Inefficient delivery of yeast cells as an explanation for reduced plating efficiency of Candida albicans.

The plating efficiency for fungal yeast cells is usually less than that expected from microscopic counts, and a number of explanations for this phenomenon have been proposed. The present study was undertaken to explore possible reasons for reduced plating efficiency of Candida albicans. Explanations that we evaluated and found unlikely included: ineffectiveness of different culture media and/or incubation temperatures for growing colonies, insufficient area of the plate available for expression of individual colonies, production of microcolonies, and inaccurate counting of the organisms in the inoculum. An assay for delivery of the inoculum into tissue culture plate wells indicated that reduced delivery of the organisms accounted for lower than expected plating efficiency. C. albicans yeast cells grown under low glucose conditions and expected to have reduced adhesiveness were found to have higher values for both delivery and plating efficiency in our assays. In summary, our results indicate that reduced plating efficiency for C. albicans under the conditions used for these experiments is best explained by the loss of some yeast cells during preparation of the inocula or delivery of the yeast cells onto the plates.

Effect of bone biopsy in guiding antimicrobial therapy for osteomyelitis complicating open wounds.

BACKGROUND: Osteomyelitis associated with infected overlying wounds represents a difficult diagnostic and therapeutic problem; bone biopsies can be done during debridement of the overlying wounds, but it is unclear how often the results of these bone cultures actually affect subsequent antibiotic decisions. The present study was undertaken to evaluate the usefulness of bone biopsies in guiding antibiotic therapy for this type of osteomyelitis. METHODS: Culture results of 44 bone biopsies taken during surgical debridement in 41 patients over the period from June 1994 to August 1998 were compared with those from the overlying wounds to determine whether the data affected the subsequent choice of antibiotics. The study design was that of a retrospective chart review in which the standard operative and microbiological procedures in place at the Milwaukee Veterans Affairs Medical Center were used. RESULTS: Sixty-one wound and 55 bone isolates were obtained during this study. Thirty-one isolates were found in bone, but not the overlying wound; diphtheroids were the most common organism obtained in this fashion. Correlation between wound and bone isolates was generally poor. Antibiotics were subsequently changed in 20 of the 44 cases after results of the bone biopsy became known, with the bone isolates already being covered in 10 cases and the bone biopsy results ignored in 14 cases. CONCLUSION: Because bone biopsy results seem to aid in tailoring antibiotic therapy in almost half the cases when bone is sampled during wound debridement surgery, this technique may be very helpful in certain cases and should be regularly undertaken when these procedures are carried out.

Effect of metals on Candida albicans growth in the presence of chemical chelators and human abscess fluid.

Calprotectin is a calcium- and zinc-binding protein that is present in abscess fluid supernatants and appears to inhibit microbial growth through competition for zinc. In the present study, growth inhibition by chemical chelators was compared with that produced by human abscess fluid to determine whether other chelators, perhaps with different metal specificities, would have the same or different patterns of metal reversibility as abscess fluid. Zinc was found to be more potent than the other metals tested in reversing C. albicans growth inhibition by human abscess fluid and three chemical chelators, even though in some cases the stability constants of certain of these chelators were higher for other metals. For example, in the presence of the chelator diethylenetriaminopentaacetic acid, zinc stimulated Candida growth at a 10-fold lower concentration than did iron, even though this chelator has a stability constant for iron that is almost 10(10) higher than that for zinc. These results suggest that the zinc specificity of calprotectin's C. albicans growth inhibition can best be explained by the marked sensitivity of this organism to zinc deprivation rather than by selective binding of this metal by the protein.

Zinc-reversible antimicrobial activity of recombinant calprotectin (migration inhibitory factor-related proteins 8 and 14).

Recombinant calprotectin, consisting of 2 individual peptide chains also called migration inhibitory factor-related protein (MRP)-8 and MRP14, was tested for antimicrobial activity in a Candida albicans growth inhibition assay. Both chains contain HEXXH zinc-binding sites and might be expected to manifest zinc-reversible, antimicrobial activity similar to that of native calprotectin. When tested alone, neither MRP8 nor MRP14 showed activity in the Candida growth assay. A synthetic 20-amino acid peptide containing the HEXXH sequence of MRP14, along with a nearby HHH sequence, was also inactive in this assay. However, equimolar concentrations of MRP8 and MRP14 demonstrated a potent growth inhibitory effect that was reversible by 30 microM zinc. Truncated MRP14 (missing the C-terminal GHHHKPGLGEGTP tail) used in combination with MRP8 demonstrated zinc-reversible activity that was somewhat less than that with complete MRP14. These results suggest that intact calprotectin, consisting of a heterodimer of MRP8 and MRP14, is necessary to form a zinc-binding site capable of inhibiting microbial growth.

Effect of zinc-reversible growth-inhibitory activity in human empyema fluid on antibiotic microbicidal activity.

Abscess fluid supernatants have zinc-reversible microbial growth-inhibitory activity that is mediated by calprotectin, a zinc-binding protein. Because it inhibits microbial growth, this activity might interfere with killing by antibiotics that require their target organisms to be proliferating. In the present study, we cultured bacteria in human empyema fluid and used zinc to overcome the growth-inhibitory effect of calprotectin. We then compared the effect of zinc on killing by the beta-lactams ampicillin and cefazolin with that of the fluoroquinolone trovafloxacin, since the latter may be better able to kill nonproliferating organisms. In empyema fluid diluted 1:5 in normal saline, addition of zinc (30 microM) increased growth of two strains of Staphyloccocus aureus and two strains of Escherichia coli but did not affect the MICs or MBCs of the three antibiotics in Mueller-Hinton broth. For one strain of S. aureus, no effect of zinc was found on killing by either ampicillin or cefazolin. However, with the other strain of S. aureus and both strains of E. coli, significant enhancement of killing by both drugs was observed with zinc addition. On the other hand, no effect on the killing of any of the organisms was observed for trovafloxacin when zinc was added. These results suggest that the zinc-reversible growth-inhibitory activity of abscess fluid may interfere with the microbicidal activity of antibiotics requiring proliferating target organisms, although antibiotics better able to kill nonproliferating organisms may be less affected by this phenomenon.

Analysis of fluconazole effect on Candida albicans viability during extended incubations.

Fluconazole is an azole agent with primarily fungistatic activity in standard in vitro susceptibility tests. However, recent work has demonstrated that this drug can reduce Candida albicans viability during prolonged incubations under non-growing conditions. The present study was undertaken to examine more closely some of the parameters of this killing activity. Fungicidal effects of 1.0 microg ml-1 of fluconazole were found during 7-14-day exposures in each of two media that prevented proliferation, distilled water and metal-depleted RPMI 1640 tissue-culture medium. Fluconazole appeared to be stable after being incubated at 37 degreesC for either 7 or 14 days. Strains of C. albicans resistant to fluconazole in standard short-term growth-inhibition assays were also found to be resistant to fluconazole's effect on viability in prolonged culture, suggesting similar mechanisms of action for these effects. C. albicans yeast cells pre-incubated for 7 days in distilled water were not more sensitive to the drug in short-term susceptibility assays. Although all proliferation of the organisms in distilled water cultures appeared to cease after 3 days, fluconazole added at 7 days still reduced C. albicans viability. Therefore, the drug appeared to kill the non-proliferating organisms directly rather than preventing growth and thereby the emergence of younger organisms that would live longer. Transmission electron microscopy demonstrated damage to the cell wall-cell membrane complex and interior contents of yeast cells incubated in distilled water alone; fluconazole appeared to increase the percentages of cells so affected. In summary, extended-incubation susceptibility tests demonstrated that fluconazole has direct fungicidal activity of non-proliferating C. albicans yeast cells. These results may be relevant to the manner in which this drug promotes clearance of chronic fungal infections.

Histidine-based zinc-binding sequences and the antimicrobial activity of calprotectin.

Calprotectin is a protein in neutrophil cytoplasm and abscess fluids that appears to inhibit microbial growth through competition for zinc. This study was undertaken to identify specific sites that might be responsible for the protein's zinc-binding antimicrobial activity. A review of published calprotectin amino acid sequences revealed the HEXXH motif of thermolysin-type metalloproteases and an HHH polyhistidine sequence near the C-terminus of the protein's heavy chain. Reagent polyhistidine had antimicrobial activity against Candida albicans similar to that of calprotectin. Also, one type of HEXXH-containing thermolysin was inactive in the C. albicans assay, whereas a protein tagged with six C-terminal histidines did have calprotectin-like zinc-reversible antimicrobial activity. The activity of polyhistidine, as well as that of calprotectin itself, was reversed by addition of zinc or treatment with the histidine-modifying compound diethylpyrocarbonate. These results suggest that calprotectin's antimicrobial activity may be related to certain histidine-based zinc-binding sequences.

Dermatophytes and host defence in cutaneous mycoses.

Dermatophytosis is the infection of keratinized tissues such as hair, nails and the stratum corneum of the skin by dermatophyte fungi. These fungi are onygenalean anamorphs and anamorphic species belonging to the genera Trichophyton, Microsporum and Epidermophyton. An important characteristic of the dermatophytes as parasites is their restriction to the dead keratinized tissues, except in rare cases where the patient is immunosuppressed. In contrast to many fungi, including normally non-pathogenic species, which can invade systemically in severely immunocompromised (e.g. neutropenic) patients, dermatophytes appear to be unable to cause systemic infection in this population. Thus, these fungi appear to have an unique interaction with the immune system. A better understanding of this interaction will contribute significantly to our knowledge of mammalian host defences.

Comparison of fungal viability assays using Candida albicans yeast cells undergoing prolonged incubation in the absence of nutrients.

Staining methods for determining fungal viability are usually assessed by comparisons with enumeration of colony-forming units (CFU) on solid media. The purpose of the present study was to compare viability as assessed by the acridine orange (AO) and MTT methods with the numbers of CFUs obtained for Candida albicans yeast cells undergoing prolonged incubation in distilled water. In initial assessments of the assays using various proportions of control and heat-killed C. albicans, the AO and MTT methods consistently indicated significantly higher values for viability than did CFU determinations. Experiments using organisms cultured overnight revealed that approximately 95% of the cells were capable of dividing at least once in a microscopic proliferation assay, whereas only 69% were capable of forming colonies. Parallel assays comparing AO uptake and MTT reduction gave excellent agreement with the microscopic proliferation assay, but not with CFU determinations. Using organisms undergoing prolonged incubations in distilled water, much lower viabilities were obtained with the CFU method at 7 and 10 days than with the microscopic proliferation assay or the two staining methods. These results indicate that the AO and MTT assays correlate well with the ability of C. albicans to divide at least once, but may not accurately indicate the percentage of organisms actually able to form colonies.

The use of dimethylsulfoxide for fixation of yeasts for electron microscopy.

Conventional methods of chemical fixation are often inadequate for preserving yeast ultrastructure. The thick cell wall severely limits penetration of fixatives rendering poor detail of the cell wall, membranes, and overall anatomy. Dimethylsulfoxide (DMSO) enhances penetration of chemicals and has been added to fixatives to improve cell preservation. At high concentrations (5 to 50%), however, it affects ultrastructure unpredictably. We found that adding 0.1% DMSO to fixatives greatly improved retention of yeast ultrastructure. Candida albicans, C. glabrata and Aspergillus fumigatus were fixed for 3 hr in 3% paraformaldehyde, 1% glutaraldehyde, 1 mM MgCl2, 1 mM CaCl2, 0.1% DMSO in 0.1 M sodium cacodylate buffer followed by 1% OsO4, 1% K2Cr2O7, 0.85% NaCl, 0.1% DMSO in the same buffer. Thin epoxy sections were post-stained in uranyl acetate and lead citrate. The multilayered character of the cell wall was distinct and well structured. Addition of ruthenium red or alcian blue to the fixatives further enhanced the outer fibrillar layer. The plasma membrane was contiguous and tightly adjacent to the inner mannoprotein layer of the cell wall. The cytoplasm was well preserved and the overall preservation of the yeast ultrastructure was significantly improved.

Inhibition of Candida albicans growth by calprotectin in the absence of direct contact with the organisms.

Calprotectin is a calcium- and zinc-binding protein that is present in neutrophil cytoplasm and abscess fluid supernatants. This protein appears to inhibit microbial growth through competition for zinc; however, experiments to show that calprotectin can inhibit growth of microorganisms across filter membranes have yielded conflicting results to date. To prevent recontamination of the filtrate by zinc in this type of experiment, Candida albicans was cultured on filter membranes placed on top of an agarose gel containing calprotectin. In these studies, calprotectin in the gels underneath did suppress growth on top of the filters, an effect reversible by 30 microM ZnSO4. In other experiments, the protein did not adhere to the organisms and later suppress their growth. These results indicate that calprotectin inhibits C. albicans growth in the absence of direct contact with the organisms; the findings support a zinc-deprivation mechanism of antimicrobial activity for this protein.

Effect of fluconazole on viability of Candida albicans over extended periods of time.

The treatment of chronic mycoses may expose the infecting organisms to antimicrobial agents for extended periods of time. It is possible that an azole antifungal drug such as fluconazole, with primarily fungistatic activity in standard in vitro susceptibility tests, might be able to damage the fungal cells and reduce their viability over prolonged incubations under nonproliferating conditions. To test this possibility, Candida albicans yeast cells were exposed to various concentrations of fluconazole in RPMI 1640 tissue culture medium for 4 h at 37 degrees C, washed free of the drug, and then incubated at 37 degrees C for a 28-day period; enumeration of the remaining CFU at various times during this period revealed no increased loss of viability for the fluconazole-exposed organisms. However, when fluconazole was added to the organisms maintained in distilled water (with or without pretreatment with the drug), a marked reduction of viability was found. At 14 days of incubation with two strains of C. albicans, negative cultures were found for 7 of 10 and 10 of 11 samples, respectively, containing 1.0 microgram of fluconazole per ml versus 0 of 10 and 1 of 11 control samples (P of < 0.01 and 0.001, respectively). The effect of fluconazole on fungal viability under these conditions became noticeable at approximately 7 days and was greater when the samples were incubated at 37 degrees C rather than 25 degrees C. These findings suggest that fluconazole may have fungicidal effects on fungal cells during prolonged exposures under conditions in which the organisms are prevented from proliferating by lack of nutrients.

Use of a microtiter plate assay to detect the rate of killing of adherent Candida albicans by antifungal agents.

OBJECTIVES: Candida albicans may become adherent to prosthetic devices of various kinds and thereby produce infections that are difficult to treat with standard antifungal therapy. The objective of the present work was to study the effectiveness of antifungal agents against adherent C. albicans yeast cells. STUDY DESIGN: A microtiter plate assay was developed to assess the time required for killing of the fungal cells by three antifungal agents. RESULTS: The assay initially was validated by demonstrating that the percentage of organisms adhering to the test wells was relatively constant and that exposure to the antifungal agents caused only minimal dislodgement of viable organisms from the plates. In studies that used this assay to determine the time required for killing the adherent yeast cells, chlorhexidine was found to be the most effective; in fact, in comparing the minimal lethal concentrations of the agents for exposures of 2 minutes versus 4 hours, a ratio of 2.9 was obtained for chlorhexidine versus 1050 for amphotericin B and 556 for nystatin. CONCLUSION: The microtiter plate assay used in these studies may therefore be useful as a screening test to determine which antifungal agents have the most rapid fungicidal effects on adherent fungal organisms.

Antimicrobial activity of calprotectin isolated from human empyema fluid supernatants.

Abscess and empyema fluid supernatants have zinc-reversible antimicrobial activity that is thought to be due to calprotectin, a calcium- and zinc-binding protein present within neutrophil cytoplasm. The present studies were undertaken to determine if calprotectin isolated from human empyema fluid supernatants demonstrated similar antimicrobial activity to that of the original specimens. The characteristics of the calprotectin complex on SDS-PAGE and Western blotting with specific antisera were similar in neutrophil lysates and in empyema fluid supernatants. Ion-exchange and size-exclusion chromatography were used to obtain highly purified preparations of calprotectin from empyema fluids, and these preparations demonstrated zinc-reversible anti-Candida albicans activity which was similar to that observed in the original specimens. These findings suggest that calprotectin is responsible for most of the growth-inhibitory activity of empyema fluid supernatants against this organism.

Cutaneous defenses against dermatophytes and yeasts.

Predispositions to the superficial mycoses include warmth and moisture, natural or iatrogenic immunosuppression, and perhaps some degree of inherited susceptibility. Some of these infections elicit a greater inflammatory response than others, and the noninflammatory ones are generally more chronic. The immune system is involved in the defense against these infections, and cell-mediated immunity appears to be particularly important. The mechanisms involved in generating immunologic reactions in the skin are complex, with epidermal Langerhans cells, other dendritic cells, lymphocytes, microvascular endothelial cells, and the keratinocytes themselves all participating in one way or another. A variety of defects in the immunologic response to the superficial mycoses have been described. In some cases the defect may be preexistent, whereas in others the infection itself may interfere with protective cell-mediated immune responses against the organisms. A number of different mechanisms may underlie these immunologic defects and lead to the development of chronic superficial fungal infection in individual patients. Although the immunologic defects appear to be involved in the chronicity of certain types of cutaneous fungal infections, treatment of these defects remains experimental at the present time.

Resistance of zinc-supplemented Candida albicans cells to the growth inhibitory effect of calprotectin.

Calprotectin is a neutrophil cytoplasmic protein with significant microbistatic activity. This protein may compete for zinc, or the metal may inactivate a different microbistatic activity of the protein. To distinguish between these possibilities, the sensitivity to calprotectin was determined for zinc-supplemented Candida albicans cells. The latter had increased growth in cultures containing either human empyema fluid as a source of calprotectin or purified calprotectin itself. This increased growth did not appear to be due to leakage of zinc into the medium. In other experiments, empyema fluid supernatants did not suppress C. albicans growth across a dialysis membrane; however, other studies suggested that it is difficult to significantly suppress C. albicans growth by zinc depletion unless the depleting agent remains in the cultures. These results indicate that calprotectin inhibits C. albicans growth through competition for zinc.

Effect of abscess fluid supernatants on the kinetics of Candida albicans growth.

Abscess fluid supernatants have been found to inhibit microbial growth, an effect that appears to be due to a protein originating in the cytoplasm of neutrophils. The antimicrobial activity of calprotectin, the responsible protein, is reversible by the addition of zinc to the culture medium. To more carefully analyze this type of antimicrobial activity supernatants of fluids from experimental subcutaneous Candida albicans abscesses in mice were studied to determine how they affect the in vitro growth kinetics of C. albicans. The abscess fluid supernatants inhibited growth in a dose-dependent fashion and more at early times than at later ones. Instability of the abscess fluid antimicrobial activity did not appear to explain the timing of the growth inhibition. A marked lengthening of the lag phase of growth was observed in cultures containing the supernatants (from approximately 6 hr in the control cultures to 15-20 hr in those with the supernatants). On the other hand, the abscess fluid supernatants had only minimal effects on the generation times of actively proliferating C. albicans yeast cells. In addition, these supernatants did not appear to significantly affect the percentage of inoculated organisms undergoing cell division, as determined by a limiting dilution assay. Therefore, these results indicate that abscess fluid supernatants extend the lag phase of C. albicans growth, an effect similar to that seen with zinc-deprived organisms.

Use of a new assay technique for quantification of antifungal activity of nystatin incorporated in denture liners.

Denture-induced stomatitis with concurrent candidal infection is the most commonly encountered intraoral abnormality among individuals who wear dentures. The institutionalized elderly demonstrate increased susceptibility and could benefit from its management with a fungicidal denture liner. As an integral part of the prosthesis, the efficacy of the fungicidal liner would be independent of patient compliance and/or nursing involvement and would provide a predictable therapeutic modality. In this study a "slant agar assay" was developed to evaluate the in vitro antimycotic activity of Visco-gel and Lynal liners impregnated with various concentrations of nystatin over a 14-day period in nonaqueous and aqueous environments. The results were as follows: preparations incorporating higher concentrations of nystatin resulted in greater inhibition of Candida albicans growth; Visco-gel liner-nystatin preparations exhibited a greater fungicidal activity than equivalent Lynal preparations; loss of potency by all of the reline-nystatin preparations consisted of an initial rapid loss between days 0 and 2, followed by a plateau during which the preparations gradually continued to lose inhibitory activity; and 1 million units of nystatin were necessary to maintain an adequate level of antifungal activity in an aqueous environment, where the liners demonstrated decreasing antifungal activity proportional to the duration of exposure to water.

Comparison of the metal-binding anticandidal activities of serum and abscess fluid supernatants.

Serum transferrin appears to play a role in host defense by competing with invading microorganisms for iron. The purpose of the present study was to compare this activity to a similar one recently described in abscess fluids and based on a calcium- and zinc-binding protein called calprotectin. Serum and abscess fluid supernatants were collected and pooled from groups of five to 10 C57BL/6 mice with experimental Candida albicans abscesses; serum was also collected from normal animals. In four experiments, serum was found to reduce in vitro C. albicans growth in Sabouraud glucose broth by a mean of 97.9% at 10 mg ml-1 of protein; this effect was reversed by adding 3-10 microM FeCl3, but not by similar amounts of ZnSO4. Abscess fluid supernatants had a greater effect, reducing growth by 99.9% at 1 mg ml-1 and 76.1% at 0.1 mg ml-1 of total protein; this effect was reversed by 3-10 microM ZnSO4, but not FeCl3. Although abscess fluid supernatants were effective when high inocula (10,000 yeast cells) were used, serum from the infected mice inhibited growth only with lower inocula (10-100 yeast cells). In a separate study, serum from infected mice (eight pools) reduced growth (by a range of 36 to 97%), whereas serum from normal mice (five pools) actually enhanced growth in this system (by a range of 173 to 595%).(ABSTRACT TRUNCATED AT 250 WORDS)

Arrays of Candida albicans pseudohyphae that protect the organisms from neutrophil fungicidal mechanisms in experimental infections of mice.

Experimental subcutaneous Candida albicans infections in mice were used to examine the manner in which this pathogen is cleared in animals recovering from cyclophosphamide-induced leucopenia. In this system, infections at the inoculation sites progressed rapidly during a 6 day period of leucopenia to form arrays of parallel filamentous organisms that effectively isolated those in the interior from contact by neutrophils, even when the leucopenia had resolved. Dense collections of organisms also developed at sites of metastatic infection in the kidneys. A majority of the organisms were found to be viable when they were retrieved from the infected subcutaneous sites of animals that had recovered from leucopenia and whose abscesses had begun to drain spontaneously. Removal of the protective arrays of fungal cells appeared to be accomplished by drainage of abscess contents through the surface of the skin or into the collecting system of the kidney. Drainage of the subcutaneous abscesses did not occur in the cyclophosphamide-treated animals until after the neutrophilic infiltrates had developed, suggesting that this drainage process was mediated by neutrophils rather than by the organisms themselves. In summary, the above findings demonstrate that C. albicans infections in leucopenic hosts may progress to the extent that they would be very difficult to clear solely through the microbicidal processes of returning neutrophils. However, neutrophils also appear to promote the removal of masses of viable fungal cells to the exterior of the body.