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.

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.

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.

Mechanism of ciprofloxacin resistance in Pseudomonas aeruginosa.

We studied the mechanism(s) of resistance to ciprofloxacin arising in a clinical strain of Pseudomonas aeruginosa and in a laboratory-derived isolate of that strain. Higher concentrations of ciprofloxacin were required to interfere with DNA synthesis in resistant isolates compared with the parent strain, a finding indicating a relative insensitivity of DNA gyrase to ciprofloxacin. Reduced uptake of ciprofloxacin was seen in one isolate and may have contributed to its ciprofloxacin resistance but was not associated with alterations in outer or cytoplasmic membrane proteins, a result suggesting that such changes are not required to decrease uptake of fluoroquinolones into cells. No evidence for plasmid-mediated resistance was found, and no ciprofloxacin-inactivating activity was detected in sonic extracts of resistant organisms. In these isolates, resistance to ciprofloxacin was likely the result of more than one mutation, because single-step mutations conferring such high-level resistance were not found in the parent strain.

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.

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.

WIN 57273, a new fluoroquinolone with enhanced in vitro activity versus gram-positive pathogens.

WIN 57273 is a new fluoroquinolone with excellent in vitro activity versus gram-positive pathogens, including methicillin-susceptible and -resistant Staphylococcus aureus and Staphylococcus epidermidis and gentamicin-susceptible and -resistant Enterococcus faecalis. We compared the microdilution MICs and MBCs of this compound to those of other antimicrobial agents for more than 30 clinical isolates of each of these groups of organisms and found that with few exceptions, it was at least 10 times more active than all other drugs tested. Selection for resistance to ciprofloxacin (greater than or equal to 5 micrograms/ml) or WIN 57273 (greater than or equal to 0.16 microgram/ml) by the gradient plate method produced mutants with diminished susceptibility to the other fluoroquinolone; however, the MICs and MBCs of WIN 57273 for such strains were still quite low and remained below the preliminary susceptibility breakpoint (less than or equal to 2 micrograms/ml). Spontaneous mutations conferring resistance to two and five times the WIN 57273 MIC were detectable at low frequencies for S. aureus and S. epidermidis; such mutations were virtually undetectable for E. faecalis. Further testing is necessary to establish if the effectiveness of WIN 57273 is maintained in vivo, first in animals and then in humans with infections caused by methicillin-susceptible and -resistant strains of S. aureus and S. epidermidis or gentamicin-susceptible and -resistant strains of E. faecalis.

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.

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.

Efflux-mediated fluoroquinolone resistance in Staphylococcus aureus.

Transport processes are used by all organisms to obtain essential nutrients and to expel wastes and other potentially harmful substances from cells. Such processes are important means by which resistance to selected antimicrobial agents in bacteria is achieved. The recently described Staphylococcus aureus norA gene encodes a membrane-associated protein that mediates active efflux of fluoroquinolones from cells. SA-1199B is a fluoroquinolone-resistant strain of S. aureus from which we cloned an allele of norA (norA1199). Similar to that of norA, the protein product of norA1199 preferentially mediates efflux of hydrophilic fluoroquinolones in both S. aureus and an Escherichia coli host, a process driven by the proton motive force. Determination of the nucleotide sequence of norA1199 revealed an encoded 388-amino-acid hydrophobic polypeptide 95% homologous with the norA-encoded protein. Significant homology with other proteins involved in transport processes also exists, but especially with tetracycline efflux proteins and with the Bacillus subtilis Bmr protein that mediates active efflux of structurally unrelated compounds, including fluoroquinolones. In S. aureus, the norA1199-encoded protein also appears to function as a multidrug efflux transporter. Southern hybridization studies indicated that norA1199 (or an allele of it) is a naturally occurring S. aureus gene and that related sequences are present in the S. epidermidis genome. The nucleotide sequence of the wild-type allele of norA1199, cloned from the fluoroquinolone-susceptible parent strain of SA-1199B, did not differ from that of norA1199 throughout the coding region. Northern (RNA) and Southern hybridization studies showed that increased transcription, and not gene amplification, of norA1199 is the basis for fluoroquinolone resistance in SA-1199B.

Enhanced elimination of ciprofloxacin after multiple-dose administration of rifampin to rabbits.

The combination of ciprofloxacin and rifampin is potentially useful for the treatment of selected infections. However, rifampin may induce the metabolism of ciprofloxacin. Ciprofloxacin was given in single doses to healthy rabbits before and after six daily doses of intramuscular rifampin. Total clearance of ciprofloxacin increased from 0.96 +/- 0.32 (standard deviation) to 1.57 +/- 0.63 liters/h per kg (P less than 0.05). This change in elimination is potentially significant for the outcome of experimental infections in rabbits.

Fluoroquinolone resistance protein NorA of Staphylococcus aureus is a multidrug efflux transporter.

The gene of the Staphylococcus aureus fluoroquinolone efflux transporter protein NorA confers resistance to a number of structurally dissimilar drugs, not just to fluoroquinolones, when it is expressed in Bacillus subtilis. NorA provides B. subtilis with resistance to the same drugs and to a similar extent as the B. subtilis multidrug transporter protein Bmr does. NorA and Bmr share 44% sequence similarity. Both the NorA- and Bmr-conferred resistances can be completely reversed by reserpine.

Comparative effect of protein binding on the killing activities of teicoplanin and vancomycin.

The effect of protein binding on the activity of teicoplanin against Staphylococcus aureus was evaluated. Bactericidal rates of teicoplanin in cation-supplemented Mueller-Hinton broth (SMHB) and in a 1:1 mixture of pooled human serum and cation-supplemented Mueller-Hinton broth (PHS-SMHB) were compared with those of vancomycin. Eight concentrations of each drug ranging from 15 to 150 micrograms/ml were studied in two series which correspond to the concentrations in serum achieved with low- (6 mg/kg of body weight once daily) and high-dose (30 mg/kg once daily) teicoplanin. Overall, the bactericidal rate of teicoplanin was lower than that of vancomycin. In the presence of serum, the bactericidal rate of teicoplanin in PHS-SMHB was lower than that in SMHB, often resulting in only one log10 drop in CFU over a 24-h period. There was no statistical difference in the bactericidal rates of high- and low-concentration teicoplanin in either medium. Additionally, concentration-dependent killing in SMHB was not evident with either agent. The bactericidal rates of teicoplanin and vancomycin in a 1:1 mixture of serum ultrafiltrate and SMHB at 60 micrograms/ml were also studied. It was noted that the bactericidal rate of neither agent was affected by the presence of serum ultrafiltrate. This finding is consistent with teicoplanin's high degree of protein binding (reported to be greater than 90% in undiluted serum) and further substantiates the hypothesis that only the free drug is active against microorganisms. These data support protein binding as being a factor in teicoplanin activity against S. aureus.

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.

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.

Mechanisms of fluoroquinolone resistance in Staphylococcus aureus.

Fluoroquinolone resistance that arose in the test strain during ciprofloxacin therapy of experimental Staphylococcus aureus endocarditis was studied. In two isolates, resistance was due to a decreased sensitivity of the process of DNA synthesis to fluoroquinolones, suggesting the presence of an altered DNA gyrase. Another isolate had an enhanced energy-dependent mechanism, possibly an efflux system, by which cell-associated [3H]norfloxacin was reduced. When a 2.7-kb SphI-KpnI chromosomal fragment from this organism was cloned into pUC19, fluoroquinolone resistance was expressed in an Escherichia coli host, and such organisms acquired an energy-dependent ability to reduce cell-associated [3H]norfloxacin. Lack of homology between this DNA and other cloned gyrA genes indicated that its protein products are distinct from the gyrA protein. S. aureus has the capability of decreasing the quantity of cell-associated fluoroquinolone. An enhancement of this system by an as yet undefined mechanism and an alteration in DNA gyrase are two means by which this organism can develop resistance to fluoroquinolones.

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.