Paradoxical Growth Effect Research Articles

Paradoxical growth effect of caspofungin on Candida spp. sessile cells not only at high drug concentrations.

According to the available literature, echinocandins display high anti-Candida spp. activity. Paradoxical growth (PG) of Candida spp. planktonic cells promoted by echinocandins is widely reported. Here we report on the ability of Candida spp. sessile cells to display PG when they are exposed to caspofungin in vitro, even at relatively low drug concentrations. Clinical significance of PG during echinocandin therapy of candidiasis remains uncertain. We assessed in vitro susceptibilities of Candida spp. sessile cells to caspofungin and analyzed the frequency of PG. The minimum inhibitory concentrations of caspofungin for sessile cells (SMICs) were determined for 70 clinical Candida spp. isolates (29 Candida albicans, 26 Candida parapsilosis, and 15 Candida glabrata isolates) and were defined as the lowest drug concentrations that resulted in at least 50% reduction in metabolic activity. PG was defined as a resurgence of growth (>50% of that in the drug-free growth control well) at drug concentrations above the MIC. The caspofungin SMICs ranged from ≤0.015 to >256 µg ml-1. We observed PG in 26.9-93.1% of isolates tested, depending on the Candida species and age of sessile cells. Antibiofilm activity of caspofungin is species-specific, and strongly strain-depending among C. albicans and C. parapsilosis isolates. Interestingly, PG was present also at relatively low caspofungin concentrations.

Understanding Echinocandin Resistance in the Emerging Pathogen Candida auris.

Candida auris has simultaneously emerged on five continents as a fungal pathogen causing nosocomial outbreaks. The challenges in the treatment of C. auris infections are the variable antifungal susceptibility profiles among clinical isolates and the development of resistance to single or multiple classes of available antifungal drugs. Here, the in vitro susceptibility to echinocandin antifungal drugs was determined and FKS1 sequencing was performed on 106 C. auris clinical isolates. Four isolates were identified to be resistant to all tested echinocandins (MIC ≥ 4 mg/liter) and harbored an S639F mutation in FKS1 hot spot region 1. All remaining isolates were FKS1 wild type (WT) and echinocandin susceptible, with micafungin being the most potent echinocandin (MIC50 = 0.125 mg/liter). Antifungal susceptibility testing with caspofungin was challenging due to the fact that all FKS1 WT isolates exhibited an Eagle effect (also known as the paradoxical growth effect), which occurred at various intensities. To assess whether the Eagle effect resulted in pharmacodynamic resistance, 8 representative isolates were evaluated for their in vivo drug response in a murine model of invasive candidiasis. All isolates were susceptible to caspofungin at a human therapeutic dose, except for those harboring the S639F mutation. The data suggest that only isolates carrying mutations in FKS1 are echinocandin resistant and that routine in vitro testing of C. auris isolates for susceptibility to caspofungin by the broth microdilution method should be viewed cautiously or avoided.

In vitro antibacterial effects of statins against bacterial pathogens causing skin infections.

With financial considerations impeding research and development of new antibiotics, drug repurposing (finding new indications for old drugs) emerges as a feasible alternative. Statins are extensively prescribed around the world to lower cholesterol, but they also possess inherent antimicrobial properties. This study identifies statins with the greatest potential to be repurposed as topical antibiotics and postulates a mechanism of action for statins' antibacterial activity. Using broth microdilution, the direct antibacterial effects of all seven parent statins currently registered for human use and three selected statin metabolites were tested against bacterial skin pathogens Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Serratia marcescens. Simvastatin and pitavastatin lactone exerted the greatest antibacterial effects (minimum inhibitory concentrations of 64 and 128μg/mL, respectively) against S. aureus. None of the statins tested were effective against E. coli, P. aeruginosa, or S. marcescens, but simvastatin hydroxy acid acid might be active against S. aureus, E. coli, and S. marcescens at drug concentrations > 256μg/mL. It was found that S. aureus may exhibit a paradoxical growth effect when exposed to simvastatin; thus, treatment failure at high drug concentrations is theoretically probable. Through structure-activity relationship analysis, we postulate that statins' antibacterial action may involve disrupting the teichoic acid structures or decreasing the number of alanine residues present on Gram-positive bacterial cell surfaces, which could reduce biofilm formation, diminish bacterial adhesion to environmental surfaces, or impede S. aureus cell division.

Potential Microbiological Effects of Higher Dosing of Echinocandins.

The antifungal "paradoxical effect" has been described as the reversal of growth inhibition at high doses of echinocandins, most usually caspofungin. This microbiological effect appears to be a cellular compensatory response to cell wall damage, resulting in alteration of cell wall content and structure as well as fungal morphology and growth. In vitro studies demonstrate this reproducible effect in a certain percentage of fungal isolates, but animal model and clinical studies are less consistent. The calcineurin and Hsp90 cell signaling pathways appear to play a major role in regulating these cellular and structural changes. Regardless of the clinical relevance of this paradoxical growth effect, understanding the specific actions of echinocandins is paramount to optimizing their use at either standard or higher dosing schemes, as well as developing future improvements in our antifungal arsenal.

Characterization of the FKBP12-Encoding Genes in Aspergillus fumigatus.

Invasive aspergillosis, largely caused by Aspergillus fumigatus, is responsible for a growing number of deaths among immunosuppressed patients. Immunosuppressants such as FK506 (tacrolimus) that target calcineurin have shown promise for antifungal drug development. FK506-binding proteins (FKBPs) form a complex with calcineurin in the presence of FK506 (FKBP12-FK506) and inhibit calcineurin activity. Research on FKBPs in fungi is limited, and none of the FKBPs have been previously characterized in A. fumigatus. We identified four orthologous genes of FKBP12, the human FK506 binding partner, in A. fumigatus and designated them fkbp12-1, fkbp12-2, fkbp12-3, and fkbp12-4. Deletional analysis of the four genes revealed that the Δfkbp12-1 strain was resistant to FK506, indicating FKBP12-1 as the key mediator of FK506-binding to calcineurin. The endogenously expressed FKBP12-1-EGFP fusion protein localized to the cytoplasm and nuclei under normal growth conditions but also to the hyphal septa following FK506 treatment, revealing its interaction with calcineurin. The FKBP12-1-EGFP fusion protein didn’t localize at the septa in the presence of FK506 in the cnaA deletion background, confirming its interaction with calcineurin. Testing of all deletion strains in the Galleria mellonella model of aspergillosis suggested that these proteins don’t play an important role in virulence. While the Δfkbp12-2 and Δfkbp12-3 strains didn’t show any discernable phenotype, the Δfkbp12-4 strain displayed slight growth defect under normal growth conditions and inhibition of the caspofungin-mediated “paradoxical growth effect” at higher concentrations of the antifungal caspofungin. Together, these results indicate that while only FKBP12-1 is the bona fide binding partner of FK506, leading to the inhibition of calcineurin in A. fumigatus, FKBP12-4 may play a role in basal growth and the caspofungin-mediated paradoxical growth response. Exploitation of differences between A. fumigatus FKBP12-1 and human FKBP12 will be critical for the generation of fungal-specific FK506 analogs to inhibit fungal calcineurin and treat invasive fungal disease.

In vitro efficacy of liposomal amphotericin B, micafungin and fluconazole against non-albicans Candida species biofilms

Non-albicans Candida species are being isolated with increasing frequency. In this study, biofilm formation by Candida tropicalis, Candida parapsilosis and Candida glabrata was evaluated and the activities of liposomal amphotericin B (LAB), micafungin (MFG) and fluconazole (FLC) against these biofilms were assessed using a clinically relevant in vitro model system. LAB exhibited strong activities against the three non-albicans Candida species and showed dose-dependent efficacy. MFG displayed a paradoxical growth effect against the C. tropicalis biofilm. FLC was ineffective for non-albicans biofilms. This study shows that Candida biofilms have unique susceptibility to LAB. The dose-dependent effects of LAB indicate that this drug may be a useful treatment for biofilm formation by non-albicans Candida species in cases in which the catheter cannot be removed for clinical reasons.

The Eagle-Like Effect of the Echinocandins: Is It Relevant for Clinical Decisions?

The Eagle effect is a paradoxical growth phenomenon long known to exist in vitro with specific antimicrobial agents, particularly the penicillins. More recent reports have indicated that this effect also exists when Candida or Aspergillus species are exposed to the echinocandin antifungals. While the majority of data for this effect exists for caspofungin, there are also reports that micafungin and anidulafungin may have a paradoxical growth effect in certain media or with particular pathogens. In efforts to reproduce the paradoxical growth curve in vivo, results with Candida have been varied. In vivo studies suggest an effect with Aspergillus, but were not reproducible in vitro. Because of these results, questions have been raised about the clinical relevance of this phenomenon. Aside from select scenarios, including late clinical failure or catheter lock therapy, current data suggest that the clinical relevance of the paradoxical growth effect is not likely to be significant.

In vitro effectiveness of Anidulafungin against Candida sp. biofilms

This study furnishes deeper insights to previous works on anidulafungin, demonstrating the potent activity against Candida strains planktonic cells and biofilms. Candida sp., associated with many biomaterial-related infections, give rise to infective pathologies typically associated with biofilm formation. We recently determined the in vitro antifungal activities of echinocandin anidulafungin in association with some antifungal drugs against some Candida strains in their planktonic states. A total of 11 Candida strains biofilms were tested in this study: six Candida albicans, three C. parapsilosis and two C. tropicalis. All yeast isolates and ATCC strains were stored at -20°C in glycerol stocks and were subcultured on antimicrobial agent-free Sabouraud dextrose agar plates. MIC endpoints were determined colorimetrically by using the indicator 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) with menadione as electron-coupling agent. The activity of anidulafungin was assessed using in vitro microbiological model relevant for clinical practice. Anidulafungin showed a strong activity in vitro against both planktonic and biofilms cells, and our study confirms that high anidulafungin concentrations might establish paradoxical growth effect in C. albicans and C. tropicalis biofilms.

Species-specific and drug-specific differences in susceptibility of Candida biofilms to echinocandins: characterization of less common bloodstream isolates.

Candida species other than Candida albicans are increasingly recognized as causes of biofilm-associated infections. This is a comprehensive study that compared the in vitro activities of all three echinocandins against biofilms formed by different common and infrequently identified Candida isolates. We determined the activities of anidulafungin (ANID), caspofungin (CAS), and micafungin (MFG) against planktonic cells and biofilms of bloodstream isolates of C. albicans (15 strains), Candida parapsilosis (6 strains), Candida lusitaniae (16 strains), Candida guilliermondii (5 strains), and Candida krusei (12 strains) by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay. Planktonic and biofilm MICs were defined as ≥ 50% fungal damage. Planktonic cells of all Candida species were susceptible to the three echinocandins, with MICs of ≤ 1 mg/liter. By comparison, differences in the MIC profiles of biofilms in response to echinocandins existed among the Candida species. Thus, C. lusitaniae and C. guilliermondii biofilms were highly recalcitrant to all echinocandins, with MICs of ≥ 32 mg/liter. In contrast, the MICs of all three echinocandins for C. albicans and C. krusei biofilms were relatively low (MICs ≤ 1 mg/liter). While echinocandins exhibited generally high MICs against C. parapsilosis biofilms, MFG exhibited the lowest MICs against these isolates (4 mg/liter). A paradoxical growth effect was observed with CAS concentrations ranging from 8 to 64 mg/liter against C. albicans and C. parapsilosis biofilms but not against C. krusei, C. lusitaniae, or C. guilliermondii. While non-albicans Candida planktonic cells were susceptible to all echinocandins, there were drug- and species-specific differences in susceptibility among biofilms of the various Candida species, with C. lusitaniae and C. guilliermondii exhibiting profiles of high MICs of the three echinocandins.

Determination of chitin content in fungal cell wall: An alternative flow cytometric method

The conventional methods used to evaluate chitin content in fungi, such as biochemical assessment of glucosamine release after acid hydrolysis or epifluorescence microscopy, are low throughput, laborious, time-consuming, and cannot evaluate a large number of cells. We developed a flow cytometric assay, efficient, and fast, based on Calcofluor White staining to measure chitin content in yeast cells. A staining index was defined, its value was directly related to chitin amount and taking into consideration the different levels of autofluorecence. Twenty-two Candida spp. and four Cryptococcus neoformans clinical isolates with distinct susceptibility profiles to caspofungin were evaluated. Candida albicans clinical isolate SC5314, and isogenic strains with deletions in chitin synthase 3 (chs3Δ/chs3Δ) and genes encoding predicted GlycosylPhosphatidylInositol (GPI)-anchored proteins (pga31Δ/Δ and pga62Δ/Δ), were used as controls. As expected, the wild-type strain displayed a significant higher chitin content (P < 0.001) than chs3Δ/chs3Δ and pga31Δ/Δ especially in the presence of caspofungin. Ca. parapsilosis, Ca. tropicalis, and Ca. albicans showed higher cell wall chitin content. Although no relationship between chitin content and antifungal drug susceptibility phenotype was found, an association was established between the paradoxical growth effect in the presence of high caspofungin concentrations and the chitin content. This novel flow cytometry protocol revealed to be a simple and reliable assay to estimate cell wall chitin content of fungi.

Changes in Cell Wall Synthesis and Ultrastructure during Paradoxical Growth Effect of Caspofungin on Four Different Candida Species

Paradoxical growth (PG) has been described for echinocandins and is characterized by cell growth at drug concentrations above the MIC. In this study, two isolates each of Candida albicans, C. tropicalis, C. orthopsilosis, and C. parapsilosis, all of which displaying PG in response to caspofungin, were subjected to MIC, minimal fungicidal concentration (MFC), and time-kill curve assays to evaluate the levels of PG. Cell wall components and ultrastructural modifications of the PG cells were also investigated. The results showed that when cell growth and survival were evaluated by MFC or time-kill curve assays, high concentrations of caspofungin did not show fungicidal activity against PG cells. Furthermore, for C. parapsilosis and C. orthopsilosis, time-kill curves were more discriminatory than MFCs in detecting the PG effect. The four different Candida species studied demonstrated similar alterations in cell wall components and ultrastructure associated with PG. In PG cells, β-1,3-glucan content decreased from 2.7- to 7.8-fold, whereas chitin content increased from 4.0- to 6.6-fold. An electron microscopy study of the PG cells revealed morphological alterations, clumping of cells, enlarged cells, the absence of filamentation, abnormal septa, and accumulation of chitin in the cell wall. Also, PG cells basically exhibited a single dark high-density layer in the cell wall, indicating the loss of the β-1,3-glucan layer. Our results present novel details about the ultrastructural alterations that occur in C. albicans, C. parapsilosis, C. orthopsilosis, and C. tropicalis during PG and show that chitin is the major component of the cell walls of PG cells. Stimulation of chitin synthesis may represent a rescue mechanism against caspofungin activity.

Frequency of paradoxical effect with caspofungin in Candida albicans

To the editor, In their article regarding the paradoxical growth effect with echinocandins, Fleischhacker et al. [1], discussing their findings with caspofungin, state: “Stevens et al. did not demonstrate [a paradoxical effect, i.e., growth in concentrations above an apparent MIC] in a ... group of C. albicans isolates...,” citing two of our papers [2, 3]. However, in our original description of the paradoxical effect with caspofungin [2], we reported the phenomenon in two sets of C. albicans isolates, 16% of 25 isolates and 18% of 17 isolates, and in the later paper studying the mechanism [3], we had expanded the study of frequency to 65 isolates, of which 22% had shown the effect. In our most recently published update [4], we reported the frequency as 17% of 102 C. albicans isolates studied at that point. We have presently studied 113 isolates, and have found the caspofungin paradoxical effect (as we defined it [2]) in 15% of C. albicans isolates. Fleischhacker et al. [1] reported it in 14% of 101 C. albicans isolates studied with caspofungin.

Stimulation of Kaposi's sarcoma cell growth by urine from women in early pregnancy, the current source for clinical-grade human chorionic gonadotropin preparations.

Clinical-grade preparations of human chorionic gonadotropin (hCG) have been shown to be toxic to Kaposi's sarcoma (KS) cells. However, the results of clinical studies using commercial hCG preparations KS remain highly contradictory. More particularly, some hCG preparations could have a paradoxical growth effect on KS. Such discrepant results may be explained by the fact that the anti-KS activity is not associated with hCG itself but with one or more factors that are co-purified with the hormone. We found here that crude urine from first trimester pregnant women, the current source for commercial hCG, had a growth stimulatory effect on KS cells. By contrast, urine from last trimester pregnant women, from non-pregnant young women, from menopausal women and from men exhibited neither a growth stimulatory nor a growth inhibitory effect on KS cells. The amplitude of this pregnancy urine-associated pro-KS activity/hCG unit was higher than that achieved with clinical-grade hCG preparations. Partial co-purification of pregnancy-associated factors during the extraction procedure of commercial hCG from urine may explain the pro-KS activity achieved with some hCG preparations. We, therefore, suggest a cautious use of hCG purified from pregnancy urine for the treatment of KS.

Comparative Activity in Vitro of Cilofungin (LY 121019) with Other Agents Used for Treatment of Deep‐seated Candida Infections Die Aktivität in vitro von Cilofungin (LY 121019) und anderen Wirkstoffen zur Behandlung tieflokalisierter Candidosen im Vergleich

Cilofungin (LY 121019) is a semi-synthetic lipopeptide antifungal agent that inhibits beta-1,3-D-glucan synthase activity in the cell wall of yeasts. Clinical strains of Candida species were tested for susceptibility to cilofungin and seven other antifungal agents. Candida albicans and Candida tropicalis were susceptible to cilofungin with mean MICs of 0.3 and 0.08 microgram/ml respectively. For most other species the mean MICs were greater than 1-2 micrograms/ml of cilofungin. Studies on the paradoxical growth effect observed with cilofungin in Sabouraud broth showed that, at high concentrations of cilofungin, sufficient damage occurred to make damaged cells highly susceptible to killing by fresh cilofungin. The damaged cells also had increased susceptibility to other antifungal agents to which they were normally resistant. These observations indicate that cilofungin may be a useful agent for the treatment of many invasive Candida infections, either alone or in combination with certain other antifungal agents.