In Vitro Analysis of Finasteride Activity against Candida albicans Urinary Biofilm Formation and Filamentation

Candida albicans is the 3rd most common cause of catheter-associated urinary tract infections, with a strong propensity to form drug-resistant catheter-related biofilms. Due to the limited efficacy of available antifungals against biofilms, drug repurposing has been investigated in order to identify novel agents with activities against fungal biofilms. Finasteride is a 5-α-reductase inhibitor commonly used for the treatment of benign prostatic hyperplasia, with activity against human type II and III isoenzymes. We analyzed the Candida Genome Database and identified a C. albicans homolog of type III 5-α-reductase, Dfg10p, which shares 27% sequence identity and 41% similarity to the human type III 5-α-reductase. Thus, we investigated finasteride for activity against C. albicans urinary biofilms, alone and in combination with amphotericin B or fluconazole. Finasteride alone was highly effective in the prevention of C. albicans biofilm formation at doses of ≥16 mg/liter and the treatment of preformed biofilms at doses of ≥128 mg/liter. In biofilm checkerboard analyses, finasteride exhibited synergistic activity in the prevention of biofilm formation in a combination of 4 mg/liter finasteride with 2 mg/liter fluconazole. Finasteride inhibited filamentation, thus suggesting a potential mechanism of action. These results indicate that finasteride alone is highly active in the prevention of C. albicans urinary biofilms in vitro and has synergistic activity in combination with fluconazole. Further investigation of the clinical utility of finasteride in the prevention of urinary candidiasis is warranted.     

A case of bilateral emphysematous pyelonephritis caused by Candida albicans

Emphysematous pyelonephritis (EPN) is a life-threatening renal infection characterized by the formation of gas within the renal parenchyma and collecting duct system, as well as perinephric tissues. We herein report a case of bilateral EPN accompanied by the urinary tract infection caused by spherical growth of Candida albicans in a patient with underlying diabetes mellitus and prostate cancer. The diagnosis was assisted by computed tomography, urography, and gram staining. Despite immediate percutaneous catheter drainage and a 4-week course of antifungal treatment, the C. albicans infection was refractory, as indicated by continuous isolation from the urine, and the patient eventually died. A local autopsy of urinary organs revealed C. albicans in the renal tissue. EPN caused by Candida species frequently occurs in men, and almost all cases have underlying poorly controlled diabetes. This condition is subject to delayed diagnosis, which may lead to extended disease and high mortality. Candida species should be considered as causative microorganism for refractory EPN in patients with poorly controlled diabetes who are receiving antibiotic treatment. Gram staining may contribute to an early diagnosis of EPN caused by Candida species, which may require long-term antifungal therapy.     

Colonization of the Intestinal Tract of Conventional Mice with Candida albicans and Treatment with Antifungal Agents

Conventional mice inoculated with Candida albicans per os were unable to maintain this organism in the intestinal tract as judged by decreasing numbers of yeast recoverable from feces. After inoculation with 10⁷ cells/mouse, fecal counts ranged from 10⁵ cells per g of feces to 5 × 10³ cells per g of feces during a 12-day experimental period. Addition of various antibiotics to the drinking water did not result in any improvement in maintenance or stability of the gut population. A combination of X irradiation and administration of tobramycin or gentamicin, however, resulted in a stable population of C. albicans in the intestinal tract, with cell counts in the feces remaining constant at a level of about 10⁶/g of feces for a period of 10 to 15 days. The usefulness of this model in assessing the effect of experimental drugs on C. albicans infections of the gut was demonstrated by the fact that treatment with a new antifungal antibiotic (A9145), amphotericin B, 5-fluorocytosine, or nystatin resulted in a reduction in the fecal counts of C. albicans from experimentally infected animals.     

In vitro and in vivo antibacterial activity of nitrofurantoin against clinical isolates of E. coli in Japan and evaluation of biological cost of nitrofurantoin resistant strains using a mouse urinary tract infection model

IntroductionNitrofurantoin is a well-established antibiotic, and is an important first-line oral treatment for uncomplicated urinary tract infections. However, little information is available with respect to its antibacterial activity in Japan, in vivo efficacy, or the in vivo biological cost of resistant strains.MethodsWe compared the susceptibility of six representative antibacterial agents—nitrofurantoin, sulfamethoxazole/trimethoprim, fosfomycin, mecillinam, ciprofloxacin, and cefdinir—against E. coli clinically isolated in Japan during 2017. We evaluated the in vivo efficacy of nitrofurantoin using a model of mouse urinary tract infection caused by ciprofloxacin resistant E. coli. We obtained nitrofurantoin resistant isolates through tests generating spontaneous mutations, and assessed the in vivo fitness of nitrofurantoin resistant isolates.ResultsThe MIC₉₀ of nitrofurantoin was 16 μg/mL, and was the lowest among the drugs tested. It was found that, in the mouse urinary tract infection model, 30 mg/kg and 100 mg/kg of nitrofurantoin reduced the count of viable bacterial cells in the kidney, while 100 mg/kg of ciprofloxacin did not. All spontaneous bacterial mutants resistant to nitrofurantoin had deletions in the nfsA gene, and we found that the resistant strain had lower growth in the mouse urinary tract infection model than in the parent strain.ConclusionsWe demonstrated promising in vitro and in vivo activity of nitrofurantoin against E. coli clinical isolates in Japan, and lower in vivo fitness of the resistant strain of nitrofurantoin.     

Antimicrobial Photodynamic Inactivation Inhibits Candida albicans Virulence Factors and Reduces In Vivo Pathogenicity

The objective of this study was to evaluate whether Candida albicans exhibits altered pathogenicity characteristics following sublethal antimicrobial photodynamic inactivation (APDI) and if such alterations are maintained in the daughter cells. C. albicans was exposed to sublethal APDI by using methylene blue (MB) as a photosensitizer (0.05 mM) combined with a GaAlAs diode laser (λ 660 nm, 75 mW/cm², 9 to 27 J/cm²). In vitro, we evaluated APDI effects on C. albicans growth, germ tube formation, sensitivity to oxidative and osmotic stress, cell wall integrity, and fluconazole susceptibility. In vivo, we evaluated C. albicans pathogenicity with a mouse model of systemic infection. Animal survival was evaluated daily. Sublethal MB-mediated APDI reduced the growth rate and the ability of C. albicans to form germ tubes compared to untreated cells (P < 0.05). Survival of mice systemically infected with C. albicans pretreated with APDI was significantly increased compared to mice infected with untreated yeast (P < 0.05). APDI increased C. albicans sensitivity to sodium dodecyl sulfate, caffeine, and hydrogen peroxide. The MIC for fluconazole for C. albicans was also reduced following sublethal MB-mediated APDI. However, none of those pathogenic parameters was altered in daughter cells of C. albicans submitted to APDI. These data suggest that APDI may inhibit virulence factors and reduce in vivo pathogenicity of C. albicans. The absence of alterations in daughter cells indicates that APDI effects are transitory. The MIC reduction for fluconazole following APDI suggests that this antifungal could be combined with APDI to treat C. albicans infections.     

Quinacrine Inhibits Candida albicans Growth and Filamentation at Neutral pH

Candida albicans is a common cause of catheter-related bloodstream infections (CR-BSI), in part due to its strong propensity to form biofilms. Drug repurposing is an approach that might identify agents that are able to overcome antifungal drug resistance within biofilms. Quinacrine (QNC) is clinically active against the eukaryotic protozoan parasites Plasmodium and Giardia. We sought to investigate the antifungal activity of QNC against C. albicans biofilms. C. albicans biofilms were incubated with QNC at serially increasing concentrations (4 to 2,048 μg/ml) and assessed using a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay in a static microplate model. Combinations of QNC and standard antifungals were assayed using biofilm checkerboard analyses. To define a mechanism of action, QNC was assessed for the inhibition of filamentation, effects on endocytosis, and pH-dependent activity. High-dose QNC was effective for the prevention and treatment of C. albicans biofilms in vitro. QNC with fluconazole had no interaction, while the combination of QNC and either caspofungin or amphotericin B demonstrated synergy. QNC was most active against planktonic growth at alkaline pH. QNC dramatically inhibited filamentation. QNC accumulated within vacuoles as expected and caused defects in endocytosis. A tetracycline-regulated VMA3 mutant lacking vacuolar ATPase (V-ATPase) function demonstrated increased susceptibility to QNC. These experiments indicate that QNC is active against C. albicans growth in a pH-dependent manner. Although QNC activity is not biofilm specific, QNC is effective in the prevention and treatment of biofilms. QNC antibiofilm activity likely occurs via several independent mechanisms: vacuolar alkalinization, inhibition of endocytosis, and impaired filamentation. Further investigation of QNC for the treatment and prevention of biofilm-related Candida CR-BSI is warranted.     

Fluoroquinolone and Third-Generation-Cephalosporin Resistance among Hospitalized Patients with Urinary Tract Infections Due to Escherichia coli: Do Rates Vary by Hospital Characteristics and Geographic Region?

This analysis of nearly 10,000 hospital-associated urinary tract infection (UTI) episodes due to Escherichia coli showed that fluoroquinolone and third-generation-cephalosporin resistance rates were 34.5% and 8.6%, respectively; the rate of concurrent resistance to both agents was 7.3%. Fluoroquinolone resistance rates exceeded 25% regardless of geographic location or hospital characteristics. The findings suggest that fluoroquinolones should be reserved and third-generation cephalosporins be used with caution as empirical agents for hospitalized patients with UTIs due to E. coli.     

Synergistic Activity of the Tyrocidines,Antimicrobial Cyclodecapeptides from Bacillus aneurinolyticus,with Amphotericin B and Caspofungin against Candida albicans Biofilms

Tyrocidines are cationic cyclodecapeptides from Bacillus aneurinolyticus that are characterized by potent antibacterial and antimalarial activities. In this study, we show that various tyrocidines have significant activity against planktonic Candida albicans in the low-micromolar range. These tyrocidines also prevented C. albicans biofilm formation in vitro. Studies with the membrane-impermeable dye propidium iodide showed that the tyrocidines disrupt the membrane integrity of mature C. albicans biofilm cells. This membrane activity correlated with the permeabilization and rapid lysis of model fungal membranes containing phosphatidylcholine and ergosterol (70:30 ratio) induced by the tyrocidines. The tyrocidines exhibited pronounced synergistic biofilm-eradicating activity in combination with two key antifungal drugs, amphotericin B and caspofungin. Using a Caenorhabditis elegans infection model, we found that tyrocidine A potentiated the activity of caspofungin. Therefore, tyrocidines are promising candidates for further research as antifungal drugs and as agents for combinatorial treatment.     

Emergence of Ciprofloxacin Resistance in Escherichia coli Isolates after Widespread Use of Fluoroquinolones

The evolution (from 1990 to 1996) of fluoroquinolone consumption and resistance and the current patterns of fluoroquinolone usage were examined in a 250-bed community hospital in Spain. Fluoroquinolone consumption increased from 1392 g in 1990 to 3203 g in 1996 (p < 0.05). A significant increase in ciprofloxacin resistance was observed in Escherichia coli isolated from urine samples (from 3 to 20%, p < 0.00001), but not in those E. coli isolated from blood or other sample cultures. In 69 randomly selected clinical charts, fluoroquinolone was used as prophylaxis, empirical therapy, and specific directed therapy in 20%, 65%, and 15%, respectively. Evaluation of quinolone indication was: first choice agents (29%), alternative agents (49%), experimental agents (4.3%) and, agents with no role (1.4%). Our study shows that the increase in the use of fluoroquinolones is associated with the emergence of ciprofloxacin-resistant E. coli from urinary tract sources. Based on their indications, current quinolone usage can be greatly reduced.     

High-Throughput Screening of a Collection of Known Pharmacologically Active Small Compounds for Identification of Candida albicans Biofilm Inhibitors

Candida albicans is the most common etiologic agent of systemic fungal infections with unacceptably high mortality rates. The existing arsenal of antifungal drugs is very limited and is particularly ineffective against C. albicans biofilms. To address the unmet need for novel antifungals, particularly those active against biofilms, we have screened a small molecule library consisting of 1,200 off-patent drugs already approved by the Food and Drug Administration (FDA), the Prestwick Chemical Library, to identify inhibitors of C. albicans biofilm formation. According to their pharmacological applications that are currently known, we classified these bioactive compounds as antifungal drugs, as antimicrobials/antiseptics, or as miscellaneous drugs, which we considered to be drugs with no previously characterized antifungal activity. Using a 96-well microtiter plate-based high-content screening assay, we identified 38 pharmacologically active agents that inhibit C. albicans biofilm formation. These drugs were subsequently tested for their potency and efficacy against preformed biofilms, and we identified three drugs with novel antifungal activity. Thus, repurposing FDA-approved drugs opens up a valuable new avenue for identification and potentially rapid development of antifungal agents, which are urgently needed.     

Fungal β-1,3-Glucan Increases Ofloxacin Tolerance of Escherichia coli in a Polymicrobial E. coli/Candida albicans Biofilm

In the past, biofilm-related research has focused mainly on axenic biofilms. However, in nature, biofilms are often composed of multiple species, and the resulting polymicrobial interactions influence industrially and clinically relevant outcomes such as performance and drug resistance. In this study, we show that Escherichia coli does not affect Candida albicans tolerance to amphotericin or caspofungin in an E. coli/C. albicans biofilm. In contrast, ofloxacin tolerance of E. coli is significantly increased in a polymicrobial E. coli/C. albicans biofilm compared to its tolerance in an axenic E. coli biofilm. The increased ofloxacin tolerance of E. coli is mainly biofilm specific, as ofloxacin tolerance of E. coli is less pronounced in polymicrobial E. coli/C. albicans planktonic cultures. Moreover, we found that ofloxacin tolerance of E. coli decreased significantly when E. coli/C. albicans biofilms were treated with matrix-degrading enzymes such as the β-1,3-glucan-degrading enzyme lyticase. In line with a role for β-1,3-glucan in mediating ofloxacin tolerance of E. coli in a biofilm, we found that ofloxacin tolerance of E. coli increased even more in E. coli/C. albicans biofilms consisting of a high-β-1,3-glucan-producing C. albicans mutant. In addition, exogenous addition of laminarin, a polysaccharide composed mainly of poly-β-1,3-glucan, to an E. coli biofilm also resulted in increased ofloxacin tolerance. All these data indicate that β-1,3-glucan from C. albicans increases ofloxacin tolerance of E. coli in an E. coli/C. albicans biofilm.     

évaluation de l’activité antifongique de différents extraits de la cannelle de Chine (Cinnamomum cassia)

The incidence of invasive fungal infections due to Candida albicans has dramatically increased since 25 years. The amphotericin B remains the best treatment despite its severe toxicity. Our work is inscribed in the frame of finding of new natural antifungals agents from a condiment widely used in our diet: the Chinese cinnamon (Cinnamomum cassia). This study is focused on the qualitative determination of different families of secondary metabolites from the bark of Cinnamon. It is also focused on assessing the antifungal activity of some extracts of Cinnamon. The plant material was extracted by exhaustion using increased polarity solvents (chloroform, ethyl acetate, methanol and water). We made five exhaustions for each solvent, each one was tested separately. The phytochemical study revealed the presence of terpenes, alkaloids and polyphenols mainly represented by flavonoids. Evaluation of antimicrobial activity of the various extracts was carried out against references yeasts strains Candida albicans ATCC 10231 and Candida albicans 444IP. Results showed that in the exception of aqueous extracts, all other extracts have an interesting activity, with inhibition zone diameters between 19 and 60 mm for the chloroform extract. Similar results were obtained for the other organic extracts. Indeed, extracts obtained from low or medium polarity solvents are the most active. In addition, the MIC and MFC of the first fraction of the chloroform extract were respectively 0.10 and 0.20 μg/ml against Candida albicans ATCC 10231. They remain lower than those of amphotericin B against the same strain (MIC = 0.2 μg/ml and MFC = 0.4 μg/ml).     

Antifungal Lock Therapy

The widespread use of intravascular devices, such as central venous and hemodialysis catheters, in the past 2 decades has paralleled the increasing incidence of catheter-related bloodstream infections (CR-BSIs). Candida albicans is the fourth leading cause of hospital-associated BSIs. The propensity of C. albicans to form biofilms on these catheters has made these infections difficult to treat due to multiple factors, including increased resistance to antifungal agents. Thus, curing CR-BSIs caused by Candida species usually requires catheter removal in addition to systemic antifungal therapy. Alternatively, antimicrobial lock therapy has received significant interest and shown promise as a strategy to treat CR-BSIs due to Candida species. The existing in vitro, animal, and patient data for treatment of Candida-related CR-BSIs are reviewed. The most promising antifungal lock therapy (AfLT) strategies include use of amphotericin, ethanol, or echinocandins. Clinical trials are needed to further define the safety and efficacy of AfLT.     

In Vitro Antifungal Activities of a Series of Dication-Substituted Carbazoles,Furans, and Benzimidazoles

Aromatic dicationic compounds possess antimicrobial activity against a wide range of eucaryotic pathogens, and in the present study an examination of the structures-functions of a series of compounds against fungi was performed. Sixty-seven dicationic molecules were screened for their inhibitory and fungicidal activities against Candida albicans and Cryptococcus neoformans. The MICs of a large number of compounds were comparable to those of the standard antifungal drugs amphotericin B and fluconazole. Unlike fluconazole, potent inhibitory compounds in this series were found to have excellent fungicidal activities. The MIC of one of the most potent compounds against C. albicans was 0.39 μg/ml, and it was the most potent compound against C. neoformans (MIC, ≤0.09 μg/ml). Selected compounds were also found to be active against Aspergillus fumigatus, Fusarium solani, Candida species other than C. albicans, and fluconazole-resistant strains of C. albicans and C. neoformans. Since some of these compounds have been safely given to animals, these classes of molecules have the potential to be developed as antifungal agents.The incidence of fungal infections in the immunocompromised population has significantly increased over the past two decades. Frequent infections caused by molds which may be primarily resistant to azoles and azole-resistant isolates of Candida albicans and Cryptococcus neoformans, which have developed recently, have increasingly been reported (7, 13, 23, 30). In light of these developments, new antifungal agents with various mechanisms of action and fungicidal activities are needed for the effective management of these clinically important infections.Recently, we reported on the antifungal activities of analogues and metabolites of pentamidine and a series of dicationic substituted bis-benzimidazoles (11). Those in vitro studies uncovered a number of compounds with potent activity against C. albicans and C. neoformans. Several compounds were found to have inhibitory activity against these two fungi more potent than that of either fluconazole or amphotericin B. In addition, the dicationic molecules, unlike fluconazole, proved to have potent fungicidal activity, with the most potent compounds having minimum fungicidal concentrations (MFCs) below 1.0 μg/ml. These initial studies also found that dicationic molecules were effective against Aspergillus fumigatus, Fusarium solani, several Candida species other than C. albicans, and fluconazole-resistant strains of C. albicans and C. neoformans.On the basis of the initial promising results reported above, the current work expands our studies on the antifungal activities of dication-substituted molecules by screening 67 additional compounds against C. albicans and C. neoformans. The criteria used to choose the structures for the current studies were based on years of testing dicationic molecules against the fungus Pneumocystis carinii in a rat model of disease (5, 14, 17, 18, 24, 26–28). Compounds in the current studies include molecules with the cationic moieties linked by carbazole, furan, and benzimidazole bridges. In addition to screening all compounds against C. albicans and C. neoformans, selected compounds were tested against other yeasts, molds, and azole-resistant strains of C. albicans and C. neoformans.     

α–Intercalated cells defend the urinary system from bacterial infection

α–Intercalated cells (A-ICs) within the collecting duct of the kidney are critical for acid-base homeostasis. Here, we have shown that A-ICs also serve as both sentinels and effectors in the defense against urinary infections. In a murine urinary tract infection model, A-ICs bound uropathogenic E. coli and responded by acidifying the urine and secreting the bacteriostatic protein lipocalin 2 (LCN2; also known as NGAL). A-IC–dependent LCN2 secretion required TLR4, as mice expressing an LPS-insensitive form of TLR4 expressed reduced levels of LCN2. The presence of LCN2 in urine was both necessary and sufficient to control the urinary tract infection through iron sequestration, even in the harsh condition of urine acidification. In mice lacking A-ICs, both urinary LCN2 and urinary acidification were reduced, and consequently bacterial clearance was limited. Together these results indicate that A-ICs, which are known to regulate acid-base metabolism, are also critical for urinary defense against pathogenic bacteria. They respond to both cystitis and pyelonephritis by delivering bacteriostatic chemical agents to the lower urinary system.     

Development and validation of a user-friendly risk nomogram for the prediction of catheter-associated urinary tract infection in neuro-intensive care patients

ObjectiveThis study aimed to develop a user-friendly nomogram model to evaluate the risk of catheter-associated urinary tract infections in neuro-critically ill patients.MethodsA retrospective cohort analysis was conducted on 537 patients with indwelling catheters admitted to the neuro-intensive care unit. Patients’ general information, laboratory examination findings, and clinical characteristics were collected. Multivariate regression analysis was applied to develop the nomogram for the prediction of catheter-associated urinary tract infections in this group of patients. The discriminative capacity, calibration ability, and clinical effectiveness of the nomogram were evaluated.ResultsThe occurrence of catheter-associated urinary tract infections was 3.91 % and Escherichia coli was the major causative pathogen. Multivariate regression analysis showed that age ≥ 60 years (odds ratio: 35.2, 95 % confidence interval: 2.3–550.8), epilepsy (39.3, 5.1–301.4), a length of neuro-intensive care stay > 30 days (272.2, 8.3–8963.5), and low albumin levels (<35 g/L) (12.1, 2.1–69.9) were independent risk factors associated with catheter-associated urinary tract infection in neuro-intensive care patients. The nomogram demonstrated good calibration and discrimination in both the training and the validation sets. The model exhibited good clinical use since the decision curve analysis covered a large threshold probability.ConclusionsWe developed a user-friendly nomogram to predict catheter-associated urinary tract ibfection in neuro-intensive care patients. The nomogram incorporated clinical variables collected on admission (age, admission diagnosis, and albumin levels) and the length of stay and enabled the effective prediction of the likelihood of catheter-associated urinary tract infections.