Streptococcus pneumoniae is low but steadily increasing, which raises concerns regarding the clinical impact of potential cross-resistance with newer fluoroquinolones. To investigate this problem, we utilized an in vitro pharmacodynamic model and compared the activities of gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, and trovafloxacin to that of ciprofloxacin against two laboratory-derived, ciprofloxacin-resistant derivatives of S. pneumoniae (strains R919 and R921). Ciprofloxacin resistance in these strains involved the activity of a multidrug efflux pump and possibly, for R919, a mutation resulting in an amino acid substitution in GyrA. Gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, and trovafloxacin achieved 99.9% killing of both R919 and R921 in < or =28 h. With respect to levofloxacin, significant regrowth of both mutants was observed at 48 h (P < 0.05). For gatifloxacin, grepafloxacin, moxifloxacin, and trovafloxacin, regrowth was minimal at 48 h, with each maintaining 99.9% killing against both mutants. No killing of either R919 or R921 was observed with exposure to ciprofloxacin. During model experiments, resistance to gatifloxacin, grepafloxacin, moxifloxacin, and trovafloxacin did not develop but the MICs of ciprofloxacin and levofloxacin increased 1 to 2 dilutions for both R919 and R921. Although specific area under the concentration-time curve from 0 to 24 h (AUC(0--24))/MIC and maximum concentration of drug in serum (C(max))/MIC ratios have not been defined for the fluoroquinolones with respect to gram-positive organisms, our study revealed that significant regrowth and/or resistance was associated with AUC(0-24)/MIC ratios of < or =31.7 and C(max)/MIC ratios of < or =3.1. It is evident that the newer fluoroquinolones tested possess improved activity against S. pneumoniae, including strains for which ciprofloxacin MICs were elevated.

Oxazolidinones: new players in the battle against multi-resistant Gram-positive bacteria.

For many years the pharmaceutical industry did not pursue the development of antimicrobial agents that specifically targeted Gram-positive bacteria. Semi-synthetic penicillins and vancomycin were the mainstays of therapy for methicillin-susceptible and -resistant strains of staphylococci, respectively, as was penicillin for Streptococcus pneumoniae and beta-lactam-aminoglycoside combinations for serious enterococcal infections. In the 1980s enterococci resistant to glycopeptides emerged, followed shortly thereafter by a dissemination of penicillin-insensitive S. pneumoniae and, more recently, the occurrence of vancomycin-intermediately susceptible Staphylococcus aureus. The emergence of fully glycopeptide-resistant S. aureus is clearly on the horizon. Multi-resistant Gram-positive bacteria now pose an important therapeutic challenge for clinicians. New drugs with activity against some of these dangerous pathogens have recently been pursued, and linezolid, the first member of the oxazolidinone class, has now been licensed for clinical use in many countries. This drug has excellent in vitro and in vivo activity against all clinically relevant multi-resistant Gram-positive cocci and fills an important void in infectious disease chemotherapy.

Comparison of a rabbit model of bacterial endocarditis and an in vitro infection model with simulated endocardial vegetations.

Animal models are commonly used to determine the efficacy of various antimicrobial agents for treatment of bacterial endocarditis. Previously we have utilized an in vitro infection model, which incorporates simulated endocardial vegetations (SEVs) to evaluate the pharmacodynamics of various antibiotics. In the present study, we compared four experimental rabbit endocarditis protocols to an in vitro infection model in an effort to determine if these models are comparable. We have evaluated the activity of clinafloxacin, trovafloxacin, sparfloxacin, and ciprofloxacin in rabbit models against Staphylococcus aureus and Enterococcus spp. In vitro models were performed simulating the antibiotic pharmacokinetics obtained in the in vivo studies. Models were dosed the same as rabbit models, and SEVs were evaluated at the same time the rabbit vegetations were examined. Clinafloxacin and trovafloxacin were evaluated against methicillin-susceptible (MSSA1199) and -resistant (MRSA494) strains of S. aureus. Ciprofloxacin was studied against MSSA1199 and MSSA487. Sparfloxacin and clinafloxacin were evaluated against Enterococcus faecium SF2149 and Enterococcus faecalis WH245, respectively. We found that reductions in SEV bacterial density obtained in the in vitro model were similar to those obtained in rabbit vegetations, indicating that the SEV model may be a valuable tool for assessing antibiotic potential in the treatment of bacterial endocarditis.

Evidence for the existence of a multidrug efflux transporter distinct from NorA in Staphylococcus aureus.

A Staphylococcus aureus norA disruption mutant was created by allelic replacement. Exposure of this mutant to norfloxacin produced SA K1748, a derivative with raised fluoroquinolone MICs, found to be the result of a grlA mutation, and raised organic cation MICs. Ethidium and enoxacin uptake was identical in SA K1748 and its parent, but pre-exposure of SA K1748 to organic cations caused a reduction in ethidium uptake as a result of increased efflux. Altered ethidium uptake and efflux, as well as increased MICs of other organic cations, suggest that SA K1748 possesses a non-NorA multidrug efflux transporter that is inducible by its substrates.

The effects of NorA inhibition on the activities of levofloxacin, ciprofloxacin and norfloxacin against two genetically related strains of Staphylococcus aureus in an in-vitro infection model.

NorA is a membrane-associated multidrug efflux protein that can decrease susceptibility to fluoroquinolones in Staphylococcus aureus. We have previously determined that NorA inhibition can increase fluoroquinolone killing activity and post-antibiotic effect. In the current investigation, we studied the killing activity and development of resistance for levofloxacin, ciprofloxacin and norfloxacin with or without the H+/K+ ATPase inhibitor omeprazole, in a wild-type strain of S. aureus (SA-1199) and its NorA hyperproducing mutant (SA-1199-3) in an in-vitro pharmacodynamic model with infected fibrin-platelet matrices. Each drug was administered every 12-24 h for 72 h and human pharmacokinetics were simulated. Levofloxacin was the most potent fluoroquinolone against both strains and its activity was not significantly affected by combination with omeprazole. The addition of omeprazole to ciprofloxacin significantly lowered colony counts at all time-points against both strains and decreased the time to 99.9% kill from 72.2 h to 33.8 h against SA-1199. The addition of omeprazole minimally increased norfloxacin activity against both strains. Omeprazole decreased the frequency of ciprofloxacin resistance nearly 100-fold at the 24 h time-point, but the frequency of resistance was not significantly different for any of the fluoroquinolone regimens after this time-point. No resistance was detected during levofloxacin regimens. The hydrophobic fluoroquinolones such as levofloxacin appear to circumvent NorA efflux, which may contribute to their better activity and decreased resistance rates against staphylococci. More durable and potent NorA inhibitor compounds are needed that can improve killing activity and prevent resistance.

Introduction of a norA promoter region mutation into the chromosome of a fluoroquinolone-susceptible strain of Staphylococcus aureus using plasmid integration.

It has been postulated that a mutation 11 bp 3' to the -10 motif of the norA promoter is involved in the increased expression of the gene observed in some strains of Staphylococcus aureus exhibiting efflux-related fluoroquinolone resistance. Introduction of this mutation into the chromosome of a fluoroquinolone-susceptible strain by plasmid integration resulted in the minimum inhibitory concentrations of NorA substrates being increased, fluoroquinolone uptake being reduced, and norA expression being enhanced. Diffuse hybridization of norA and integrating vector probes at a similar molecular weight range, higher than that of the norA transcript, was observed in the integrant, suggesting the possibility of a plasmid-based promoter contributing to norA expression. The ratio of the quantity of this transcript, which was also observed in the parent strain of the integrant, to the quantity of primary norA transcript was 0.14, demonstrating that it was unlikely that this mRNA species contributed significantly to the results observed. It is more likely that the introduced promoter region mutation does affect the expression of norA.

Effects of NorA inhibitors on in vitro antibacterial activities and postantibiotic effects of levofloxacin, ciprofloxacin, and norfloxacin in genetically related strains of Staphylococcus aureus.

NorA is a membrane-associated multidrug efflux protein that can decrease susceptibility to fluoroquinolones in Staphylococcus aureus. To determine the effect of NorA inhibition on the pharmacodynamics of fluoroquinolones, we evaluated the activities of levofloxacin, ciprofloxacin, and norfloxacin with and without various NorA inhibitors against three genetically related strains of S. aureus (SA 1199, the wild-type; SA 1199B, a NorA hyperproducer with a grlA mutation; and SA 1199-3, a strain that inducibly hyperproduces NorA) using susceptibility testing, time-kill curves, and postantibiotic effect (PAE) methods. Levofloxacin had the most potent activity against all three strains and was minimally affected by addition of NorA inhibitors. In contrast, reserpine, omeprazole, and lansoprazole produced 4-fold decreases in ciprofloxacin and norfloxacin MICs and MBCs for SA 1199 and 4- to 16-fold decreases for both SA 1199B and SA 1199-3. In time-kill experiments reserpine, omeprazole, or lansoprazole increased levofloxacin activity against SA 1199-3 alone by 2 log10 CFU/ml and increased norfloxacin and ciprofloxacin activities against all three strains by 0.5 to 4 log10 CFU/ml. Reserpine and omeprazole increased norfloxacin PAEs on SA 1199, SA 1199B, and SA 1199-3 from 0.9, 0.6, and 0.2 h to 2.5 to 4.5, 1.1 to 1.3, and 0.4 to 1.1 h, respectively; similar effects were observed with ciprofloxacin. Reserpine and omeprazole increased the levofloxacin PAE only on SA 1199B (from 1.6 to 5.0 and 3.1 h, respectively). In conclusion, the NorA inhibitors dramatically improved the activities of the more hydrophilic fluoroquinolones (norfloxacin and ciprofloxacin). These compounds may restore the activities of these fluoroquinolones against resistant strains of S. aureus or may potentially enhance their activities against sensitive strains.

Efficacy of LY333328 against experimental methicillin-resistant Staphylococcus aureus endocarditis.

The in vivo efficacy of LY333328, a new glycopeptide antibiotic, was compared with that of vancomycin by using the rabbit model of left-sided methicillin-resistant Staphylococcus aureus endocarditis. Animals received LY333328 or vancomycin (25 mg/kg of body weight every 24 or 8 h, respectively) for 4 days. These drugs were equally effective in clearing bacteremia and in reducing bacterial counts in vegetations and tissues. We conclude that in this model, LY333328 was microbiologically effective and may be a therapeutic alternative to vancomycin.

Efficacy of trovafloxacin against experimental Staphylococcus aureus endocarditis.

Trovafloxacin is a new fluoronaphthyridone chemically and functionally related to members of the fluoroquinolone class of antimicrobial agents. The in vivo efficacy of the drug was compared with that of vancomycin by using the rabbit model of left-sided endocarditis. Rabbits infected with either a nafcillin-susceptible or -resistant test strain were treated with trovafloxacin (13.3 mg/kg of body weight every 12 h) or vancomycin (25 mg/kg of body weight every 8 h) for 4 days. In comparison with untreated controls, both antimicrobial agents effectively cleared bacteremia and significantly reduced bacterial counts in vegetations and tissues of animals infected with either test strain. No resistance to trovafloxacin emerged in test strains during therapy. We conclude that in this model trovafloxacin is as efficacious as vancomycin is and may serve as a viable alternative to vancomycin for use in humans with similar infections.

Comparative in vitro activities and postantibiotic effects of the oxazolidinone compounds eperezolid (PNU-100592) and linezolid (PNU-100766) versus vancomycin against Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus faecalis, and Enterococcus faecium.

The activities of the oxazolidinone antibacterial agents eperezolid (PNU-100592) and linezolid (PNU-100766) were compared with that of vancomycin against clinical isolates of methicillin-susceptible and -resistant Staphylococcus aureus (n = 200), coagulase-negative staphylococci (n = 100), and vancomycin-susceptible and -resistant Enterococcus faecalis and Enterococcus faecium (n = 50). Eperezolid and linezolid demonstrated good in vitro inhibitory activity, regardless of methicillin susceptibility for staphylococci (MIC at which 90% of the isolates are inhibited [MIC90] range, 1 to 4 microg/ml) or vancomycin susceptibility for enterococci (MIC90 range, 1 to 4 microg/ml). In time-kill studies, eperezolid and linezolid were bacteriostatic in action. A postantibiotic effect of 0.8+/-0.5 h was demonstrated for both eperezolid and linezolid against S. aureus, S. epidermidis, E. faecalis, and E. faecium.

Topoisomerase mutations in fluoroquinolone-resistant and methicillin-susceptible and -resistant clinical isolates of Staphylococcus aureus.

The incidence of the various mutations in the genes encoding topoisomerase IV and DNA gyrase in fluoroquinolone-resistant clinical isolates of Staphylococcus aureus is not known. Using restriction fragment length polymorphism analysis and DNA sequencing, we found that in fluoroquinolone- and methicillin-resistant strains, mutations in grlA and gyrA are quite likely to be present together. For fluoroquinolone-resistant but methicillin-susceptible strains, mutations in grlA alone are more common.

Pharmacodynamics of RP 59500 (quinupristin-dalfopristin) administered by intermittent versus continuous infusion against Staphylococcus aureus-infected fibrin-platelet clots in an in vitro infection model.

We evaluated the bactericidal activity of RP 59500 (quinupristin-dalfopristin) against fibrin-platelet clots (FPC) infected with two clinical isolates of Staphylococcus aureus, one constitutively erythromycin and methicillin resistant (S. aureus AW7) and one erythromycin and methicillin susceptible (S. aureus 1199), in an in vitro pharmacodynamic infection model. RP 59500 was administered by continuous infusion (peak steady-state concentration of 6 microg/ml) or intermittent infusion (simulated regimens of 7.5 mg/kg of body weight every 6 h (q6h) q8h, and q12h. FPCs were infected with S. aureus to achieve an initial bacterial density of 10(9) CFU/g. Model experiments were run in duplicate over 72 h. Two FPCs were removed from each model at 0, 12, 24, 36, 48, and 72 h, and the bacterial densities (in CFU per gram) were determined and compared to those of growth control experiments. Additional samples were also removed from the model over the 72-h period for pharmacokinetic evaluation. All regimens significantly (P < or = 0.01) decreased bacterial densities in the infected FPCs for both isolates compared to growth controls. This occurred even though MBCs were equal to or greater than the RP 59500 concentrations achieved in the models. There were no significant differences found between the dosing frequencies and levels of killing when examining each isolate separately. However, examination of the residual bacterial densities (CFU per gram at 72 h) and visual inspection of the overall killing effect (killing curve plots over 72 h) clearly demonstrated a more favorable bactericidal activity against 1199 than against the AW7 isolate. This was most apparent when the q8h and the q12h AW7 regimens were compared to all 1199 treatment regimens by measuring the 72-h bacterial densities (P < or = 0.01). Killing (99.9%) was not achieved against the AW7 isolate. However, a 99.9% kill was demonstrated for all dosing regimens against the 1199 isolate. The area under the concentration-time curve from 0 to 24 h was found to be significantly correlated with reduction in bacterial density for the AW7 isolate (r = 0.74, P = 0.04). No resistance was detected during any experiment for either isolate. RP 59500 efficacy against constitutively erythromycin- and methicillin-resistant S. aureus may be improved by increasing organism exposure to RP 59500 as a function of dosing frequency.

Mechanisms of fluoroquinolone resistance in genetically related strains of Staphylococcus aureus.

Fluoroquinolone resistance in Staphylococcus aureus results from amino acid substitutions at particular locations in the DNA gyrase A and B subunits as well as in the topoisomerase IV A subunit and from NorA-mediated efflux. More than one resistance mechanism may be present in a single strain. Fluoroquinolone-resistant derivatives of SA-1199, a methicillin-susceptible S. aureus strain, were selected in vivo or in vitro, and their mechanisms of fluoroquinolone resistance were identified. We found that many of the resistance mechanisms described above can develop in derivatives of a single parent strain, either singly or in combination, and can arise in a single step. Variances in MICs for strains with the same apparent resistance mechanisms likely are due to the presence of new or undetected but established means of fluoroquinolone resistance. NorA-mediated resistance can occur in the apparent absence of topoisomerase mutations and in some strains may be the result of a promoter region mutation causing increased expression of norA. However, increased expression of norA can occur independently of this mutation, suggesting that a regulatory locus for this gene exists elsewhere on the chromosome.

In vitro activities of oxazolidinone compounds U100592 and U100766 against Staphylococcus aureus and Staphylococcus epidermidis.

The new oxazolidinone antimicrobial agents U100592 and U100766 demonstrated good in vitro inhibitory activity against clinical strains of Staphylococcus aureus and Staphylococcus epidermidis regardless of methicillin susceptibility. Both agents appeared bacteriostatic by time-kill analysis. Stable resistance to low multiples of the MIC of either drug could be produced only in methicillin-resistant S. aureus.

Inducible NorA-mediated multidrug resistance in Staphylococcus aureus.

The NorA protein of Staphylococcus aureus mediates the active efflux of hydrophilic fluoroquinolones from the cell, conferring low-level resistance upon the organism. The protein also is capable of transporting additional structurally diverse compounds, indicating that it has a broad substrate specificity. Increased transcription of the norA gene, leading to a greater quantity of the NorA protein within the cytoplasmic membrane, is felt to be the mechanism by which strains possessing such changes resist fluoroquinolones. S. aureus SA-1199 and its in vivo-selected derivative SA-1199B are fluoroquinolone-susceptible and -resistant isolates, respectively; SA-1199B resists hydrophilic fluoroquinolones via a NorA-mediated mediated mechanism in a constitutive manner. SA-1199-3 is an in vitro-produced derivative of SA-1199 in which NorA-mediated multidrug resistance is expressed inducibly. Compared with organisms exposed to subinhibitory concentrations of a NorA substrate for the first time, preexposure of SA-1199-3 to such a compound followed by growth in the presence of that substrate results in the elimination of a 2- to 6-h period of organism killing that occurs prior to the onset of logarithmic growth. The uptake of radiolabeled fluoroquinolone is markedly reduced by preexposure of SA-1199-3 to NorA substrates: such prior exposure also results in a dramatic increase in RNa transcripts that hybridize with a norA probe. Preexposure of SA-1199 and SA-1199B to such substrates results in small increases or no increases in these transcripts. No sequence differences between SA-1199 and SA-1199-3 within the norA gene or flanking DNA were found. It appears likely that the regulation of norA in SA-1193, which may be effected by one or more genetic loci outside the norA region of the chromosome, differs from that of SA-1199 and SA-1199B.

Effect of aerobic and anaerobic environments on antistaphylococcal activities of five fluoroquinolones.

A previously established in vitro pharmacodynamic system was used to evaluate the antistaphylococcal activities of five fluoroquinolones under both aerobic and anaerobic conditions. Staphylococcus aureus ATCC 29213 was exposed to a 5-micrograms/ml concentration of each of the following fluoroquinolones: ciprofloxacin, ofloxacin, temafloxacin, sparfloxacin, and clinafloxacin. Terminal elimination half-lives of 4, 6, 8, 8, and 13 h were simulated for the respective drugs. Each fluoroquinolone was bactericidal under both aerobic and anaerobic conditions. However, the bactericidal activity of each fluoroquinolone was delayed by anaerobiosis. This difference in fluoroquinolone activity under aerobic and anaerobic conditions could not be attributed to any particular parameter or physiochemical property but was most likely caused by a combination of factors (e.g., variations in hydrophobicity, intracellular pH, antibiotic concentration, and structure-activity relationships). Fluoroquinolone uptake studies were also performed to investigate the possibility of active, energy-dependent transport mechanisms in S. aureus ATCC 29213. Uptake studies indicated that active efflux does occur in S. aureus ATCC 29213.