Erika J. Ernst

Antimicrobial Resistance Trends and Outbreak Frequency in United States Hospitals

We assessed resistance rates and trends for important antimicrobial-resistant pathogens (oxacillin-resistant Staphylococcus aureus [ORSA], vancomycin-resistant Enterococcus species [VRE], ceftazidime-resistant Klebsiella species [K-ESBL], and ciprofloxacin-resistant Escherichia coli [QREC]), the frequency of outbreaks of infection with these resistant pathogens, and the measures taken to control resistance in a stratified national sample of 670 hospitals. Four hundred ninety-four (74%) of 670 surveys were returned. Resistance rates were highest for ORSA (36%), followed by VRE (10%), QREC (6%), and K-ESBL (5%). Two-thirds of hospitals reported increasing ORSA rates, whereas only 4% reported decreasing rates, and 24% reported ORSA outbreaks within the previous year. Most hospitals (87%) reported having implemented measures to rapidly detect resistance, but only approximately 50% reported having provided appropriate resources for antimicrobial resistance prevention (53%) or having implemented antimicrobial use guidelines (60%). The most common resistant pathogen in US hospitals is ORSA, which accounts for many recognized outbreaks and is increasing in frequency in most facilities. Current practices to prevent and control antimicrobial resistance are inadequate.

Influence of Test Conditions on Antifungal Time-Kill Curve Results: Proposal for Standardized Methods

ABSTRACT This study was designed to examine the effects of antifungal carryover, agitation, and starting inoculum on the results of time-kill tests conducted with various Candida species. Two isolates each of Candida albicans, Candida tropicalis, and Candida glabrata were utilized. Test antifungal agents included fluconazole, amphotericin B, and LY303366. Time-kill tests were conducted in RPMI 1640 medium buffered with morpholinepropanesulfonic acid (MOPS) to a pH of 7.0 and incubated at 35°C. Prior to testing, the existence of antifungal carryover was evaluated at antifungal concentrations ranging from 1× to 16× MIC by four plating methods: direct plating of 10, 30, and 100 μl of test suspension and filtration of 30 μl of test suspension through a 0.45-μm-pore-size filter. Time-kill curves were performed with each isolate at drug concentrations equal to 2× MIC, using a starting inoculum of approximately 105 CFU/ml, and incubated with or without agitation. Last, inoculum experiments were conducted over three ranges of starting inocula: 5 × 102 to 1 × 104, >1 × 104 to 1 × 106, and >1 × 106 to 1 × 108 CFU/ml. Significant antifungal carryover (>25% reduction in CFU/milliliter from the control value) was observed with amphotericin B and fluconazole; however, carryover was eliminated with filtration. Agitation did not appreciably affect results. The starting inoculum did not significantly affect the activity of fluconazole or amphotericin B; however, the activity of LY303366 may be influenced by the starting inoculum. Before antifungal time-kill curve methods are routinely employed by investigators, methodology should be scrutinized and standardized procedures should be developed.

Postantifungal Effects of Echinocandin, Azole, and Polyene Antifungal Agents against Candida albicans and Cryptococcus neoformans

ABSTRACT The postantifungal effect (PAFE) of fluconazole, MK-0991, LY303366, and amphotericin B was determined against isolates of Candida albicans and Cryptococcus neoformans. Concentrations ranging from 0.125 to 4 times the MIC were tested following exposure to the antifungal for 0.25 to 1 h. Combinations of azole and echinocandin antifungals (MK-0991 and LY303366) were tested againstC. neoformans. Fluconazole displayed no measurable PAFE against Candida albicans or Cryptococcus neoformans, either alone or in combination with either echinocandin antifungal. MK-0991, LY303366, and amphotericin B displayed a prolonged PAFE of greater than 12 h againstCandida spp. when tested at concentrations above the MIC for the organism and 0 to 2 h when tested at concentrations below the MIC for the organism.

In Vitro Activity of Micafungin (FK-463) against Candida spp.: Microdilution, Time-Kill, and Postantifungal-Effect Studies

ABSTRACT 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 [MIC80]) and complete visual inhibition of growth (MIC100). 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 MIC80 to assess the correlation between MIC80 and fungicidal activity. PAFE studies were conducted with each isolate by using concentrations ranging between 0.25 and 4 times the MIC80. The MIC80s 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 MIC80. 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).

Osmitopsis asteriscoides(Asteraceae)-the antimicrobial activity and essential oil composition of a Cape-Dutch remedy

The essential oil composition and antimicrobial activity of Osmitopsis asteriscoides, a medicinal plant used in traditional herbal preparations in South Africa has been investigated. Three different antimicrobial methods (disc diffusion, minimum inhibitory concentration by micro-titer plate and time-kill studies) were comparatively evaluated against Candida albicans, Staphylococcus aureus and Pseudomonas aeruginosa. A preliminary screening was done using the disc diffusion method on nine bacterial and four fungal isolates. Minimum inhibitory concentrations showed some correlation with the disc diffusion method. However, time-kill studies appear to be a more superior method for determining antimicrobial activity of volatile compounds such as essential oils. Two moderately susceptible and one resistant organism were selected to further demonstrate the variability between the three methods. The antimicrobial activity of the essential oil, tested by means of time-kill methodology at concentrations ranging from 0.5 to 2% (v/v) indicate a strong fungicidal activity against Candida albicans and the oil was also found to be bacteriostatic against Staphylococcus aureus in a concentration-dependent manner. The essential oil rapidly reduced viable counts of Pseudomonas aeruginosa, but regrowth was noted after 240 min. The results have been generated in duplicate in separate microbiology laboratories using different time-kill methods and the results are congruent. The two major essential oil components camphor and 1,8-cineole were investigated indicating the positive antimicrobial efficacy of 1,8-cineole independently and in combination with camphor. In addition to (-)-camphor and 1,8-cineole, 40 compounds were identified by GC-MS in the hydro-distilled essential oil. The high concentration of cineole and camphor and their synergistic effect is presented as a possible explanation for the traditional use of Osmitopsis asteriscoides for treating microbe-related illnesses.

Novel triazole antifungal agents

The risk of opportunistic infections is greatly increased in patients who are immunocompromised due to AIDS, cancer chemotherapy and organ or bone marrow transplantation. Candida albicans is often associated with serious systemic fungal infections, however other Candida species such as Candida krusei, Candida tropicalis and Candida glabrata, as well as Cryptococcus neoformans and filamentous fungi such as Aspergillus, have also emerged as clinically significant fungal pathogens. Two triazole antifungal agents, fluconazole and itraconazole, were introduced over a decade ago and since then have been used extensively for the prophylaxis and treatment of a variety of fungal infections. Although both drugs are effective and have their place in therapy, limitations regarding the utility of these agents do exist. For example, fluconazole is not effective for the prophylaxis or treatment of Aspergillus species and has limited activity against C. krusei and C. glabrata. The use of itraconazole has been limited secondary to concerns regarding unpredictable bioavailability. The rising incidence of fungal infections and the reported increase of non-albicans candidal infections noted over the past two decades highlight the need for new antifungal agents with improved spectra of activity. Several new triazole agents are in various phases of preclinical and clinical trials and may be available for human use in the near future. Three such agents voriconazole, posaconazole and ravuconazole are reviewed and compared with existing agents.

Evaluation of Voriconazole Pharmacodynamics Using Time-Kill Methodology

ABSTRACT Voriconazole is an investigational azole antifungal agent with activity against a variety of fungal species, including fluconazole-susceptible and -resistant Candida species andCryptococcus neoformans. In this study, we employed in vitro time-kill methods to characterize the relationship between concentrations of voriconazole and its fungistatic activity againstCandida albicans, Candida glabrata,Candida tropicalis, and C. neoformans. Isolates were exposed to voriconazole concentrations ranging from 0.0625 to 16 times the MIC, and the viable colony counts were determined over time. The 50 and 90% effective concentrations (EC50 and EC90, respectively) were determined at 8, 12, and 24 h following the addition of voriconazole. At each time point, near-maximal fungistatic activity, as indicated by the EC90, was noted at a drug concentration of approximately three times the MIC. Additionally, EC50 and EC90 did not change over time, thus suggesting that the rate of activity was not improved by increasing concentrations. Voriconazole exhibits non-concentration-dependent pharmacodynamic characteristics in vitro.

Antifungal activities of fluconazole, caspofungin (MK0991), and anidulafungin (LY 303366) alone and in combination against Candida spp. and Crytococcus neoformans via time-kill methods.

The activities of the echinocandins caspofungin and anidulafungin were evaluated alone and in combination with fluconazole using time-kill methods against isolates of Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei, and Cryptococcus neoformans. Antifungal concentrations tested against each isolate were 0.5 microg/mL and 20 microg/mL of fluconazole and 0.007 microg/mL and 2 microg/mL of both caspofungin and anidulafungin. In addition, 20 microg/mL of fluconazole was tested with 2 microg/mL of caspofungin and anidulafungin to test for additive or antagonistic activity. Finally 0.5 microg/mL of fluconazole was tested with 0.007 microg/mL of caspofungin and anidulafungin to test for synergy. Combinations of fluconazole and caspofungin or anidulafungin resulted in indifference. Azole-echinocandin combinations do not produce antagonistic effects; therefore, combinations of these agents may warrant future clinical evaluation.

Update on antifungal resistance

Overuse of antifungal agents has resulted in the selection of naturally resistant Candida species, as well as expression of resistance from previously susceptible species resulting from genetic mutations and/or selection of resistant subpopulations. Strategies for the appropriate use of antifungal agents need to be developed to prevent further development of resistance.

Investigational antifungal agents.

Several new antifungal agents, including novel compounds in familiar classes and entirely new classes targeting previously untapped mechanisms, are in various stages of the drug development process. Many new triazole antifungal agents are being studied, including voriconazole, posaconazole, and ravuconazole. The echinocandin antifungals, which represent a new class of antifungal agents, possess activity against a variety of fungal pathogens. The sodarin derivatives and nikkomycins are two additional classes of antifungals in early stages of development; future studies will determine their therapeutic usefulness.