Killing Curve Studies Research Articles

Pharmacokinetic/Pharmacodynamic (PK/PD) Evaluation of a Once-Daily Treatment Using Ciprofloxacin in an Extended-Release Dosage Form*

To evaluate the suitability of a once-a-day dosing regimen of ciprofloxacin using a new extended-release dosage form based on PK/PD principles. Ciprofloxacin's serum concentrations were measured after administration of 500 mg immediate-release twice-daily, and 1,000 mg extended-release once-daily to 19 healthy volunteers. Pharmacokinetic parameters were determined using non-compartmental and compartmental data analysis. Measured serum concentration profiles were linked to ciprofloxacin's effect against Escherichia coli (MIC 0.013 mg/l) from in vitro kill curve studies where the pharmacokinetics of ciprofloxacin were simulated and change in number of bacteria (CFU/ml) versus time was monitored. Resulting parameters were used to compare expected kill curves for the two dosing regimens based on measured ciprofloxacin concentrations. Fitting the data using an appropriate PK/PD model resulted in a set of mean pharmacodynamic parameters (bacterial growth rate constant, k0, maximum kill rate constant, Kmax, and EC50). The model included a novel term to account for a change in kill rate after approximately 4 h when Kmax decreased in concentration-dependent matter. The model allowed excellent curve fits of all ciprofloxacin concentrations investigated. Comparison of expected kill curves with the immediate-release versus extended-release treatments showed similar outcome. Both treatments resulted in a decrease in CFU/ml > 5 log units over 24 h. Results indicate that once-a-day dosing of equal total daily doses with the new and more compliance-friendly extended-release dosing form will be therapeutically equivalent to once-a-day dosing with traditional immediate-release dosage forms for treatment of infections with this microorganism.

Evaluation of PPI-0903M (T91825), a novel cephalosporin: bactericidal activity, effects of modifying in vitro testing parameters and optimization of disc diffusion tests

To evaluate bactericidal activity of PPI-0903M and in vitro testing parameters for this compound. A total of 110 strains were tested to determine MIC and MBC values of PPI-0903M. MIC values were determined by reference broth microdilution whereas MBC was assessed by plating the broth from the MIC well and from those wells above the MIC for each organism onto appropriate drug-free growth media. Seventeen strains were tested by the kill-curve methodology to again evaluate the bactericidal activity of PPI-0903M. Broth microdilution results for 15 strains (in triplicate) were compared with the standard, reference method after modifying several testing parameters. Optimal disc content was assessed by testing seven reference strains with five PPI-0903M disc concentrations (5, 10, 30, 50 and 100 microg). PPI-0903MMBC/MIC ratios were < or = 4 in 90% of strains tested. Kill-curve studies showed >99.9% killing at concentrations < or = 4x MIC within 8 to 24 h against staphylococci and Enterobacteriaceae tested. PPI-0903M MIC results were rarely influenced by the test or medium changes evaluated [high inoculum, low calcium or reduced pH (5.0)]. Human serum had no effect on in vitro antimicrobial activity of PPI-0903M. The 10 microg disc content would be recommended for optimal correlation of MIC breakpoints of < or = 1-4 mg/L. PPI-0903M was generally bactericidal against tested species, and the MIC results were stable across numerous susceptibility test condition changes.

A comparative study of fungicidal activities of voriconazole and amphotericin B against hyphae of Aspergillus fumigatus

To study the in vitro fungicidal activity of voriconazole against hyphae of Aspergillus fumigatus and compare the results with those obtained for the known fungicidal drug amphotericin B. A. fumigatus mycelia were grown on Sabouraud dextrose agar and in peptone yeast extract glucose broth until the cultures reached a mid-logarithmic growth phase. The fungicidal activities of voriconazole and amphotericin B against actively growing hyphae of A. fumigatus were examined by a kill-curve experiment and a fungal cell viability test. For the kill-curve study, the drug-treated hyphae were washed, homogenized and resuspended in 1 mL of sterile water, diluted 10-1000 fold and aliquots of 0.1 mL were spread on Sabouraud dextrose agar and allowed to grow for 48 h at 35 degrees C. The cfu were determined and plotted against drug concentrations for each time of exposure to obtain the kill curve. The viability of drug-treated A. fumigatus hyphae was determined by their ability to reduce tetrazolium compound 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide. Exposure of A. fumigatus hyphae to several concentrations (1-16 mg/L) of voriconazole or amphotericin B for various time intervals killed the hyphae in a time- and drug concentration-dependent manner. Voriconazole at 1 mg/L killed >95% of the hyphae grown on Sabouraud dextrose agar after 48 h of exposure, whereas amphotericin B at the same concentration killed approximately 70% of the hyphae after exposure for the same duration. Approximately 99% killing of hyphae grown in peptone yeast extract glucose broth was obtained for voriconazole at 1 mg/L after 48 h of exposure, whereas amphotericin B at 1 mg/L yielded approximately 82% killing after 48 h. The fungal cell viability test by tetrazolium reduction assay showed that mycelia exposed to > or =1 mg/L (Sabouraud dextrose agar blocks) and > or =2 mg/L (broth cultures) of voriconazole for 48 h completely failed to reduce 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide. At low concentrations (1-2 mg/L) amphotericin B had no detectable effect on 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction by drug-treated mycelia, whereas mycelia treated with 16 mg/L for 48 h showed approximately 50% inhibition of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction compared with the control. Voriconazole possesses excellent fungicidal activity against actively growing hyphae of A. fumigatus. A comparison of results with those obtained for the known fungicidal drug amphotericin B shows that, in peptone yeast extract glucose broth, voriconazole has superior fungicidal activity against A. fumigatus hyphae compared with that of amphotericin B.

Antibacterial activity of polygodial

Polygodial was found to possess moderate antibacterial activity against gram-positive bacteria including Bacillus subtilis, Staphylococcus aureus and gram-negative bacteria including Escherichia coli and Salmonella choleraesuis with minimum bactericidal concentrations (MBC) of 100 and 100 microg/mL (0.34 mm) and 100 and 50 microg/mL, respectively. The time kill curve study showed that polygodial was bactericidal against B. subtilis and S. choleraesuis. However, there was a difference in its bactericidal action against endospore-forming B. subtilis and food-borne S. choleraesuis.

Lack of vancomycin tolerance in Streptococcus pneumoniae strains isolated in Barcelona, Spain, from 1999 to 2001.

To evaluate the incidence of vancomycin tolerance among Streptococcus pneumoniae isolates, we performed killing curve studies with 633 isolates. The penicillin MIC was > or = 0.12 mg/liter for 481 (76%) of the isolates. All strains were susceptible to vancomycin. Killing curve studies were performed with a vancomycin concentration of 2.5 mg/liter. The Tupelo strain was used for quality control. No vancomycin-tolerant strain was detected.

An approach for the evaluation of synergy between antimicrobials

Synergy can be assessed by a variety of microbiological techniques. Mathematical modelling of synergy data can accomplish similar results and may provide more information than currently available techniques. In this study a combination of 1/4 MIC of aztreonam (AZTR) and 1 MIC of ciprofloxacin (CIPX) showed synergy against Pseudomonas aeruginosa in killing curve studies (based on the two log different criteria) at the end of 24 h. However, re-growth was always observed even when bactericidal concentrations of antibiotics were present. The surviving organisms showed a two-fold increase of MIC to CIPX after the studies and this resistance was not reversible. A mathematical model to quantitatively describe this observation is proposed.

Modes of antifungal action of alkanols against Saccharomyces cerevisiae

Primary aliphatic alcohols from C 6 to C 13 were tested for their antifungal activity against Saccharomyces cerevisiae. Undecanol was found to be the most potent fungicide followed by decanol. The time–kill curve study showed that undecanol was fungicidal against S. cerevisiae at any growth stages. This fungicidal activity was not influenced by pH values. The alcohols tested inhibited glucose-induced acidification by inhibiting the plasma membrane H +-ATPase. The primary antifungal action of amphipathic medium-chain (C 9–C 12) alkanols comes mainly from their ability as nonionic surfactants to disrupt the native membrane-associated function of the integral proteins. Hence, the antifungal activity of alkanols is mediated by biophysical process, and the maximum activity can be obtained when balance between hydrophilic and hydrophobic portions becomes the most appropriate.

Non-antibiotic antibacterial activity of dodecyl gallate

Dodecyl (C 12) gallate (3,4,5-trihydroxybenzoate) ( 1) was found to possess antibacterial activity specifically against Gram-positive bacteria, in addition to its potent antioxidant activity. The time–kill curve study indicates that this amphipathic gallate exhibits bactericidal activity against methicillin resistant Staphylococcus aureus (MRSA) strains. Dodecyl (lauryl) gallate inhibited oxygen consumption in whole cells and oxidation of NADH in membrane preparation. The antibacterial activity of this gallate comes in part from its ability to inhibit the membrane respiratory chain. As far as alkyl gallates are concerned, their antimicrobial spectra and potency depend in part on the hydrophobic portion of the molecule.

Controlled release of water-soluble polymeric complexes of sorbic acid with antifungal activities.

We synthesized six water-soluble polymeric complexes of sorbic acid with polyvinylpyrrolidone of different molecular weight (mol wt). As shown by infrared absorption spectrum analysis, the complexes were formed by hydrogen bonding. The complexes (SC1, with mol wt=10 kDa, SC2 with mol wt=25 kDa, SC3 with mol wt=30 kDa, SC4 with mol wt=40 kDa, SC5 with mol wt=90 kDa, and SC6 with mol wt=360 kDa) were characterized as low mol wt (SC1, SC2, and SC3) and high mol wt (SC4, SC5, and SC6). The antifungal potencies of the complexes were tested by the macrodilution susceptibility method against environmental and clinically important fungi. Sorbic acid as well as the complexes exhibited minimum inhibitory concentrations (MICs) lower than potassium sorbate against all the strains tested. MICs of SC1, SC2, and SC3 were shown to be 2- to 4-fold lower for yeast and 1.5- to 3-fold lower than those of sorbic acid for moulds, respectively. The MICs of SC4 and SC5 against both of the Candida species tested ranged from 500 to 800 microg/ml, whereas for SC6 and sorbic acid they were about 1 mg/ml. The potencies of the high mol wt complexes against moulds were decreased by increasing the mol wt. For both of the moulds tested, the MICs of SC4 were slightly lower than those of sorbate. The MICs of sorbic acid and SC5 were equal to 300 microg/ml and 500 microg/ml respectively for Aspergillus parasiticus and for Penicillum viridicatum. The susceptibility to SC6 of all of the hyphomycetes tested was higher than that to sorbic acid. The low mol wt complexes and the sorbic acid exhibited minimal fungicidal concentrations (MFCs) 2 and 3 times higher respectively than the MICs. Sorbic acid and SC3 at a concentration of 2.5 mg/ml in an in vitro time kill curve study of Candida tropicalis were shown to be fungistatic, whereas SC1 and SC2 were fungicidal at the same concentrations. For Aspergillus parasiticus sorbic acid at 2.5 mg/ml was fungistatic for a 24-h period, whereas SC1, SC2, and SC3 were fungicidal.

Naturally occurring anti-Salmonella agents.

Polygodial and (2E)-hexenal were found to possess antibacterial activity against Salmonella choleraesuis with the minimum bactericidal concentrations (MBC) of 50 microg/mL (0.17 mM) and 100 microg/mL (0.98 mM), respectively. The time kill curve study showed that these two alpha,beta-unsaturated aldehydes were bactericidal against this food-borne bacterium at any stage of growth. However, they showed different effects on the growth of S. choleraesuis. The combination of polygodial and anethole exhibited strong synergism on their bacteriostatic action but only marginal synergism on their bactericidal action.

Anti-MRSA activity of alkyl gallates

A series of alkyl gallates (3,4,5-trihydroxybenzoates) was found to show antibacterial activity against Gram-positive bacteria including methicillin resistant Staphylococcus aureus (MRSA) strains. For example, dodecyl (C 12) gallate ( 1) exhibited bactericidal activity against MRSA ATCC 33591 strain with the minimum bactericidal concentration (MBC) of 25 μg/mL (74 μM). The time–kill curve study showed that dodecyl gallate is bactericidal against this MRSA strain. This bactericidal activity comes in part from its ability to inhibit respiratory electron transport systems. The length of the alkyl chain is not a major contributor but plays an important role in eliciting the activity.

In vitro activities of nontraditional antimicrobials against multiresistant Acinetobacter baumannii strains isolated in an intensive care unit outbreak.

Fifteen multiresistant Acinetobacter baumannii isolates from patients in intensive care units and 14 nonoutbreak strains were tested to determine in vitro activities of nontraditional antimicrobials, including cefepime, meropenem, netilmicin, azithromycin, doxycycline, rifampin, sulbactam, and trovafloxacin. The latter five drugs were further tested against four of the strains for bactericidal or bacteriostatic activity by performing kill-curve studies at 0.5, 1, 2, and 4 times their MICs. In addition, novel combinations of drugs with sulbactam were examined for synergistic interactions by using a checkerboard configuration. MICs at which 90% of the isolates tested were inhibited for antimicrobials showing activity against the multiresistant A. baumannii strains were as follows (in parentheses): doxycycline (1 microg/ml), azithromycin (4 microg/ml), netilmicin (1 microg/ml), rifampin (8 microg/ml), polymyxin (0.8 U/ml), meropenem (4 microg/ml), trovafloxacin (4 microg/ml), and sulbactam (8 microg/ml). In the kill-curve studies, azithromycin and rifampin were rapidly bactericidal while sulbactam was more slowly bactericidal. Trovafloxacin and doxycycline were bacteriostatic. None of the antimicrobials tested were bactericidal against all strains tested. The synergy studies demonstrated that the combinations of sulbactam with azithromycin, rifampin, doxycycline, or trovafloxacin were generally additive or indifferent.

In-vitro isolation and antifungal susceptibility of amphotericin B-resistant mutants of Aspergillus fumigatus.

Aspergillus fumigatus mutants resistant to amphotericin B were selected in the laboratory following UV irradiation. A total of 18 colonies (frequency 1.8 x 10(-7)) that grew in the presence of amphotericin B (8 mg/L and 16 mg/L) on peptone yeast extract glucose agar were tested for their susceptibility to amphotericin B, nystatin, azoles, and the echinocandin L-743872. Ten of the 18 isolates showed an eight-fold rise in amphotericin B MIC (4 mg/L) compared with the susceptible parent whereas the remaining isolates showed a 16 to 32-fold rise in amphotericin B MIC (8-16 mg/L). Subculturing of three representatives from each of the groups that had MIC values of 4 mg/L and 8-16 mg/L for six cycles revealed that the resistance trait was stably expressed. All amphotericin B-resistant isolates showed a significant level of cross-resistance to nystatin but not to azoles and L-743872. Kill-curve studies with amphotericin B revealed that the killing of the resistant isolates was significantly less than that of the susceptible parent strain. These results show that amphotericin B-resistant mutants of A. fumigatus can be isolated in the laboratory following a single-step UV-induced mutagenesis and suggest that similar mechanisms could operate in nature for the emergence of resistance in clinical isolates.

Influence of pulmonary surfactant on in vitro bactericidal activities of amoxicillin, ceftazidime, and tobramycin.

The influence of a natural pulmonary surfactant on antibiotic activity was investigated to assess the possible use of exogenous surfactant as a vehicle for antibiotic delivery to the lung. The influence of surfactant on the bactericidal activity of amoxicillin was tested against Staphylococcus aureus and Streptococcus pneumoniae, and the influence of surfactant on the activities of ceftazidime and tobramycin was tested against Klebsiella pneumoniae, Pseudomonas aeruginosa, S. aureus, and S. pneumoniae. In vitro antibiotic activity was determined by killing curve studies in media with and without surfactant. Amoxicillin and ceftazidime activities were not changed in the presence of surfactant, except for a decreased killing rate of S. pneumoniae by ceftazidime in medium with additional rabbit serum. In contrast, killing curves with low concentrations of tobramycin (0.25x and 1x the MIC) showed a decreased level of activity of tobramycin against all pathogens tested in the presence of surfactant. With higher tobramycin concentrations (4x the MIC) killing rates were decreased less or were unchanged in the presence of surfactant. Concluding from the results of the study, both amoxicillin and ceftazidime can be combined with surfactant without the loss of activity. For mixing surfactant with tobramycin, dosages should be adjusted to overcome the partial inactivation of tobramycin by surfactant.

Triple combination penicillin-vancomycin-gentamicin for experimental endocarditis caused by a highly penicillin-and glycopeptide-resistant isolate of Enterococcus faecium.

A combination of low-dose penicillin (75,000 IU/kg twice daily [b.i.d.]) vancomycin (30 mg/kg b.i.d.) and gentamicin (6 mg/kg b.i.d.) has been shown to be as effective as a combination of high-dose penicillin (500,000 IU/kg b.i.d.) and gentamicin (6 mg/kg b.i.d.) in the treatment of rabbit endocarditis caused by an Enterococcus faecium strain moderately resistant to beta-lactams and highly resistant to glycopeptides. The same regimens were evaluated against an E. faecium strain highly resistant to both penicillin (MIC, 128 micrograms/mL) and vancomycin (MIC, 512 micrograms/mL). High doses of penicillin-gentamicin and vancomycin-gentamicin had no effect in in vitro killing-curve studies or in rabbits after treatment for 5 days. High doses of penicillin-vancomycin were only bacteriostatic in killing curves and provided a small reduction in the bacterial titers of the vegetations. In contrast, high-dose penicillin-vancomycin-gentamicin was bactericidal in vitro and highly effective in treating rabbits. However, the emergence of a bacterial subpopulation resistant to the synergistic effect of penicillin and vancomycin could reduce the clinical utility of this combination.

Clarithromycin in vitro activity enhanced by its major metabolite, 14-hydroxyclarithromycin

14-Hydroxyclarithromycin, an active metabolite of clarithromycin, was compared for antimicrobial activity alone and in combination with the parent compound. The 14-hydroxyclarithromycin potency was comparable to that of clarithromycin, but was more active against Haemophilus influenzae (MIC 50, 1 μg/ml). Combination MICs at pharmacokinetic ratios produced end points equal to the most active component of the combination. However, checkerboard MICs and kill-curve studies suggested enhanced interactive effects. Partial synergy and additive interactions were demonstrated in 96% of strains tested with synergy (partial) most often observed among the Enterococcus faecalis, H. influenzae, and staphylococci. To determine the best in vitro test methods for predicting the value of 14-hydroxyclarithromycin, combination disks or ratio MIC tests may not be practical. A modification of the proposed clarithromycin-susceptible breakpoint (⩽2 μg/ml) upward to ⩽ μg/ml (⩾14 mm) was suggested to recognize the additional activity contributed by the 14-hydroxy metabolite. This modification should be applied, limited to susceptibility tests of H. influenzae and possibly the enterococci.

Antimicrobial activity of subinhibitory concentrations of ciprofloxacin against Pseudomonas aeruginosa as determined by the killing curve method and the postantibiotic effect.

This investigation used the postantibiotic effect (PAE) and killing curves to examine the antimicrobial activity of subinhibitory (1/8x, 1/4x and 1/2x MIC) and inhibitory (1x MIC) concentrations of ciprofloxacin against mucoid (M) and nonmucoid (NM) urinary isolates of Pseudomonas aeruginosa. Subinhibitory concentrations (1/8x, 1/4x and 1/2x MIC) of ciprofloxacin produced PAEs with no difference between M and NM strains. For NM strains, those with low MICs (< or = 1.0 mg/l) to ciprofloxacin produced significantly longer PAEs than isolates with high MICs (> 1 mg/l). Killing curve studies demonstrated that subinhibitory concentrations of ciprofloxacin produce little effect (1/8x MIC) or stasis (1/4x and 1/2x MIC) of growth for several hours. Only 1x MIC was bactericidal for several strains. At 1/2x and 1x MIC, bacterial inhibition was greater against NM versus M isolates. The M phenotype of P. aeruginosa reduces killing by ciprofloxacin but not the PAE.

Antimicrobial Activity of Subinhibitory Concentrations of Aminoglycosides against &lt;i&gt;Pseudomonas aeruginosa&lt;/i&gt; as Determined by the Killing-Curve Method and the Postantibiotic Effect

This investigation used the postantibiotic effect (PAE) and killing curves to provide data on the antimicrobial activity of subinhibitory (1/8x, 1/4x and 1/2x minimum inhibitory concentration; MIC) and inhibitory (1x MIC) concentrations of amikacin, gentamicin and tobramycin against Pseudomonas aeruginosa. Subinhibitory concentrations (1/4x and 1/2x MIC) of aminoglycosides demonstrated a reproducible PAE. At 1/4x MIC, the order of duration of the PAE was approximately 15 min for all aminoglycosides, while at 1/2x MIC all three aminoglycosides displayed a similar PAE of approximately 40 min. Killing-curve studies demonstrated that subinhibitory concentrations of aminoglycosides either decrease bacterial growth for several hours (1/4x and 1/2x MIC) or produce stasis of growth for several hours (1/8x MIC). Only inhibitory aminoglycoside concentrations (1x MIC) proved to be bactericidal. Subinhibitory concentrations of aminoglycosides decrease bacterial growth and produce a PAE against P. aeruginosa.

In vitro and in vivo effects of combinations of cefotaxime or other beta-lactams with rolitetracycline on methicillin-resistant Staphylococcus aureus.

Combinations of cefotaxime (CTX) or other five beta-lactams with rolitetracycline (RTC) were examined using the checkerboard method for their synergistic effects against 27 strains of methicillin-resistant Staphylococcus aureus (MRSA), and the combination of CTX and RTC was further evaluated for its synergistic effect in vitro and in vivo against 1 or 2 strains. Synergy occurred against 44% of the strains when RTC was combined with CTX, 22 to 30% with cefazolin, methicillin, and ceftizoxime, and 4 to 11% with latamoxef and cefmetazole. No antagonism was found with any combinations tested. Killing curve studies also showed that CTX/RTC was synergistic between 3 and 24 hours after the beginning of exposure, and the synergy was especially strong at 24 hours and potencies of combined bactericidal effect determined at 24 hours were in the following order: the 2 antibiotics given simultaneously, CTX given 2 hours before RTC, and CTX given 2 hours after RTC. In addition, the 2 drugs in combination synergistically inhibited (a) mortality in mice infected intraperitoneally with MRSA and (b) formation of subcutaneous abscess induced by MRSA in mice. The results of our study indicate that beta-lactams, especially CTX, had synergistic effects in vitro when combined with RTC against MRSA and that the combination of CTX and RTC was also synergistic in vivo.

Time kill curve analysis of vancomycin and rifampin alone and in combination against nine strains of nutritionally deficient streptococci.

Endocarditis caused by nutritionally deficient streptococci has a high bacteriologic and clinical failure rate, despite appropriate antimicrobial therapy. We investigated by time kill curve methodology nine clinical endocarditis isolates of nutritionally deficient streptococci to determine the in vitro efficacies of vancomycin and rifampin alone and in combination. The combination of vancomycin and rifampin demonstrated synergy and bactericidal activity in five of the strains. In one strain, this combination inhibited growth by greater than 2 log10 CFU/ml when compared to the growth control or either antibiotic alone, but it failed to be bactericidal. Indifference, defined as less than or equal to 2 log10 CFU per milliliter increase in killing of the combination compared to the next most active single agent, was demonstrated with the remaining three isolates. Changing the antibiotic concentrations in the time kill curve studies for these latter strains failed to demonstrate synergistic activity of the antibiotic combination. The vancomycin and rifampin combination may be a promising therapeutic modality for which in vivo correlation is indicated.