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The weaker diffusion of echinocandins in the peritoneal fluid (PF) could promote Candida-resistant isolates. The aim of this study was to analyze the pharmacokinetics (PK)/pharmacodynamics (PD) of caspofungin in plasma and PF samples from liver transplant recipients. Liver transplant patients received caspofungin as postoperative prophylaxis. Caspofungin concentrations were quantified in plasma and PF samples on days 1, 3, and 8. Data were analyzed using nonlinear mixed-effect modeling and Monte Carlo simulations. Area under the curve (AUC) values for plasma and PF were simulated under three dosing regimens. Probabilities of target attainment (PTAs) were calculated using area under the unbound plasma concentration-time curve from 0 to 24 h at steady state (fAUC(0-24))/MIC ratios, with MICs ranging from 0.008 to 8 mg/L. All of the patients included were monitored weekly for Candida colonization and for Candida infections. Twenty patients were included. The median daily dose of caspofungin was 0.81 mg/kg. Plasma (n = 395) and PF (n = 50) concentrations at steady state were available. A two-compartment model with first-order absorption and elimination was described. Our two-compartment model with first-order absorption and elimination produced an effective PK/PD relationship in plasma, achieving a PTA of ≥90% with MICs ranging from 0.008 to 0.12 mg/L for Candida albicans and Candida glabrata. In PF, PTAs at D8 were optimal only for a MIC of 0.008 mg/L in patients weighing 60 kg under the three dosing regimens. Among the 16 patients colonized, all MIC values were below the maximal concentration (C(max)) in plasma but not in PF. PF concentrations of caspofungin were low. Simulations showed that the PTAs for Candida spp. in PF were not optimal, which might suggest a potential risk of resistance.

Ethionamide is recommended as part of regimens to treat multidrug-resistant and rifampicin-resistant tuberculosis. This study was conducted to (i) describe the distribution of ethionamide MICs, (ii) describe the pharmacokinetics of ethionamide, and (iii) determine the probability of attaining target area under the concentration-time curve from 0 to 24 h (AUC(0–24))/MIC values associated with suppression of resistant subpopulation and microbial kill. Participants received 15 to 20 mg of drug/kg of body weight of ethionamide daily (in 500- or 750-mg doses) as part of a multidrug regimen. Pretreatment MICs of ethionamide for Mycobacterium tuberculosis sputum isolates were determined using Sensititre MYCOTB MIC plates. Plasma concentrations of ethionamide (measured predose and at 2, 4, 6, 8, and 10 h postdose) were available for 84 patients. A one-compartment disposition model, including a liver compartment capturing hepatic extraction, best described ethionamide pharmacokinetics. Clearance and volume were allometrically scaled using fat-free mass. Isoniazid coadministration reduced ethionamide clearance by 31%, resulting in a 44% increase in AUC(0–24). The median (range) MIC (n = 111) was 2.5 mg/liter (40 mg/liter). Simulations showed increased daily doses of ethionamide (1,250 mg, 1,500 mg, and 1,750 mg for patients weighing ≤45 kg, 46 to 70 kg, and >70 kg, respectively) resulted in the probability of attaining an area under the concentration-time curve from 0 to 24 h for the free, unbound fraction of a drug (fAUC(0–24))/MIC ratio of ≥42 in more than 90% of patients only at the lowest MIC of 0.3 mg/liter. The WHO-recommended doses of ethionamide do not achieve target concentrations even for the lowest MIC measured in the cohort.

Use of carbapenem antibiotics to treat infections caused by Enterobacterales expressing increasingly aggressive extended-spectrum β-lactamases (ESBL) has contributed to the emergence of carbapenem resistance. Enmetazobactam is a novel ESBL inhibitor being developed in combination with cefepime as a carbapenem-sparing option for infections caused by ESBL-producing Enterobacterales. Cefepime-enmetazobactam checkerboard minimum inhibitory concentration (MIC) profiles were obtained for a challenge panel of cefepime-resistant ESBL-producing clinical isolates of Klebsiella pneumoniae. Sigmoid Emax modelling described cefepime MIC as a function of enmetazobactam concentration with no bias. A concentration of 8 μg/ml enmetazobactam proved sufficient to restore >95% of cefepime antibacterial activity in vitro against >95% of isolates tested. These results support a fixed concentration of 8 μg/ml of enmetazobactam for MIC testing.

Ciprofloxacin is one of the most widely used antibiotics for treating Pseudomonas aeruginosa infections. However P. aeruginosa acquires mutations that confer ciprofloxacin resistance, making treatment more difficult. Resistance is multifactorial, with mutations in multiple genes influencing the resistance phenotype. However the contributions of individual mutations and mutation combinations to the amounts of ciprofloxacin that P. aeruginosa can tolerate are not well understood. Engineering P. aeruginosa strain PAO1 to contain mutations in any one of the resistance-associated genes gyrA, nfxB, rnfC, parC and parE showed that only gyrA mutations increased the Minimum Inhibitory Concentration (MIC) for ciprofloxacin. Mutations in parC and parE increased the MIC of a gyrA mutant, making the bacteria ciprofloxacin resistant. Mutations in nfxB and rnfC increased the MIC, conferring resistance, only if both were mutated in a gyrA background. Mutations in all of gyrA, nfxB, rnfC and parC/E further increased the MIC. These findings reveal an epistatic network of gene-gene interactions in ciprofloxacin resistance. We used this information to predict ciprofloxacin resistance/susceptibility for 274 isolates of P. aeruginosa from their genome sequences. Antibiotic susceptibility profiles were predicted correctly for 84% of the isolates. The majority of isolates for which prediction was unsuccessful were ciprofloxacin resistant, demonstrating the involvement of additional as yet unidentified genes and mutations in resistance. Our data show that gene-gene interactions can play an important role in antibiotic resistance and can be successfully incorporated into models predicting resistance phenotype.

Candida auris is a novel Candida species that has spread in all continents causing nosocomial outbreaks of invasive candidiasis. C. auris has the ability to develop resistance to all antifungal drug classes. Notably, many C. auris isolates are resistant to the azole drug fluconazole, a standard therapy of invasive candidiasis.Azole resistance in C. auris can result from mutations in the azole target gene ERG11 and/or overexpression of the efflux pump Cdr1. TAC1 is a transcription factor controlling CDR1 expression in C. albicans The role of TAC1 homologs in C. auris (TAC1a and TAC1b) remains to be better defined.In this study, we compared sequences of ERG11, TAC1a and TAC1b between a fluconazole-susceptible and five fluconazole-resistant C. auris isolates of clade IV. Among four of the resistant isolates, we identified a similar genotype with concomitant mutations in ERG11 (F444L) and TAC1b (S611P). The simultaneous deletion of tandemly arranged TAC1a/TAC1b resulted in a decrease of minimal inhibitory concentration (MIC) for fluconazole. Introduction of the ERG11 and TAC1b mutations separately and/or combined in the wild-type azole susceptible isolate resulted in a significant increase of azole resistance with a cumulative effect of the two combined mutations. Interestingly, CDR1 expression was not significantly affected by TAC1a/TAC1b deletion or by the presence of the TAC1b S611P mutation, suggesting the existence of Tac1-dependent and Cdr1-independent azole resistance mechanisms.We demonstrated the role of two previously unreported mutations responsible for azole resistance in C. auris, which were a common signature among four azole-resistant isolates of clade IV.

OPS-2071 is a novel quinolone antibacterial agent characterized by low oral absorption that reduces the risk of adverse events typical of fluoroquinolone class antibiotics. The in vitro and in vivo antibacterial activities of OPS-2071 against Clostridioides difficile were evaluated in comparison to vancomycin and fidaxomicin., OPS-2071 is a novel quinolone antibacterial agent characterized by low oral absorption that reduces the risk of adverse events typical of fluoroquinolone class antibiotics. The in vitro and in vivo antibacterial activities of OPS-2071 against Clostridioides difficile were evaluated in comparison to vancomycin and fidaxomicin. OPS-2071 exhibited potent antibacterial activity against 54 clinically isolated C. difficile strains with a MIC of 0.125 μg/ml (MIC50) and 0.5 μg/ml (MIC90), making it more active than vancomycin on a concentration basis (MIC50, 2 μg/ml; MIC90, 4 μg/ml) and comparable to fidaxomicin (MIC50, 0.063 μg/ml; MIC90, 8 μg/ml). OPS-2071 showed equally potent antibacterial activity against both hypervirulent and nonhypervirulent strains, while a significant difference in susceptibility to fidaxomicin was observed. Spontaneous resistance to OPS-2071 and vancomycin was not observed; however, resistance to fidaxomicin was observed at 4× MIC. The mutant prevention concentration of OPS-2071 was 16-fold lower than those of fidaxomicin and vancomycin, and the postantibiotic effect of OPS-2071 was longer than those of fidaxomicin and vancomycin. Also, OPS-2071 showed low systemic exposure, with OPS-2071 having 2.9% oral bioavailability at 1 mg/kg in rats. Furthermore, OPS-2071 showed significant in vivo efficacy at 0.0313 mg/kg/day (50% effective doses), 39.0-fold and 52.1-fold lower than those of vancomycin and fidaxomicin, respectively, in a hamster model of C. difficile infection. OPS-2071 has the potential to become a new therapeutic option for treating C. difficile infection.

Pseudomonas aeruginosa bacteremia is an infection associated with a high mortality rate. Piperacillin-tazobactam is a β-lactam–β-lactamase inhibitor combination that is frequently used for the management of Pseudomonas aeruginosa infections. The pharmacokinetic-pharmacodynamic index associated with in vitro maximal bacterial killing for piperacillin-tazobactam is the percentage of the time between doses at which the free fraction concentration remains above the MIC (%fT(>MIC)). However, the precise %fT(>MIC) target associated with improved clinical outcomes is unknown. The aim of this study was to investigate the correlation between the survival of patients with Pseudomonas aeruginosa bacteremia and the threshold of the piperacillin-tazobactam %fT(>MIC). This retrospective study included all adult patients hospitalized over an 82-month period with Pseudomonas aeruginosa bacteremia and treated with piperacillin-tazobactam. Patients with a polymicrobial infection or those who died within 72 h of the time of collection of a sample for culture were excluded. The %fT(>MIC) of piperacillin-tazobactam associated with in-hospital survival was derived using classification and regression tree analysis. After screening 270 patients, 78 were eligible for inclusion in the study; 18% died during hospitalization. Classification and regression tree analysis identified a %fT(>MIC) of >60.68% to be associated with improved survival, and this remained statistically significant after controlling for clinical covariates (odds ratio = 7.74, 95% confidence interval = 1.32 to 45.2). In conclusion, the findings recommend dosing of piperacillin-tazobactam with the aim of achieving a pharmacodynamic target %fT(>MIC) of at least 60% in these patients.

The antimicrobial triclosan is used in a wide range of consumer products ranging from toothpaste, cleansers, socks, and baby toys. A bacteriostatic inhibitor of fatty acid synthesis, triclosan is extremely stable and accumulates in the environment. Approximately 75% of adults in the United States have detectable levels of the compound in their urine, with a sizeable fraction of individuals (>10%) having urine concentrations equal to or greater than the minimal inhibitory concentration for Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). Previous work has identified connections between defects in fatty acid synthesis and accumulation of the alarmone guanosine tetraphosphate (ppGpp), which has been repeatedly associated with antibiotic tolerance and persistence. Based on these data, we hypothesized that triclosan exposure may inadvertently drive bacteria into a state in which they are able to tolerate normally lethal concentrations of antibiotics. Here we report that clinically relevant concentrations of triclosan increased E. coli and MRSA tolerance to bactericidal antibiotics as much as 10,000-fold in vitro and reduced antibiotic efficacy up to 100-fold in a mouse urinary tract infection model. Genetic analysis indicated that triclosan-mediated antibiotic tolerance requires ppGpp synthesis but is independent of growth. These data highlight an unexpected and certainly unintended consequence of adding high concentrations of antimicrobials in consumer products, supporting an urgent need to reevaluate the costs and benefits of the prophylactic use of triclosan and other bacteriostatic compounds.

A recent surveillance study in South Korea revealed that 14% (7/50) of Aspergillus flavus clinical isolates had a voriconazole minimum inhibitory concentration of ≥4 μg/ml. Of seven non-wild-type (non-WT) isolates, six ear isolates from four hospitals shared the same microsatellite genotype. None of the non-WT isolates showed cyp51 mutations associated with azole resistance. However, the mean expression levels of efflux pump (MDR2, atrF, and mfs1) and target (cyp51A) genes exhibited significant differences between non-WT and other isolates.

Stool samples from 38 travelers returning from India were screened for extended-spectrum cephalosporin- and carbapenem-resistant Enterobacteriaceae implementing standard selective plates. Twenty-six (76.3%) people were colonized with CTX-M or DHA producers, but none of the strains was colistin resistant and/or mcr-1 positive. Nevertheless, using overnight enrichment and CHROMagar Orientation plates supplemented with colistin, four people (10.5%) were found to be colonized with colistin-resistant Escherichia coli . One cephalosporin-susceptible sequence type 10 (ST10) strain carried a 4,211-bp IS Apl1-mcr-1 -IS Apl1 element in an IncHI2 plasmid backbone.

Solithromycin (CEM-101) is a novel fluoroketolide antimicrobial agent with activity against typical and atypical pathogens associated with community-acquired bacterial pneumonia. Using a neutropenic murine lung infection model, the objectives of this study were to identify the pharmacokinetic/pharmacodynamic (PK/PD) index most closely associated with efficacy and the magnitude of such indices associated with solithromycin efficacy against Streptococcus pneumoniae. Plasma and epithelial lining fluid (ELF) samples for pharmacokinetics (PK) were collected serially over 24 hours from healthy mice administered single doses of solithromycin (0.625 to 40 mg/kg). Neutropenic CD-1 mice infected with 10(8) CFUs of one of five S. pneumoniae isolates were administered solithromycin (0.156 to 160 mg/kg/day) via oral gavage. Doses were administered in a fractionated manner for mice infected with one isolate, while mice infected with the remaining four isolates received solithromycin as either a regimen every 6 hours or every 12 hours. A three-compartment model best described solithromycin PK in the plasma and ELF (r(2) = 0.935 and 0.831, respectively). The ratio of total-drug ELF to free-drug plasma area under the concentration-time curve (AUC) from time 0 to 24 hours was 2.7. Free-drug plasma and total-drug ELF AUC to minimum inhibitory concentration ratios (AUC/MIC ratios) were most predictive of efficacy (r(2) = 0.851 and 0.850, respectively). The magnitude of free-drug plasma/total-drug ELF AUC/MIC ratios associated with net bacterial stasis and a 1- and 2-log(10) CFU reduction from baseline was 1.65/1.26, 6.31/15.1, and 12.8/59.8, respectively. These data provided dose selection support for solithromycin for clinical trials in patients with community-acquired bacterial pneumonia.

The determination of antibiotic potency against bacterial strains by assessment of their minimum inhibitory concentration normally uses a standardized broth microdilution assay procedure developed more than 50 years ago. However, certain antibiotics require modified assay conditions in order to observe optimal activity., The determination of antibiotic potency against bacterial strains by assessment of their minimum inhibitory concentration normally uses a standardized broth microdilution assay procedure developed more than 50 years ago. However, certain antibiotics require modified assay conditions in order to observe optimal activity. For example, daptomycin requires medium supplemented with Ca2+, and the lipoglycopeptides dalbavancin and oritavancin require Tween 80 to be added to the growth medium to prevent the depletion of free drug via adsorption to the plastic microplate. In this report, we examine systematically the effects of several different plate types on microdilution broth MIC values for a set of antibiotics against Gram-positive and Gram-negative bacteria, both in medium alone and in medium supplemented with the commonly used additives Tween 80, lysed horse blood, and 50% human serum. We observed very significant differences in measured MICs (up to 100-fold) for some lipophilic antibiotics, such as the Gram-positive lipoglycopeptide dalbavancin and the Gram-negative lipopeptide polymyxins, and found that nonspecific binding plates can replace the need for surfactant additives. Microtiter plate types and any additives should be specified when reporting broth dilution MIC values, as results can vary dramatically for some classes of antibiotics.

Oxazolidinones are efficacious in treating mycobacterial infections, including tuberculosis (TB) caused by drug-resistant Mycobacterium tuberculosis . In this study, we compared the in vitro activities and MIC distributions of delpazolid, a novel oxazolidinone, and linezolid against multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) in China. Additionally, genetic mutations in 23S rRNA, rplC , and rplD genes were analyzed to reveal potential mechanisms underlying the observed oxazolidinone resistance. A total of 240 M. tuberculosis isolates were included in this study, including 120 MDR-TB isolates and 120 XDR-TB isolates. Overall, linezolid and delpazolid MIC 90 values for M. tuberculosis isolates were 0.25 mg/liter and 0.5 mg/liter, respectively. Based on visual inspection, we tentatively set epidemiological cutoff (ECOFF) values for MIC determinations for linezolid and delpazolid at 1.0 mg/liter and 2.0 mg/liter, respectively. Although no significant difference in resistance rates was observed between linezolid and delpazolid among XDR-TB isolates ( P > 0.05), statistical analysis revealed a significantly greater proportion of linezolid-resistant isolates than delpazolid-resistant isolates within the MDR-TB group ( P = 0.036). Seven (53.85%) of 13 linezolid-resistant isolates were found to harbor mutations within the three target genes. Additionally, 1 isolate exhibited an amino acid substitution (Arg126His) within the protein encoded by rplD that contributed to high-level resistance to linezolid (MIC of >16 mg/liter), compared to a delpazolid MIC of 0.25. In conclusion, in vitro susceptibility testing revealed that delpazolid antibacterial activity was comparable to that of linezolid. A novel mutation within rplD that endowed M. tuberculosis with linezolid, but not delpazolid, resistance was identified.

Broth microdilution was compared with tube macrodilution and a simplified population analysis agar method for evaluating vancomycin and teicoplanin MICs and detecting glycopeptide-intermediate isolates of Staphylococcus aureus . Modal vancomycin and teicoplanin MICs recorded by tube macrodilution and the agar plate assay, which both used inocula of 10 6 CFU, were significantly higher (2 μg/ml) against a panel of borderline glycopeptide-susceptible and glycopeptide-intermediate methicillin-resistant S. aureus (MRSA) bloodstream isolates compared to broth microdilution (1 μg/ml). Vancomycin and teicoplanin MIC distributions by tube macrodilution and agar testing were also markedly different from those evaluated by broth microdilution. The 20-fold-lower inoculum size used for broth microdilution compared to macrodilution and agar MIC assays explained in part, but not entirely, the systematic trend toward lower vancomycin and teicoplanin MICs by microdilution compared to other methods. Broth microdilution assay led to underdetection of the vancomycin-intermediate S. aureus (VISA) phenotype, yielding only three VISA isolates, for which vancomycin MICs were 4 μg/ml compared to 8 and 19 VISA isolates detected by macrodilution and agar testing, respectively. While macrodilution and agar testing detected 7 and 22 isolates with elevated teicoplanin MICs (8 μg/ml), respectively, broth microdilution failed to detect such isolates. Detection rates of isolates with elevated vancomycin and teicoplanin MICs by macrodilution and agar testing assays were higher at 48 h than at 24 h. In conclusion, the sensitivity of broth microdilution MIC testing is questionable for reliable detection and epidemiological surveys of glycopeptide-intermediate resistance in S. aureus isolates.

We evaluated the in vitro activities of anidulafungin, micafungin, and caspofungin againstCandida kruseiby determining MIC and minimum fungicidal concentration (MFC) measurements and by the time-kill method. The geometric mean (GM)-MIC/GM-MFC values were 0.1/0.34, 0.25/0.44, and 1/2.29, respectively. The mean times to reach 99.9% growth reduction were 19.1 ± 18.2 h (mean ± standard deviation) for 2 mg/liter anidulafungin, 37.4 ± 8.8 h for 2 mg/liter caspofungin, and 30.7 ± 12.2 h for 1 mg/liter micafungin. Anidulafungin exhibited the highest time-kill rate, followed by micafungin. The three echinocandins showed fungicidal activity at concentrations reached in serum.

Retapamulin, the first pleuromutilin antimicrobial agent approved for the topical treatment of skin infections in humans, was tested against 987 clinical isolates representing 30 species and/or resistance groups. MICs were determined along with disk diffusion zone diameters using a 2-μg disk. Population distribution and MIC versus disk zone diameter scattergrams were analyzed to determine microbiological MIC cutoff values and inhibition zone correlates. Minimum bactericidal concentrations were performed on a smaller subset of key species. The retapamulin MIC 90 against 234 Staphylococcus aureus isolates and 110 coagulase-negative staphylococci was 0.12 μg/ml. Retapamulin MIC 90 s ranged from 0.03 to 0.06 μg/ml against beta-hemolytic streptococci including 102 Streptococcus pyogenes , 103 Streptococcus agalactiae , 59 group C Streptococcus , and 71 group G Streptococcus isolates. The MIC 90 against 55 viridans group streptococci was 0.25 μg/ml. Retapamulin had very little activity against 151 gram-negative bacilli and most of the Enterococcus species tested. Based on the data from this study, for staphylococci, MICs of ≤0.5, 1, and ≥2 μg/ml with corresponding disk diffusion values of ≥20 mm, 17 to 19 mm, and ≤16 mm can be proposed for susceptible, intermediate, and resistant microbiological cutoffs, respectively. For beta-hemolytic streptococci, a susceptible-only MIC of ≤0.25 μg/ml with a corresponding disk diffusion value of ≥15 mm can be proposed for susceptible-only microbiological cutoffs.

Mutations in two specific regions of the Fks1 subunit of 1,3-β- d -glucan synthase are known to confer decreased caspofungin susceptibility on Candida spp. Clinical isolates of Candida spp. (404 Candida albicans , 62 C. tropicalis , and 21 C. krusei isolates) sent to the French National Reference Center were prospectively screened for susceptibility to caspofungin in vitro by the broth microdilution reference method of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing (AFST-EUCAST). Twenty-eight isolates (25 C. albicans , 2 C. tropicalis , and 1 C. krusei isolate) for which the caspofungin MIC was above the MIC that inhibited 90% of the isolates of the corresponding species (MIC 90 ) were subjected to molecular analysis in order to identify mutations in the fks1 gene. Substitutions in the deduced protein sequence of Fks1 were found for 8 isolates, and 20 isolates had the wild-type sequence. Among the six C. albicans isolates harboring mutations, six patterns were observed involving amino acid changes at positions 641, 645, 649, and 1358. For C. tropicalis , one isolate showed an L644W mutation, and for one C. krusei isolate, two mutations, L658W and L701M, were found. Two media, RPMI medium and AM3, were tested for their abilities to distinguish between isolates with wild-type Fks1 and those with mutant Fks1. In RPMI medium, caspofungin MICs ranged from 0.25 to 2 μg/ml for wild-type isolates and from 1 to 8 μg/ml for mutant isolates. A sharper difference was observed in AM3: all wild-type isolates were inhibited by 0.25 μg/ml of caspofungin, while caspofungin MICs for all mutant isolates were ≥0.5 μg/ml. These data demonstrate that clinical isolates of C. albicans , C. tropicalis , and C. krusei with decreased susceptibility to caspofungin in vitro have diverse mutations in the fks1 gene and that AM3 is potentially a better medium than RPMI for distinguishing between mutant and wild-type isolates using the AFST-EUCAST method.

Against 447 anaerobe strains, the investigational carbapenem doripenem had an MIC50of 0.125 μg/ml and an MIC90of 1 μg/ml. Results were similar to those for imipenem, meropenem, and ertapenem. Time-kill studies showed that doripenem had very good bactericidal activity compared to other carbapenems, with 99.9% killing of 11 strains at 2× MIC after 48 h.

Contains fulltext : 53715.pdf (Publisher’s version ) (Closed access) We report on the correlation of the outcomes for two cohorts of patients who had been treated for candidemia (126 episodes) or oropharyngeal candidiasis (110 episodes) with various doses of fluconazole and the MIC of fluconazole obtained by using the EUCAST standard for fermentative yeasts. Of 145 episodes caused by an isolate with a fluconazole MIC < or =2 mg/liter, 93.7% (136 of 145) responded to fluconazole treatment. The response for those infected with a strain with a MIC of 4 mg/liter was 66% but reached 100% when the dose was greater than 100 mg/day, whereas the response for those infected with strains with MICs > or =8 mg/liter was only 12%. Hence, a MIC of 2 mg/liter or 4 mg/liter was able to predict successful treatment. A cure rate of 93.9% (140 of 149) was achieved when the dose/MIC ratio was > or =100 but fell to 14.6% (16 of 109) when the ratio was less. The dose/MIC required to achieve a response rate of 50% (the 50% effective concentration) was 43.7 for the cohort of patients with oropharyngeal candidiasis. Classification and regression analysis indicated that a dose/MIC of 35.5 was the threshold for the prediction of cure or failure. However, an increase in exposure above this threshold further increased the probability of cure, and all patients were cured when the dose/MIC exceeded 100. Monte Carlo simulations showed a probability of target attainment of 99% at MICs < or =2 mg/liter and a pharmacodynamic target of a dose/MIC ratio of 100, which was equivalent to an unbound fraction of the fluconazole area under the curve versus the MIC of 79.

The present study tested in vitro susceptibility of Candida bloodstream isolates to fluconazole to determine if the ratio of the fluconazole area under the concentration-time curve (AUC) or weight-normalized daily dose (dose wn ) to MIC correlated with mortality. Fluconazole susceptibility and outcome data were determined for 77 patients with a positive Candida blood culture between 2002 and 2005. The most commonly isolated Candida species were C. albicans (64%), C. glabrata (14%), C. parapsilosis (8%), C. tropicalis (6%), and C. lusitaniae (4%). Only two isolates were classified as fluconazole resistant by the CLSI M27-A2 method. Fluconazole MICs were highest against C. glabrata relative to other Candida species. Overall the crude mortality assessed at hospital discharge was 19.4% ( n = 15). Mortality rates by species were as follows: C. albicans , 16.3%; C. glabrata , 36.4%; C. parapsilosis , 0%; C. tropicalis , 0%; C. lusitaniae , 33.3%. A mortality rate of 50% was noted among patients infected with nonsusceptible isolates (MIC ≥ 16 μg/ml) compared to 18% for patients infected with susceptible (MIC ≤ 8 μg/ml) isolates ( P = 0.17). The fluconazole dose wn /MIC (24-h) values were significantly higher for the 62 survivors (13.3 ± 10.5 [mean ± standard deviation]) compared to the 15 nonsurvivors (7.0 ± 8.0) ( P = 0.03). The fluconazole AUC/MIC (24 h) values also trended higher for survivors (775 ± 739) compared to nonsurvivors (589 ± 715) ( P = 0.09). These data support the dose-dependent properties of fluconazole. Underdosing fluconazole against less-susceptible Candida isolates has the potential to increase the risk of mortality associated with candidemia.

Treatment of the fully vancomycin-susceptible Staphylococcus aureus strain COL with subinhibitory concentrations of vancomycin allowed its continued growth but generated a phenotype reminiscent of some S. aureus isolates with vancomycin-intermediate S. aureus (VISA)-type resistance: the bacteria grew in multicellular clusters; electron microscopy showed inhibition of cell separation and accumulation of amorphous cell wall-like material at the bacterial surface. Titration of free vancomycin showed a gradual disappearance of the drug from the medium, which—eventually—coincided with an increase in the growth rate, burst in viable titer, and dispersal of cellular clusters. Addition of inhibitory concentrations of vancomycin to the same strain at a higher cell concentration caused a very different—antibiotic-tolerant—response: an immediate halt in growth, followed by a prolonged lag, during which there was neither a loss of viable titer or optical density nor a change in cell morphology but a gradual removal of vancomycin from the medium to the cell wall of the bacterium, from which the antibiotic could be recovered in a biologically active form. Eventually, the drug-treated culture resumed normal growth. The transient appearance of both the VISA phenotype and vancomycin tolerance could be traced to the inhibition of the autolytic system of the bacterium by vancomycin molecules attached to the cell wall, blocking the access of a staphylococcal murein hydrolase(s) to its cell wall substrate.

Suppression of resistance in a dense Pseudomonas aeruginosa population has previously been shown with optimized quinolone exposures. However, the relevance to β-lactams is unknown. We investigated the bactericidal activity of meropenem and its propensity to suppress P. aeruginosa resistance in an in vitro hollow-fiber infection model (HFIM). Two isogenic strains of P. aeruginosa (wild type and an AmpC stably derepressed mutant [MIC = 1 mg/liter]) were used. An HFIM inoculated with approximately 1 × 10 8 CFU/ml of bacteria was subjected to various meropenem exposures. Maintenance doses were given every 8 h to simulate the maximum concentration achieved after a 1-g dose in all regimens, but escalating unbound minimum concentrations ( C mi n s) were simulated with different clearances. Serial samples were obtained over 5 days to quantify the meropenem concentrations, the total bacterial population, and subpopulations with reduced susceptibilities to meropenem (>3× the MIC). For both strains, a significant bacterial burden reduction was seen with all regimens at 24 h. Regrowth was apparent after 3 days, with the C min /MIC ratio being ≤1.7 (time above the MIC, 100%). Selective amplification of subpopulations with reduced susceptibilities to meropenem was suppressed with a C min /MIC of ≥6.2 or by adding tobramycin to meropenem ( C min /MIC = 1.7). Investigations that were longer than 24 h and that used high inocula may be necessary to fully evaluate the relationship between drug exposures and the likelihood of resistance suppression. These results suggest that the C min /MIC of meropenem can be optimized to suppress the emergence of non-plasmid-mediated P. aeruginosa resistance. Our in vitro data support the use of an extended duration of meropenem infusion for the treatment of severe nosocomial infections in combination with an aminoglycoside.

The present study, using an in vitro model, assessed telithromycin pharmacodynamic activity at simulated clinically achievable free-drug concentrations in serum (S) and epithelial lining fluid (ELF) against efflux ( mefE )-producing macrolide-resistant Streptococcus pneumoniae . Two macrolide-susceptible (PCR negative for both mefE and ermB ) and 11 efflux-producing macrolide-resistant [PCR-positive for mefE and negative for ermB ) S. pneumoniae strains with various telithromycin MICs (0.015 to 1 μg/ml) were tested. The steady-state pharmacokinetics of telithromycin were modeled, simulating a dosage of 800 mg orally once daily administered at time 0 and at 24 h (free-drug maximum concentration [ C max ] in serum, 0.7 μg/ml; half-life [ t 1/2 ], 10 h; free-drug C max in ELF, 6.0 μg/ml; t 1/2 , 10 h). Starting inocula were 10 6 CFU/ml in Mueller-Hinton Broth with 2% lysed horse blood. Sampling at 0, 2, 4, 6, 12, 24, and 48 h assessed the extent of bacterial killing (decrease in log 10 CFU/ml versus initial inoculum). Free-telithromycin concentrations in serum achieved in the model were C max 0.9 ± 0.08 μg/ml, area under the curve to MIC (AUC 0-24 h ) 6.4 ± 1.5 μg · h/ml, and t 1/2 of 10.6 ± 0.6 h. Telithromycin-free ELF concentrations achieved in the model were C max 6.6 ± 0.8 μg/ml, AUC 0-24 h 45.5 ± 5.5 μg · h/ml, and t 1/2 of 10.5 ± 1.7 h. Free-telithromycin S and ELF concentrations rapidly eradicated efflux-producing macrolide-resistant S. pneumoniae with telithromycin MICs up to and including 0.25 μg/ml and 1 μg/ml, respectively. Free-telithromycin S and ELF concentrations simulating C max /MIC ≥ 3.5 and AUC 0-24 h /MIC ≥ 25 completely eradicated (≥4 log 10 killing) macrolide-resistant S. pneumoniae at 24 and 48 h. Free-telithromycin concentrations in serum simulating C max /MIC ≥ 1.8 and AUC 0-24 h /MIC ≥ 12.5 were bacteriostatic (0.1 to 0.2 log 10 killing) against macrolide-resistant S. pneumoniae at 24 and 48 h. In conclusion, free-telithromycin concentrations in serum and ELF simulating C max /MIC ≥ 3.5 and AUC 0-24 h /MIC ≥ 25 completely eradicated (≥4 log 10 killing) macrolide-resistant S. pneumoniae at 24 and 48 h.

Sulfonamide resistance in meningococci is associated with mutations in the chromosomal gene folP , which encodes dihydropteroate synthase. Several mutations associated with resistance have been previously described, including amino acid substitutions at codons 31 and 194, a glycine-serine insertion at codons 195 and 196, and, recently, an additional mutation at nucleotide 682 (C682A). In this study, sulfisoxazole MICs were determined for 424 geographically diverse clinical isolates of Neisseria meningitidis , including all major subtypes. A subset of 134 isolates with MICs ranging from 0.5 to >64 μg/ml were assayed for the C682A mutation by real-time PCR, and 25 isolates were selected for folP gene sequencing. All isolates for which the sulfisoxazole MIC was ≥8 possessed the C682A mutation by real-time PCR or folP sequencing, and 34 of 35 isolates with a MIC of ≤2 lacked this mutation. Of 16 sequenced isolates for which the sulfisoxazole MIC was ≥4, 15 possessed previously described mutations, including 10 at codon 31, 1 at codon 194, and 4 with the 2-amino-acid insertion codons 195 and 196; all 16 possessed the C682A mutation. The C682A mutation predicted elevated sulfonamides MICs for a large number of geographically diverse clinical isolates of meningococci. Detection of this mutation by real-time PCR or other methods may allow more wide-scale detection of meningococcal isolates with for which the sulfonamide MICs are elevated without resorting to multiple assays or folP gene sequencing, providing a simple, high-throughput screening method for use in public health and epidemiologic settings.

In a previous study tolerance to amphotericin B (AMB) was found among Candida parapsilosis and C. dubliniensis strains by seeding the whole volumes of wells used for MIC determinations, and minimum fungicidal concentrations (MFC) for non- C. albicans Candida strains were demonstrated to be above the levels safely achievable in serum. As an extension of that study, we performed time-kill assays with 26 blood culture isolates (6 C. albicans , 5 C. parapsilosis , 5 C. krusei , 4 C. glabrata , 3 C. lusitaniae , and 3 C. tropicalis isolates), 3 oropharyngeal C. dubliniensis isolates, 3 AMB-susceptible isolates (ATCC 90028, ATCC 22019, ATCC 6254), and 6 AMB-resistant isolates (ATCC 200955, ATCC 200956, ATCC 200950, ATCC 200951, ATCC 200952, ATCC 200953) using RPMI 1640 medium and 0.12 to 32 μg of AMB per ml and determined the numbers of CFU per milliliter at 0, 2, 4, 8, 12, 24, and 48 h. MFCs and time-kill patterns were species specific (MFCs, ≤1 μg/ml for all C. dubliniensis and C. albicans isolates except AMB-resistant strain ATCC 200955; MFCs, 2 to >16 μg/ml for the other isolates). The times required to reach the fungicidal endpoint (99.9% killing) at four times the MIC were 2 h for C. albicans and C. dubliniensis , 16 h for C. glabrata , 24 h for C. parapsilosis and C. lusitaniae , and ≥40 h for C. tropicalis and C. krusei . The killing rate increased as the AMB concentration was increased up to 2 μg/ml. The highest killing rates were achieved for C. albicans , C. dubliniensis , and C. lusitaniae , while viable C. tropicalis, C. krusei , and C. parapsilosis cells were present after 48 h (MICs, ≤2 μg/ml) when AMB was used at 2 μg/ml. Time-kill curves and MFCs can detect viable cells after 48 h when AMB is used at ≥2 μg/ml. The failure of AMB treatment could be due to its poor killing activity against some species at the concentrations reached in patients' serum.

Previous studies have shown that there is considerable variation in the methods and media used to determine the susceptibility of Neisseria meningitidis to antimicrobial agents in different countries. In this study, national and regional reference laboratories used a standardized methodology to determine the MICs of antibiotics used in the management of meningococcal infection. Fourteen laboratories participated in the study, determining the susceptibility to penicillin G, rifampin, cefotaxime, ceftriaxone, ciprofloxacin, and ofloxacin of a collection of 17 meningococci, of which 11 strains were previously defined as having intermediate resistance to penicillin (Pen I ) by sequencing and restriction fragment length polymorphism analysis of the penA gene. The MIC was determined by agar dilution and Etest with Mueller-Hinton agar (MH), MH supplemented with sheep blood (MH+B), and MH supplemented with heated (chocolated) blood. Several laboratories encountered problems obtaining confluent growth with unsupplemented MH. MH+B was considered to give the most congruent and reproducible results among the study laboratories. The modal MIC for MH+B for each antibiotic and method was calculated to define the MIC consensus, allowing assessment of each individual laboratory's data in relation to the others. The agreement in each antibiotic/method/medium combination was defined as the percentage of laboratories with a result within one dilution of the modal result. For the whole study, an agreement of 90.6% was observed between agar dilution and Etest methods. The agreement in each laboratory/antibiotic/method combination ranged from 98.2% to 69.7%, with six laboratories demonstrating agreement higher than 90% and 11 more than 80%. The ability of the laboratories to detect the Pen I isolates ranged from 18.2% to 100%. The apparent difficulty in interpreting susceptibility to rifampin, particularly with the Etest method, is very interesting.

Daptomycin MICs at which 50% of isolates were inhibited (MIC 50 s) and MIC 90 s determined by the NCCLS broth microdilution method were both 0.25 μg/ml (range, 0.06 to 2 μg/ml) for 350 pneumococcal isolates. MICs determined by E test strips on commercially prepared Mueller-Hinton sheep blood agars with different calcium contents were 2 to 3 dilutions higher than those determined by strips that contained daptomycin plus calcium. Daptomycin zone diameters varied little on the same media.

During the last decade some studies have shown that the area under the curve (AUC)/MIC ratio is the pharmacodynamic index that best predicts the efficacies of quinolones, while other studies suggest that the predictive value of the peak concentration/MIC (peak / MIC) ratio is superior to the AUC/MIC ratio in explaining clinical and microbiological outcomes. In classical fractionated dose-response studies with animals, it is difficult to differentiate between the AUC/MIC ratio and the peak/MIC ratio because of colinearity. Three different levofloxacin and ciprofloxacin dosing regimens were studied in a neutropenic mouse pneumonia model. The different regimens were used with the aim of increasing the AUC/MIC ratio without changing the peak/MIC ratio and vice versa. The first regimen (RC) consisted of daily doses of 5 up to 160 mg/kg of body weight divided into one, two, or four doses. In the second regimen (R0), mice were given 1.25 mg/kg every hour from 1 to 23 h, while the dose given at 0 h was 2.5, 5, 10, 20, 40, or 80 mg/kg. In the third regimen (R11), mice also received 1.25 mg/kg every hour from 0 to 23 h; but in addition, they also received 2.5, 5, 10, 20, 40, or 80 mg/kg at 11 h. The level of protein binding was also evaluated. The results indicate that the unbound fraction ( f u ) was concentration dependent for both levofloxacin and ciprofloxacin and ranged from approximately 0.67 to 0.88 for both drugs between concentrations of 0.5 and 80 mg/liter. The relationships between the AUC/MIC ratio and the number of CFU were slightly better than those between the peak/MIC ratio and the number of CFU. There was no clear relationship between the amount of time that the concentration remained above the MIC and effect ( R 2 < 0.1). For both drugs, the peak/MIC ratio that resulted in a 50% effective concentration was lower for the R0 and R11 dosing regimens, indicating the importance of the AUC/MIC ratio. The same was true for the static doses. Survival studies showed that for mice treated with the low doses the rate of survival was comparable to that for the controls, but with the higher doses the rate of survival was better for mice receiving the R0 regimen. We conclude that for quinolones the AUC/MIC ratio best correlates with efficacy against pneumococci and that the effect of the peak/MIC ratio found in some studies could be partly explained by concentration-dependent protein binding.

The new quinolone garenoxacin (BMS-284756), which lacks a C-6 fluorine, was examined for its ability to block the growth of Staphylococcus aureus . Measurement of the MIC and the mutant prevention concentration (MPC) revealed that garenoxacin was 20-fold more potent than ciprofloxacin for a variety of ciprofloxacin-susceptible isolates, some of which were resistant to methicillin. The MPC for 90% of the isolates (MPC 90 ) was below published serum drug concentrations achieved with recommended doses of garenoxacin. These in vitro observations suggest that garenoxacin has a low propensity for selective enrichment of fluoroquinolone-resistant mutants among ciprofloxacin-susceptible isolates of S. aureus . For ciprofloxacin-resistant isolates, the MIC at which 90% of the isolates tested were inhibited was below serum drug concentrations while the MPC 90 was not. Thus, for these strains, garenoxacin concentrations are expected to fall inside the mutant selection window (between the MIC and the MPC) for much of the treatment time. As a result, garenoxacin is expected to selectively enrich mutants with even lower susceptibility.

Currently, therapy of black-grain mycetoma caused by Madurella mycetomatis consists of extensive debridement of the infected tissue combined with prolonged antifungal therapy with ketoconazole or itraconazole. In the present study, the in vitro activity of the new triazole isavuconazole toward M. mycetomatis was evaluated. Isavuconazole appeared to have high activity against M. mycetomatis , with MICs ranging from ≤0.016 to 0.125 μg/ml. Due to its favorable pharmacokinetics, isavuconazole could be a promising antifungal agent in the treatment of mycetoma.

The association between macrolide resistance mechanisms and clinical outcomes remains understudied. The present study, using an in vitro pharmacodynamic model, assessed clarithromycin (CLR) activity against mef (A)-positive and erm (B)-negative Streptococcus pneumoniae isolates by simulating free-drug concentrations in serum and both total (protein-bound and free) and free drug in epithelial lining fluid (ELF). Five mef (A)-positive and erm (B)-negative strains, one mef (A)-negative and erm (B)-positive strain, and a control [ mef (A)-negative and erm (B)-negative] strain of S. pneumoniae were tested. CLR was modeled using a one-compartment model, simulating a dosage of 500 mg, per os, twice a day (in serum, free-drug C p maximum of 2 μg/ml, t 1/2 of 6 h; in ELF, C ELF(total) maximum of 35μg/ml, t 1/2 of 6 h; C ELF(free) maximum of 14 μg/ml, t 1/2 of 6 h). Starting inocula were 10 6 CFU/ml in Mueller-Hinton broth with 2% lysed horse blood. With sampling at 0, 4, 8, 12, 20, and 24 h, the extent of bacterial killing was assessed. Achieving CLR T/MIC values of ≥90% (AUC 0-24 /MIC ratio, ≥61) resulted in bacterial eradication, while T>MIC values of 40 to 56% (AUC 0-24 /MIC ratios of ≥30.5 to 38) resulted in a 1.2 to 2.0 log 10 CFU/ml decrease at 24 h compared to that for the initial inoculum. CLR T/MIC values of ≤8% (AUC 0-24 /MIC ratio, ≤17.3) resulted in a static effect or bacterial regrowth. The high drug concentrations in ELF that were obtained clinically with CLR may explain the lack of clinical failures with mef (A)-producing S. pneumoniae strains, with MICs up to 8 μg/ml. However, mef (A) isolates for which MICs are ≥16 μg/ml along with erm (B) may result in bacteriological failures.

We evaluated the in vitro activity of the new echinocandin antifungal micafungin against Candida spp. using microdilution and time-kill methods. Additionally, we examined the postantifungal effect (PAFE) of micafungin. Finally, we evaluated the effect of the addition of serum and plasma on the MIC of micafungin. Four Candida albicans isolates and two isolates of each Candida glabrata , Candida krusei , and Candida tropicalis were selected for testing. The MICs of micafungin were determined in RPMI 1640 medium buffered with morpholinepropanesulfonic acid alone and with the addition of 10, 20, and 50% human serum and plasma. MICs were determined by using two endpoints: a prominent reduction in growth (the MIC at which 80% of isolates are inhibited [MIC 80 ]) and complete visual inhibition of growth (MIC 100 ). The minimum fungicidal concentration (MFC) of micafungin for each isolate was also determined. Time-kill curves were determined for each isolate in RPMI 1640 medium with micafungin at concentrations ranging from 0.125 to 16 times the MIC 80 to assess the correlation between MIC 80 and fungicidal activity. PAFE studies were conducted with each isolate by using concentrations ranging between 0.25 and 4 times the MIC 80 . The MIC 80 s for the test isolates ranged from 0.0039 to 0.25 μg/ml. Overall, the addition of serum or plasma increased the MIC 6 to 7 doubling dilutions for C. albicans and 3 to 4 doubling dilutions for C. krusei and C. tropicalis . Micafungin time-kill studies demonstrated fungicidal activity at concentrations ranging from 4 to 16 times the MIC 80 . Micafungin is very potent agent against a variety of Candida spp., producing fungicidal activity against 7 of 10 isolates tested. A PAFE was observed against all isolates. The PAFE was influenced by the drug concentration, with the highest concentration resulting in the longest observed PAFE in each case. The highest concentration tested, four times the MIC, resulted in a PAFE of more than 9.8 h for 5 of 10 isolates tested (range, 0.9 to ≥20.1 h).

The 6-anilinouracils (AUs) constitute a new class of bactericidal antibiotics selective against gram-positive (Gr + ) organisms. The AU family of compounds specifically inhibits a novel target, replicative DNA polymerase Pol IIIC. Like other antibiotics, AUs can be expected to engender the development of resistant bacteria. We have used a representative AU and clinically relevant strains of Staphylococcus aureu s and Enterococcus to determine the frequency and mechanism(s) of resistance development. The frequency of resistance was determined by using N 3 -hydroxybutyl 6-(3′-ethyl-4′-methylanilino) uracil (HBEMAU) and commercially available antibiotics at eight times the MICs. For all five Gr + organisms tested, the frequency of resistance to HBEMAU ranged from 1 × 10 −8 to 3 × 10 −10 . The frequencies of resistance to the antibiotics tested, including rifampin, gentamicin, and ciprofloxacin, were either greater than or equal to those for HBEMAU. In order to understand the mechanism of resistance, HBEMAU-resistant organisms were isolated. MIC assays showed that the organisms had increased resistance to AU inhibitors but not to other families of antibiotics. Inhibition studies with DNA polymerases from HBEMAU-sensitive and -resistant strains demonstrated that the resistance was associated with Pol IIIC. DNA sequence analysis of the entire polC genes from both wild-type and resistant organisms revealed that the resistant organisms had a sequence change that mapped to a single amino acid codon in all strains examined.

The MICs of triclosan for 31 clinical isolates of Staphylococcus aureus were 0.016 μg/ml (24 strains), 1 to 2 μg/ml (6 strains), and 0.25 μg/ml (1 strain). All the strains for which triclosan MICs were elevated (>0.016 μg/ml) showed three- to fivefold increases in their levels of enoyl-acyl carrier protein (ACP) reductase (FabI) production. Furthermore, strains for which triclosan MICs were 1 to 2 μg/ml overexpressed FabI with an F204C alteration. Binding studies with radiolabeled NAD + demonstrated that this change prevents the formation of the stable triclosan-NAD + -FabI complex, and both this alteration and its overexpression contributed to achieving MICs of 1 to 2 μg/ml for these strains. Three novel, potent inhibitors of FabI (50% inhibitory concentrations, ≤64 nM) demonstrated up to 1,000-fold better activity than triclosan against the strains for which triclosan MICs were elevated. None of the compounds tested from this series formed a stable complex with NAD + -FabI. Consequently, although the overexpression of wild-type FabI gave rise to an increase in the MICs, as expected, overexpression of FabI with an F204C alteration did not cause an additional increase in resistance. Therefore, this work identifies the mechanisms of triclosan resistance in S. aureus , and we present three compounds from a novel chemical series of FabI inhibitors which have excellent activities against both triclosan-resistant and -sensitive clinical isolates of S. aureus .

Animal infection models have historically been used to study pharmacodynamic relationships. Similar results could theoretically be produced by using an in vitro pharmacodynamic model as an alternative to animal models. We compared the antibiotic effects of ticarcillin administered in various doses and dosing regimens against Pseudomonas aeruginosa ATCC 27853 under conditions analogous to those previously employed in a neutropenic-mouse thigh infection model (B. Vogelman et al., J. Infect. Dis. 158: 831-847, 1988). Ticarcillin dosages of either 96, 192, or 384 mg/day were administered at 1-, 2-, 3-, 4-, 8-, 12-, or 24-h intervals into a two-compartment model in order to duplicate the concentration-time profiles of the animal model. Colony counts were enumerated at 0 and 24 h. Linear regression and sigmoidal maximum-effect (Emax) model fitting were used to assess the relationship between the percentage of time that the concentration remained above the MIC (% T >MIC) or above four times the MIC (% T >4×MIC) and the change in the log 10 CFU per milliliter (Δlog 10 CFU/ml) in the central and peripheral compartments. Statistical analysis of the Δlog 10 CFU/ml values was performed for matched regimens of the in vitro and animal models based on the % T >MICs. The slopes of the regression equations of % T >MICs relative to Δlog 10 CFU/ml values were similar for the in vitro and animal models, but the y intercept was greater with the in vitro model. The Δlog 10 CFU/ml values of the 0- to 24-h colony counts at equivalent % T >MICs in the two models were not statistically different ( P = 0.087). Overall, the peripheral compartment of the in vitro model was a better predictor of effect than the central compartment. This study, which compares pharmacodynamic principles between an in vitro and an animal model, demonstrated similar relationships between % T >MICs and effects.

Linezolid is a new oxazolidinone with activity against gram-positive cocci. We determined the in vivo activity of linezolid against four strains of Staphylococcus aureus (two methicillin-susceptible S. aureus [MSSA] strains and two methicillin-resistant S. aureus strains) and one penicillin-susceptible Streptococcus pneumoniae (PSSP) strain, two penicillin-intermediate S. pneumoniae strains, and five penicillin-resistant S. pneumoniae strains. The mice had 10 6.3 to 10 7.7 CFU/thigh before therapy and were then treated for 24 h with 5 to 1,280 mg of linezolid/kg divided into 1, 2, 4, 8, or 16 doses. The killing activities after 4 h of therapy ranged from 2.4 to 5.0 log 10 CFU/thigh against S. pneumoniae and 1.35 to 2.2 log 10 CFU/thigh against S. aureus . Increasing doses produced minimal concentration-dependent killing; doses of 20 and 80 mg/kg produced no in vivo postantibiotic effects (PAEs) with PSSP and modest PAEs (3.4 and 3.2 h) with MSSA. Pharmacokinetic studies at doses of 20 and 80 mg/kg by high-pressure liquid chromatography analysis exhibited peak dose values of 0.68 and 0.71 and elimination half-lives of 1.02 and 1.00 h. Linezolid MICs ranged from 0.5 to 1.0 μg/ml for S. pneumoniae and from 1.0 to 4.0 μg/ml for S. aureus . A sigmoid dose-response model was used to estimate the dose required to achieve a net bacteriostatic effect over 24 h. Static doses against S. pneumoniae ranged from 22.2 to 97.1 mg/kg/24 h and from 133 to 167 mg/kg/24 h for S. aureus . The 24-h area under the concentration-time curve (AUC)/MIC ratio was the major parameter determining the efficacy of linezolid against PSSP ( R 2 = 82% for AUC/MIC versus 57% for T>MIC and 59% for the peak level in serum/MIC [peak/MIC]). It was difficult to determine the most relevant pharmacokinetic/pharmacodynamic parameter with S. aureus , although the outcomes correlated slightly better with the 24-h AUC/MIC ratio ( R 2 = 75%) than with the other parameters (T>MIC R 2 = 75% and peak/MIC R 2 = 65%). The 24-h AUC/MIC ratio required for a bacteriostatic effect with linezolid varied from 22 to 97 (mean = 48) for pneumococci and from 39 to 167 (mean = 83) for staphylococci. Based upon a pharmacokinetic goal of a 24-h AUC/MIC of 50 to 100, a dosage regimen of 600 mg given either intravenously or orally twice daily would achieve success against organisms with MICs as high as 2 to 4 μg/ml.

Cethromycin (ABT-773), a new ketolide, possesses potent in vitro activity against Streptococcus pneumoniae . The objective of this study was to investigate the in vivo bactericidal activity of cethromycin against macrolide-susceptible and -resistant S. pneumoniae in a murine pneumonia model and to describe the pharmacodynamic (PD) profile of cethromycin. Eight (two macrolide susceptible, six macrolide resistant) clinical isolates of S. pneumoniae were investigated. Cyclophosphamide administration rendered ICR mice transiently neutropenic prior to intratracheal inoculation with 0.05 ml of an S. pneumoniae suspension containing 10 7 to 10 8 CFU/ml. Oral cethromycin was initiated 12 to 14 h postinoculation over a dosage range of 0.1 to 800 mg/kg of body weight/day. Lungs from seven to eight mice per treatment and control groups were collected at 0 and 24 h posttherapy to assess bacterial density. The cumulative mortality ( n = 12 to 13) was assessed at 120 h (end of therapy) and at 192 h (3 days posttherapy). Recovery of pneumococci from the lungs of infected animals prior to the initiation of therapy ranged from 4.6 to 7.2 log 10 CFU. Growth in untreated control animals over a 24-h study period increased 0.3 to 2.7 log 10 CFU. Cethromycin demonstrated a substantial bactericidal effect, regardless of macrolide susceptibility. Correlation between changes in bacterial density (24 h) and survival over both 120 and 192 h were statistically significant. All three PD parameters demonstrated a significant correlation with changes in log 10 CFU/lung (Spearman's correlation coefficient, P < 0.001); however, the goodness of fit as assessed with the maximum effect ( E max ) model revealed that the maximum concentration of free drug in serum ( C max free )/MIC and the area under the free drug concentration-time curve (AUC free )/MIC best explained the relationship between drug exposure and reductions in viable bacterial counts. These data reveal that an approximate cethromycin AUC free /MIC of 50 or C max free /MIC of 1 results in bacteriostatic effects, while higher values (twofold) maximize survival.

We identified and genetically characterized seven fluoroquinolone-resistant Streptococcus pneumoniae strains among 293 clinical strains isolated from 1999 to 2001 in Japan. The resistant strains were isolated only from adults, and 7 of 31 isolates (22.6%) were from patients more than 20 years old. Resistant strains were not found in 262 isolates from children under age 10.

In this study, we propose a simple and reproducible host-cell-based assay for the screening of antimycobacterial drugs that is suitable for drug discovery. The method evaluates both antimycobacterial activity of the drugs and their cytotoxicity to host cells. The basis of this simple fibroblast-based assay (SFA) is that cells of human lung fibroblast cell line MRC-5, which are highly sensitive to mycobacterial cytotoxicity, are killed by virulent Mycobacterium tuberculosis strain H 37 Rv bacilli in response to the viability of bacilli. Clinically used antimycobacterial drugs inhibited the mycobacterial cytotoxicity to MRC-5 cells in a dose-dependent manner. MICs of isoniazid, streptomycin, rifampin, and ethambutol determined by this SFA (0.428, 1.816, 0.013, and 3.465 μg/ml, respectively) were within 1 log of MICs determined by the broth dilution test (BDT) using Middlebrook 7H9 medium. The MIC of pyrazinamide, which exhibits bactericidal activity only at a high dose by BDT (1,231 μg/ml at pH 6.6 and 492 μg/ml at pH 5.8), was 3.847 μg/ml in the modified method of SFA. On the other hand, sodium azide, a toxic agent for both mammalian cells and bacteria, exhibited cytotoxicity to fibroblasts at a dose lower than that required to inhibit mycobacterial growth. Thus, this fibroblast-based method enabled us to evaluate both antibacterial activity of drugs and their cytotoxicity to human cells within a short period of time.

ABT-773 was tested against 317 fastidious isolates; it inhibited 99% of organisms at a concentration of 4.0 μg/ml. With ampicillin-sulbactam and levofloxacin, only 2 and 6% of these strains, respectively, were resistant at the breakpoint. With clindamycin, penicillin G, and metronidazole, 22, 26, and 58% of the strains, respectively, were resistant.

Visual determination of MIC end points for azole antifungal agents can be complicated by the trailing growth phenomenon. To determine the incidence of trailing growth, we performed testing of in vitro susceptibility to fluconazole and itraconazole using the National Committee for Clinical Laboratory Standards broth microdilution M27-A reference procedure and 944 bloodstream isolates of seven Candida spp., obtained through active population-based surveillance between 1998 and 2000. Of 429 C. albicans isolates, 78 (18.2%) showed trailing growth at 48 h in tests with fluconazole, and 70 (16.3%) showed trailing in tests with itraconazole. Of 118 C. tropicalis isolates, 70 (59.3%) showed trailing growth in tests with fluconazole, and 35 (29.7%) showed trailing in tests with itraconazole. Trailing growth was not observed with any of the other five Candida spp. tested ( C. dubliniensis , C. glabrata , C. krusei , C. lusitaniae , and C. parapsilosis ). To confirm whether or not isolates that showed trailing growth in fluconazole and/or itraconazole were resistant in vitro to these agents, all isolates that showed trailing growth were retested by the sterol quantitation method, which measures cellular ergosterol content rather than growth inhibition after exposure to azoles. By this method, none of the trailing isolates was resistant in vitro to fluconazole or itraconazole. For both agents, a 24-h visual end point or a spectrophotometric end point of 50% reduction in growth relative to the growth control after 24 or 48 h of incubation correlated most closely with the result of sterol quantitation. Our results indicate that MIC results determined by either of these end point rules may be more predictive of in vivo outcome for isolates that give unclear visual end points at 48 h due to trailing growth.

Interpretive agreements among the results of fluconazole broth microdilution tests, Etests, and disk diffusion tests were documented by evaluating 495 Candida spp. Microdilution reference test results were in agreement with 96% of the Etest results; most discrepancies were minor differences. Fluconazole resistance of Candida krusei strains often required a full 48 h of incubation in order to be observed by the standard method. For the disk diffusion tests that were performed on Mueller-Hinton agar with glucose and methylene blue, 97% of results were in agreement with those of the reference test, especially when zones of inhibition were measured after the first 24 h of incubation. Some Candida glabrata isolates failed to grow satisfactorily until a full 48 h of incubation was completed. Precision was determined by testing 50 selected isolates in triplicate in each of three laboratories. The reproducibility of results of disk diffusion tests was comparable to that of the reference method. With all procedures, determination of test results was particularly challenging with some strains, and new methods are needed in order to improve endpoint definition.

Clostridium difficile is the most frequently identified enteric pathogen in patients with nosocomially acquired, antibiotic-associated diarrhea. The drugs most commonly used to treat diseases associated with C. difficile are metronidazole and vancomycin. Most clinical laboratories assume that all C. difficile isolates are susceptible to metronidazole and vancomycin. We report on the antimicrobial susceptibilities of 415 C. difficile isolates to metronidazole and vancomycin over an 8-year period (1993 to 2000). The overall rate of resistance to metronidazole at the critical breakpoint (16 μg/ml) was 6.3%. Although full resistance to vancomycin was not observed, the overall rate of intermediate resistance was 3.1%. One isolate had a combination of resistance to metronidazole and intermediate resistance to vancomycin. Rates of resistance to metronidazole and vancomycin were higher among isolates from human immunodeficiency virus-infected patients. Molecular typing methods proved the absence of clonality among the isolates with decreased susceptibilities to the antimicrobials tested.