Extent Of Killing Research Articles

A Randomized Control Study of Oral Itraconazole versus Itraconazole with Terbinafine in the Treatment of Dermatophytosis

Background: Dermatophyte infections are one of the first known fungal infections of humankind and are most common throughout the world. The antifungal therapies over the past 30 years have improved, but the phenomenon of antifungal resistance is still of major concern. Combination therapies can show great promise in overcoming this problem to broaden the spectrum of activity, enhance the rate or extent of killing, and minimize the development of resistance. Objectives: To find out the therapeutic efficacy of itraconazole versus itraconazole with terbinafine for the treatment of dermatophytosis. Materials and Methods: A prospective study was conducted for a duration of 1 year on 64 patients diagnosed with dermatophytosis and was randomly divided into two groups. Group A was given oral itraconazole whereas Group B received oral itraconazole and oral terbinafine for 4 weeks along with levocetirizine daily for 2 weeks and miconazole 2% topically for 6 weeks. The measurability criteria were done on detailed clinical examination (itching, erythema, size, number, scaling, and margin) and through photographic assessment. Results: The mean difference and standard deviation of total scores for group A were 10.12 ± 4.09 and for group B were 9.94 and 3.75, respectively. The P value was not significant (P = 0.78). Out of 64 cases, 3 (5%) patients complained of gastric discomfort, 2 (3%) patients complained of nausea, and 1 (1%) of dizziness. Conclusion: This study revealed that there was a highly significant improvement in both groups with no significant difference in their clinical criteria.

127. Antimicrobial Efficacy Against Antibiotic-Tolerant Staphylococcus aureus Depends on the Mechanism of Antibiotic Tolerance

Abstract Background Bacteria can adopt an alternate metabolic state favoring small molecule synthesis over growth. In Staphylococcus aureus this is induced by factors present during infection including nutritional limitation and competition with other bacteria. Isogenic “tolerant” subpopulations have variable responses to antibiotics and can remain viable. Survivors resume growth upon cessation of antibiotics and cause relapse or recurrent infection. In this study we compare the capability of antibiotics to reduce viability of S. aureus made tolerant by different mechanisms. Methods Overnight cultures of S. aureus SH1000 were diluted to 106 cfu/mL. Tolerance was induced with mupirocin or 2-n-heptyl-4-hydroxypuinoline N-oxide (HQNO) simulating nutritional or competitive tolerance, respectively. Tolerant cultures were exposed to ceftaroline, daptomycin, gentamicin, levofloxacin, oritavancin or vancomycin at physiological concentrations and viability assessed by dilution plating. Minimum duration for 3-log viability reduction (“bactericidal activity”, MDK99.9) and 24h viability reduction were calculated independently for each of three biological replicates. Significance (P < 0.05) was determined using Student’s t-test. Results Time to bactericidal activity was prolonged for all antibiotics tested against nutritionally-tolerant S. aureus. Time to bactericidal activity was similarly prolonged against competitively-tolerant S. aureus, although the MDK99.9 of oritavancin was not affected. Viability reduction was mitigated for all antibiotics tested against nutritionally-tolerant S. aureus with the exception of oritavancin. In contrast, 24h viability reduction of competitively-tolerant S. aureus only occurred with ceftaroline, gentamicin and vancomycin while daptomycin, levofloxacin and oritavancin remained equally potent. Conclusion Tolerance can alter both the time to bactericidal effect and the extent of killing. Both the antibiotic and the mechanism of tolerance impact time to bacterial effect and extent of killing. Oritavancin was the only antibiotic that maintained the same extent of killing regardless of tolerance mechanism. Further studies to evaluate additional antistaphylococcal antibiotics and different inducers of tolerance are warranted. Disclosures All Authors: No reported disclosures.

Cytotoxic effect of galvanically coupled magnesium-titanium particles on Escherichia coli.

Orthopedic device-related infections (ODRIs) are difficult to control due to microbial biofilm formation and associated with high-level resistance to conventional antibiotics. In many cases, the only treatment option for ODRI is explantation. Previous studies have shown that application of cathodic potentials at the metal surface can eradicate biofilms, and Mg and Mg-Ti particles have the same effect as cathodic potentials. This study investigated the effects of Mg and Mg-Ti particles on established biofilms and planktonic cells E. coli. Bacterial cultures with developed biofilms or planktonic cells were treated with Mg or Mg-Ti particles, and the viability were assessed using flow cytometry or visual assessment methods (i.e., observation from SEM images and opacity of the solution). It was found that viability of biofilms treated with 16.67 mg/ml of Mg was 2.8 ± 0.96% at the end of 6-hr killing compared to untreated controls. This extent of killing was more significant compared to 24-hr grown biofilms treated with ofloxacin, an antibiotic known to be effective against these bacteria. Biofilms treated with 50 and 100 μg/ml of ofloxacin had 62 ± 4.6% and 52 ± 19.3% survival, respectively, where ofloxacin at these concentrations is known to kill planktonic counterparts very effectively. Inhibition zone tests revealed that biofilms within 2 mm of Mg or Mg-Ti particle clusters were effectively killed. These results demonstrated the potential of Mg or Mg-Ti particles in killing microbial biofilms and potential for controlling ODRI.

Distinct Effectiveness of Oritavancin against Tolerance-Induced Staphylococcus aureus.

Within a sufficiently large bacterial population, some members will naturally adopt an alternate, metabolically-active state that favors small molecule synthesis over cell division. These isogenic “tolerant” subpopulations have variable responses during antibiotic exposure and can remain viable in the presence of typically bactericidal concentrations. In this study, we determine the ability of typical and atypical antistaphylococcal therapies to reduce the viability of mupirocin-induced tolerant Staphylococcus aureus bacteria. Overall, tolerance-induced staphylococci exhibited a markedly decreased rate and extent of killing following antibiotic exposure. However, oritavancin remained effective at maintaining a similar extent of killing. Further studies to investigate the role of oritavancin against recurrent or relapse staphylococcal infection are warranted.

Abstract 4228: PLAP (placental alkaline phosphatase)-CAR-T cells specifically target colorectal cancer

Abstract Placental alkaline phosphatase, PLAP encoded by ALPP gene in humans is mainly expressed in placenta and testis, and not expressed in any other normal tissues. PLAP is overexpressed in a proportion of colorectal cancers which makes it an attractive target for CAR (chimeric antigen receptor)-T cell therapy. PLAP mRNA expression was detected in 21.4% (25 out of 117) of colorectal cancer cell lines and this expression was confirmed by FACS at the protein level. In addition, IHC staining on primary colorectal cancer tumors demonstrated PLAP expression in >20% of colorectal cancer tumors. We generated mouse and humanized PLAP ScFv-CAR-T cells and demonstrated high specificity against PLAP-positive colon cancer cells using RTCA (real-time cytotoxicity assay) and IFN-gamma secretion. In addition, humanized-CAR-T cells significantly decreased Lovo xenograft tumor growth in vivo. The combination of hPLAP-CAR-T cells with PD-1, PDL-1 or LAG-3 checkpoint inhibitors significantly increased the activity of hPLAP-CAR-T cells. Significance: This study demonstrates ability of novel PLAP-CAR-T cells to kill colorectal cancers and that the extent of killing can be increased by combination with checkpoint inhibitors. These preclinical results suggest that PLAP-CAR-T cells combined with checkpoint inhibitors may be developed into an effective clinical therapy for PLAP expressing colorectal and other cancers. Citation Format: Vita M. Golubovskaya, Robert Berahovich, Hua Zhou, Xianghong Liu, Feng Li, Shirley Xu, Yuehua Wei, Djamila Ouaret, Walter Bodmer, Lijun Wu. PLAP (placental alkaline phosphatase)-CAR-T cells specifically target colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4228.

PLAP -CAR Tcells mediate high specific cytotoxicity against colon cancer cells.

Placental alkaline phosphatase, PLAP encoded by ALPP gene in humans is mainly expressed in placenta and testis, and not expressed in any other normal tissues. PLAP is overexpressed in colorectal cancers which makes it an attractive target for CAR (chimeric antigen receptor)-T cell therapy. PLAP mRNA expression was detected in 21.5% (25 out of 116) of colorectal cancer cell lines and this expression was confirmed by FACS at the protein level. In addition, IHC staining on primary colorectal cancer tumors demonstrated PLAP expression in >20% of colorectal cancer tumors. We generated mouse and humanized PLAP ScFv-CAR-T cells and demonstrated high specificity against PLAP-positive colon cancer cells using RTCA (real-time cytotoxicity assay) and IFN-gamma secretion. In addition, humanized-CAR-T cells significantly decreased Lovo xenograft tumor growth in vivo. The combination of hPLAP-CAR-T cells with PD-1, PD-L1 or LAG-3 checkpoint inhibitors significantly increased the activity of hPLAP-CAR-T cells. This study demonstrates ability of novel PLAP-CAR-T cells to kill colorectal cancers and that the extent of killing can be increased by combination with checkpoint inhibitors.

Comparison of the composition and in vitro activity of polymyxin B products

A number of companies manufacture polymyxin B using United States Pharmacopeia (USP) metrics, rather than chemical composition, to report biological activity. Given that polymyxin B contains several different components, it is unknown whether pharmacokinetic and pharmacodynamic variability exists between the different brands and whether USP metrics capture this variability. Here we investigated the composition of polymyxin B obtained from four manufacturers (Sigma-Aldrich, AK Scientific, USP and MP Biomedicals) and evaluated their rate and extent of killing against multidrug-resistant Acinetobacter baumannii and Klebsiella pneumoniae using in vitro static time-kill experiments. Ultraviolet (UV) fingerprinting and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed similarities and differences between component distributions. The significant differences between products, based on UV fingerprinting and LC-MS/MS, did not translate into pharmacodynamic differences at the three concentrations evaluated. The aggregate polymyxin B concentration, rather than that of the individual components, influences overall bacterial killing.

In Vitro Assessment of Combined Polymyxin B and Minocycline Therapy against Klebsiella pneumoniae Carbapenemase (KPC)-Producing K. pneumoniae.

The multidrug resistance profiles of Klebsiella pneumoniae carbapenemase (KPC) producers have led to increased clinical polymyxin use. Combination therapy with polymyxins may improve treatment outcomes, but it is uncertain which combinations are most effective. Clinical successes with intravenous minocycline-based combination treatments have been reported for infections caused by carbapenemase-producing bacteria. The objective of this study was to evaluate the in vitro activity of polymyxin B and minocycline combination therapy against six KPC-2-producing K. pneumoniae isolates (minocycline MIC range, 2 to 32 mg/liter). Polymyxin B monotherapy (0.5, 1, 2, 4, and 16 mg/liter) resulted in a rapid reduction of up to 6 log in bactericidal activity followed by regrowth by 24 h. Minocycline monotherapy (1, 2, 4, 8, and 16 mg/liter) showed no reduction of activity of >1.34 log against all isolates, although concentrations of 8 and 16 mg/liter prolonged the time to regrowth. When the therapies were used in combination, rapid bactericidal activity was followed by slower regrowth, with synergy (60 of 120 combinations at 24 h, 19 of 120 combinations at 48 h) and additivity (43 of 120 combinations at 24 h, 44 of 120 combinations at 48 h) against all isolates. The extent of killing was greatest against the more susceptible polymyxin B isolates (MICs of ≤0.5 mg/liter) regardless of the minocycline MIC. The pharmacodynamic activity of combined polymyxin B-minocycline therapy against KPC-producing K. pneumoniae is dependent on polymyxin B susceptibility. Further in vitro and animal studies must be performed to fully evaluate the efficacy of this drug combination.

Two mechanisms of killing of Pseudomonas aeruginosa by tobramycin assessed at multiple inocula via mechanism-based modeling.

Bacterial resistance is among the most serious threats to human health globally, and many bacterial isolates have emerged that are resistant to all antibiotics in monotherapy. Aminoglycosides are often used in combination therapies against severe infections by multidrug-resistant bacteria. However, models quantifying different antibacterial effects of aminoglycosides are lacking. While the mode of aminoglycoside action on protein synthesis has often been studied, their disruptive action on the outer membrane of Gram-negative bacteria remains poorly characterized. Here, we developed a novel quantitative model for these two mechanisms of aminoglycoside action, phenotypic tolerance at high bacterial densities, and adaptive bacterial resistance in response to an aminoglycoside (tobramycin) against three Pseudomonas aeruginosa strains. At low-intermediate tobramycin concentrations (<4 mg/liter), bacterial killing due to the effect on protein synthesis was most important, whereas disruption of the outer membrane was the predominant killing mechanism at higher tobramycin concentrations (≥8 mg/liter). The extent of killing was comparable across all inocula; however, the rate of bacterial killing and growth was substantially lower at the 10(8.9) CFU/ml inoculum than that at the lower inocula. At 1 to 4 mg/liter tobramycin for strain PAO1-RH, there was a 0.5- to 6-h lag time of killing that was modeled via the time to synthesize hypothetical lethal protein(s). Disruption of the outer bacterial membrane by tobramycin may be critical to enhance the target site penetration of antibiotics used in synergistic combinations with aminoglycosides and thereby combat multidrug-resistant bacteria. The two mechanisms of aminoglycoside action and the new quantitative model hold great promise to rationally design novel, synergistic aminoglycoside combination dosage regimens.

Medical Evidence in Dowry Deaths: An Evaluation by Indian Courts

Dowry Death has been one of the most barbaric forms of cruelty inflicted on young brides in the matrimonial home. Over the years, it assumed dangerous proportions calling for immediate legislative changes. Supreme Court judgment dated 11th Oct 2006 held that the demand for dowry or money from the parents of the bride has shown a phenomenal increase in last few years. Cases are frequently coming before the Courts, where the husband or in-laws have gone to the extent of killing the bride if the demand is not met. These crimes are generally committed in complete secrecy inside the house and it becomes very difficult for the prosecution to lead evidence.

Novel rate-area-shape modeling approach to quantify bacterial killing and regrowth for in vitro static time-kill studies.

In vitro static concentration time-kill (SCTK) studies are a cornerstone for antibiotic development and designing dosage regimens. However, mathematical approaches to efficiently model SCTK curves are scarce. The currently used model-free, descriptive metrics include the log10 change in CFU from 0 h to a defined time and the area under the viable count versus time curve. These metrics have significant limitations, as they do not characterize the rates of bacterial killing and regrowth and lack sensitivity. Our aims were to develop a novel rate-area-shape modeling approach and to compare, against model-free metrics, its relative ability to characterize the rate, extent, and timing of bacterial killing and regrowth from SCTK studies. The rate-area-shape model and the model-free metrics were applied to data for colistin and doripenem against six Acinetobacter baumannii strains. Both approaches identified exposure-response relationships from 0.5- to 64-fold the MIC. The model-based approach estimated an at least 10-fold faster killing by colistin than by doripenem at all multiples of the MIC. However, bacterial regrowth was more extensive (by 2 log10) and occurred approximately 3 h earlier for colistin than for doripenem. The model-free metrics could not consistently differentiate the rate and extent of killing between colistin and doripenem. The time to 2 log10 killing was substantially faster for colistin. The rate-area-shape model was successfully implemented in Excel. This new model provides an improved framework to distinguish between antibiotics with different rates of bacterial killing and regrowth and will enable researchers to better characterize SCTK experiments and design subsequent dynamic studies.

Aggregatibacter actinomycetemcomitans biofilm killing by a targeted ciprofloxacin prodrug

A pH-sensitive ciprofloxacin prodrug was synthesized and targeted against biofilms of the periodontal pathogen Aggregatibacter actinomycetemcomitans (Aa). The dose required to reduce the viability of a mature biofilm of Aa by ∼80% was in the range of ng cm−2 of colonized area (mean biofilm density 2.33 × 109 cells cm−2). A mathematical model was formulated that predicts the temporal change in the concentration of ciprofloxacin in the Aa biofilm as the drug is released and diffuses into the bulk medium. The predictions of the model were consistent with the extent of killing obtained. The results demonstrate the feasibility of the strategy to induce mortality, and together with the mathematical model, provide the basis for design of targeted antimicrobial prodrugs for the topical treatment of oral infections such as periodontitis. The targeted prodrug approach offers the possibility of optimizing the dose of available antimicrobials in order to kill a chosen pathogen while leaving the commensal microbiota relatively undisturbed.

Genetic analysis of colistin resistance in Salmonella enterica serovar Typhimurium.

Colistin is a cyclic cationic peptide that kills gram-negative bacteria by interacting with and disrupting the outer membrane. We isolated 44 independent mutants in Salmonella enterica serovar Typhimurium with reduced susceptibility to colistin and identified 27 different missense mutations located in the pmrA and pmrB genes (encoding the regulator and sensor of a two-component regulatory system) that conferred increased resistance. By comparison of the two homologous sensor kinases, PmrB and EnvZ, the 22 missense mutations identified in pmrB were shown to be located in four different structural domains of the protein. All five pmrA mutations were located in the phosphate receiver domain of the regulator protein. The mutants appeared at a mutation rate of 0.6 x 10(-6) per cell per generation. The MICs of colistin for the mutants increased 2- to 35-fold, and the extent of killing was reduced several orders of magnitude compared to the susceptible strain. The growth rates of the mutants were slightly reduced in both rich medium and M9-glycerol minimal medium, whereas growth in mice appeared unaffected by the pmrA and pmrB mutations. The low fitness costs and the high mutation rate suggest that mutants with reduced susceptibility to colistin could emerge in clinical settings.

Quinolone-mediated bacterial death.

The fluoroquinolones are broad-spectrum antibacterial agents that are becoming increasingly popular as bacterial resistance erodes the effectiveness of other agents (fluoroquinolone sales accounted for 18% of the antibacterial market in 2006) (41). One of the attractive features of the quinolones is their ability to kill bacteria rapidly, an ability that differs widely among the various derivatives. For example, quinolones differ in rate and extent of killing, in the need for aerobic metabolism to kill cells, and in the effect of protein synthesis inhibitors on quinolone lethality. Understanding the mechanisms underlying these differences could lead to new ways for identifying the most bactericidal quinolone derivatives. Before describing the types of damage caused by the quinolones, it is useful to define lethal activity. Operationally, it is the ability of drug treatment to reduce the number of viable cells, usually measured as CFU on drug-free agar after treatment. This assay is distinct from measurements that detect inhibition of growth (e.g., MIC), since with the latter bacteria are exposed to drug throughout the measurement. The distinction between killing and blocking growth is important because it allows susceptibility determinations to be related to particular biological processes. For example, inhibition of growth is typically reversed by the removal of drug, while cell death is not. Thus, biochemical events associated with blocking growth should be readily reversible, while those responsible for cell death should be difficult to reverse. Reversibility can be used to distinguish among quinolone derivatives and assign functions to particular aspects of drug structure. Moreover, protective functions, such as repair and stress responses, can be distinguished by whether their absence affects inhibition of growth, killing, or both. The intracellular targets of the quinolones are two DNA topoisomerases: gyrase and topoisomerase IV. Gyrase tends to be the primary target in gram-negative bacteria, while topoisomerase IV is preferentially inhibited by most quinolones in gram-positive organisms (28). Both enzymes use a double-strand DNA passage mechanism, and it is likely that quinolone biochemistry is similar for both. However, physiological differences between the enzymes exist, some of which may bear on quinolone lethality. In the present minireview we consider cell death through a two-part “poison” hypothesis in which the quinolones form reversible drug-topoisomerase-DNA complexes that subsequently lead to several types of irreversible (lethal) damage. Other consequences of quinolone treatment, such as depletion of gyrase and topoisomerase IV activity, are probably less immediate (42). To provide a framework for considering quinolone lethality, we begin by briefly describing the drug-topoisomerase-DNA complexes. Readers interested in a more comprehensive discussion of quinolones are referred to a previously published work (28).

Pharmacodynamic Effects of Telavancin against Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Strains in the Presence of Human Albumin or Serum and in an In Vitro Kinetic Model

Telavancin is a novel bactericidal lipoglycopeptide with multiple mechanisms of action against gram-positive pathogens. The aim of this study was to describe the dynamics of the antimicrobial effect of telavancin against two strains of Staphylococcus aureus (methicillin susceptible and methicillin resistant) in an in vitro kinetic model with simulated human pharmacokinetics. Also, static experiments were performed to determine the rate and extent of killing by telavancin in the presence and absence of human albumin and human serum. Experiments in broth and in nutrient-depleted medium were performed to study the rate and extent of killing by telavancin of bacteria in different growth phases. In the in vitro kinetic model regrowth was noted at 24 h for both strains when exposed to initial concentrations below 5 mg/liter. There was a >3-log(10) killing at all concentrations from 0.5x MIC and above at 24 h both in broth and in the presence of 40-g/liter human albumin. In contrast to the methicillin-susceptible strain, the methicillin-resistant strain in 40-g/liter human albumin showed a regrowth at concentrations of 0.5x MIC and 1x MIC at 24 h. At all the other concentrations >3-log(10) killing was seen at 24 h. Concordant results were seen in 50% human serum. At a target area under the curve/MIC ratio of 50 (corresponding to the human dose of 10 mg/kg of body weight, administered intravenously), >3-log(10) killing was observed at 6 to 8 h. Unlike most antibiotics, telavancin was able to kill both strains in a nongrowing phase.

Candida albicans Biofilms Produce Antifungal-Tolerant Persister Cells

Fungal pathogens form biofilms that are highly recalcitrant to antimicrobial therapy. The expression of multidrug resistance pumps in young biofilms has been linked to increased resistance to azoles, but this mechanism does not seem to underlie the resistance of mature biofilms that is a model of in vivo infection. The mechanism of drug resistance of mature biofilms remains largely unknown. We report that biofilms formed by the major human pathogen Candida albicans exhibited a strikingly biphasic killing pattern in response to two microbicidal agents, amphotericin B, a polyene antifungal, and chlorhexidine, an antiseptic, indicating that a subpopulation of highly tolerant cells, termed persisters, existed. The extent of killing with a combination of amphotericin B and chlorhexidine was similar to that observed with individually added antimicrobials. Thus, surviving persisters form a multidrug-tolerant subpopulation. Interestingly, surviving C. albicans persisters were detected only in biofilms and not in exponentially growing or stationary-phase planktonic populations. Reinoculation of cells that survived killing of the biofilm by amphotericin B produced a new biofilm with a new subpopulation of persisters. This suggests that C. albicans persisters are not mutants but phenotypic variants of the wild type. Using a stain for dead cells, rare dark cells were visible in a biofilm after amphotericin B treatment, and a bright and a dim population were physically sorted from this biofilm. Only the dim cells produced colonies, showing that this method allows the isolation of yeast persisters. Given that persisters formed only in biofilms, mutants defective in biofilm formation were examined for tolerance of amphotericin B. All of the known mutants affected in biofilm formation were able to produce normal levels of persisters. This finding indicates that attachment rather than formation of a complex biofilm architecture initiates persister formation. Bacteria produce multidrug-tolerant persister cells in both planktonic and biofilm populations, and it appears that yeasts and bacteria have evolved analogous strategies that assign the function of survival to a small part of the population. In bacteria, persisters are dormant cells. It remains to be seen whether attachment initiates dormancy that leads to the formation of fungal persisters. This study suggests that persisters may be largely responsible for the multidrug tolerance of fungal biofilms.

The Effect of Temperature on the Antimicrobial Activity of Optisol-GS

To determine the survival of different bacteria inoculated in Optisol-GS at refrigerated storage temperature (6 degrees C) and after subsequent warming to room temperature (19-22 degrees C). Staphylococcus aureus, Enterococcus faecium, Streptococcus pneumoniae, and Pseudomonas aeruginosa were chosen from stock clinical isolates for inclusion in the study. The first group consisted of 12 Optisol-GS vials. The second group consisted of 12 Optisol-GS vials containing corneas inappropriate for transplantation according to the Eye Bank Association of America (EBAA) protocols. Each group was inoculated with 3 concentrations of approximately 10, 10, and 10 colony-forming units (CFU)/mL of each bacterial species and then refrigerated per EBAA protocol. After 48 hours of refrigeration, all vials were placed at room temperature (RT) and counts were performed at 48, 50 (2 hour RT), 54 (6 hour RT), 60 (12 hour RT), 72 (24 hour RT), and 96 (48 hour RT) hours. At 96 hours, the corneal tissue from 10 and 10 inocula were cultured. All samples underwent serial dilution, spiral plating on blood agar plates, and incubation at 35 degrees C. Viable colony counts were determined at 24 hours. Except for the 10 CFU/mL inocula of P. aeruginosa, all isolates were viable after 48 hours of refrigeration. Rapid bactericidal activity was observed against P. aeruginosa after 2 hours at RT, with complete sterilization by 6 hours. The rate and extent of killing against S. aureus were influenced by the initial inoculum. Bactericidal activity was achieved after 2 hours at RT with 10 CFU/mL of S. aureus versus 24 hours with the 10 inoculum. Of note, bactericidal activity was not observed against S. pneumoniae and E. faecium following 24 hours of storage at RT. The presence of corneal tissue did not affect viable counts, with counts from corneal tissue cultures reflecting the counts seen from Optisol-GS after 48 hours at RT. The antimicrobial activity of Optisol-GS was reduced at refrigerated temperature and enhanced at RT. Bactericidal activity was not observed against E. faecium at either refrigerated temperature or RT.

Significance of low-level resistance to ciprofloxacin in Klebsiella pneumoniae and the effect of increased dosage of ciprofloxacin in vivo using the rat granuloma pouch model.

This study was designed to compare the killing effect of ciprofloxacin on strains of Klebsiella pneumoniae with different MICs of ciprofloxacin in vivo using the rat granuloma pouch infection model. Five different strains were used: one ciprofloxacin-susceptible strain (MIC 0.06 mg/L); one strain highly resistant to ciprofloxacin (MIC 8 mg/L); and three nalidixic acid-resistant strains with low-level resistance to ciprofloxacin (MIC 0.25-0.5 mg/L). The efficacy of ciprofloxacin was evaluated 3 h after bacterial challenge (treating an acute infection) or after 3 days (treating a late infection) with a single intraperitoneal injection of ciprofloxacin (40 and 200 mg/kg). Ciprofloxacin was bactericidal against both growing K. pneumoniae (acute infection model) and non-growing K. pneumoniae (late infection model), but the extent of killing was significantly higher on growing bacteria and against ciprofloxacin-susceptible K. pneumoniae. A peak concentration of ciprofloxacin, at the infection site, <3 x MIC was not sufficient for optimal bacterial elimination. However, it was possible to compensate for the lower killing in low-level ciprofloxacin-resistant K. pneumoniae by increasing the dosage of ciprofloxacin from 40 to 200 mg/kg, consistent with the higher MIC.

Pharmacodynamic studies of trovafloxacin and grepafloxacin in vitro against Gram-positive and Gram-negative bacteria.

Grepafloxacin and trovafloxacin are two novel fluoroquinolones with extended Gram-positive bacterial spectra compared with older quinolones. The aim of the present study was to investigate the different pharmacodynamic parameters of grepafloxacin in comparison with those of trovafloxacin. The following studies were performed against various Gram-positive and Gram-negative bacteria: (i) determination of the rate and extent of killing at a concentration corresponding to the 1 h non-protein-bound human serum level following an oral dose of 800 mg grepafloxacin and 300 mg trovafloxacin; (ii) determination of the rate and extent of killing of the two quinolones at different concentrations; (iii) determination of the post-antibiotic effects (PAEs); (iv) determination of the post-antibiotic sub-MIC effects (PA SMEs); (iv) determination of the rate and extent of killing in an in vitro kinetic model. It was shown that both grepafloxacin and trovafloxacin exhibited concentration-dependent killing against both Gram-positive and Gram-negative bacteria. Grepafloxacin exhibited a slower bactericidal effect against all the Gram-positive strains investigated in comparison with trovafloxacin in spite of a similar C(max)/MIC in the static experiments and a similar AUC/MIC ratio in the kinetic experiments. No major differences in the extent and rate of killing were noted against the Gram-negative strains, which were killed almost completely after 3 h except for Pseudomonas aeruginosa. A PAE of both quinolones was noted for all strains investigated. Trovafloxacin induced longer PAEs against the Gram-positive strains but shorter PAEs in comparison with those of grepafloxacin against the Gram-negative strains. A prolonging of the PAEs was noted for all bacteria when exposed to sub-MICs in the post-antibiotic phase. With a similar AUC/MIC of 310 for the penicillin-sensitive strain of Streptococcus pneumoniae and 143 for the penicillin-resistant strain, the time for 99.9% eradication for both strains was 2 h for trovafloxacin and 6 h for grepafloxacin.

Comparison of the effects of bax-expression in yeast under fermentative and respiratory conditions: investigation of the role of adenine nucleotides carrier and cytochrome c

A new system for bax-expression in yeast has been devised to investigate bax’s effect under fermentative and respiro-fermentative conditions. This has allowed us to show unambiguously that the ability of bax to kill yeast is higher under respiratory conditions than under purely fermentative conditions. The extent of killing under respiro-fermentative conditions (non-repressive sugars) is intermediate. It has been proposed that the two proteins adenine nucleotides carrier (ANC) and cytochrome c play a crucial role in bax-induced cell death. We have investigated the effects of deletion of the genes encoding the two proteins on the toxicity induced by bax, using this new system. The absence of ANC did not modify bax-induced lethality in any way. Moreover, the absence of cytochrome c also did not prevent bax-induced death. Only the kinetics of lethality were altered. All these effects are prevented by co-expression of bcl-x L.