An improved protocol for bacteria identification by MALDI-TOF MS directly from positive blood cultures.

FASTinov® developed a rapid antimicrobial susceptibility test that includes the purification of a bacterial suspension directly from positive blood cultures (BC). In order to streamline laboratory workflow, the use of the bacterial suspension obtained through FASTinov® sample prep was tested for identification (ID) by matrix absorption laser deionization-time of flight mass spectrometry (MALDI-TOF MS) (Bruker) in 364 positive BC, and its accuracy assessed comparing with the MALDI-TOF MS ID of the next-day subcultured colonies. FASTinov sample prep was highly reliable for rapid ID directly from BC with proportion of agreement of 94.9% for Gram-positive and 96.3% for Gram-negative bacteria.

Performance of Flow Cytometry-Based Rapid Assay in Detection of Carbapenemase-Producing Enterobacterales.

Carbapenemase-producing Enterobacterales are increasingly being recognized in nosocomial infections. The performance of a flow cytometry-based rapid assay for their detection and differentiation was evaluated. This is a disruptive phenotypic technology, phenotypic and growth-independent, that searches for the lesions produced by drugs acting on cells after a short incubation time. Overall, 180 Gram-negative bacteria were studied, and results were compared with those obtained molecularly by PCR and phenotypically by 'KPC, MBL and OXA-48 Confirm Kit'. This phenotypic method was used as reference for comparison purposes. Susceptibility to carbapenems (imipenem, meropenem, and ertapenem) was determined by standard broth microdilution. Overall, 112 isolates (62.2%) were carbapenemase producers, 41 KPCs, 36 MßLs, and 31 OXA-48, and 4 strains were KPC + MßL co-producers. Sixty-eight isolates were carbapenemase-negative. The percentage of agreement, sensitivity, and specificity were calculated according to ISO 20776-2:2021. The FASTinov assay showed 97.7% agreement with the reference method for carbapenemase detection. Discrepant flow cytometry results were obtained in four isolates compared with both reference and PCR results. The sensitivity and specificity of this new technology were 95.3% and 98.5%, respectively, for KPCs, 97.6% and 99.3% for MßLs, and 96.9% and 98% for OXA-48 detection. In conclusion, we describe a rapid flow cytometry assay with high accuracy for carbapenemase detection and the differentiation of various carbapenemases, which should impact clinical microbiology laboratories and patient management.

Evaluation of FASTinov Ultrarapid Flow Cytometry Antimicrobial Susceptibility Testing Directly from Positive Blood Cultures.

The FASTinov flow cytometry kit, an ultrarapid antimicrobial susceptibility test, was directly evaluated on positive blood cultures (BC) at two sites: (i) FASTinov, S.A., in Porto, Portugal, using BC spiked with well-characterized bacteria, and (ii) Ramón y Cajal University Hospital in Madrid, Spain, using positive BC from patients. Two kits were evaluated, FASTgramneg (Enterobacterales, Pseudomonas, Acinetobacter) and FASTgrampos (Staphylococcus, Enterococcus). Dedicated software for cytometric data analysis and interpretative reporting, including both CLSI and EUCAST criteria, was used. The FASTgramneg kit also provides information about the presence of resistant mechanisms, including extended-spectrum beta-lactamases (ESBLs) and carbapenemases. After 1 h of incubation at 37°C, bacteria were analyzed using a CytoFLEX cytometer (Beckman, CA). Disk diffusion was performed as the reference susceptibility method. Overall, 447 positive BC were included, 100 from hospitalized patients. Categorical agreement values for the FASTgramneg panel were 96.8% based on EUCAST criteria and 96.4% based on CLSI criteria. For the FASTgrampos panel, categorical agreement was 98.6% when using both criteria. When EUCAST criteria were used, the percentages of errors for the FASTgramneg panel were 2.1% minor errors (mE), 1.3% major errors (ME), and 0.6% very major errors (VME). When CLSI criteria were used, 2.9% mE, 0.9% ME, and 0.4% VME were found. VME were mainly observed with amoxicillin-clavulanate, cefotaxime, ceftazidime, and gentamicin. The FASTgrampos panel showed 0.3% mE, 1.4% ME, and 0.4% VME when EUCAST criteria were used (VME with respect to gentamicin and Staphylococcus) and 0.4% mE, 1.4% ME, and no VME when CLSI criteria were used. The FASTinov flow cytometry kits represent a rapid alternative for direct antimicrobial susceptibility testing from positive BC, showing time to results of <2 h, and can be used to personalize antibiotic and stewardship practices.

Mechanisms of Acquired In Vivo and In Vitro Resistance to Voriconazole by Candida krusei following Exposure to Suboptimal Drug Concentration.

Five Candida krusei isolates (susceptible and resistant) recovered from the urine of a kidney transplant patient treated with voriconazole (VRC) 200 mg twice daily for 20 days were studied. Eight unrelated clinical isolates of C. krusei were exposed in vitro to VRC 0.001 µg/ml for 30 days. Development of VRC transient resistance occurred in vivo, and induction of permanent resistance occurred in vitro Mostly, ABC1 and ERG11 genes were overexpressed, and a homozygous T418C mutation in the ERG11 gene was found.

Evaluation of ultra-rapid susceptibility testing of ceftolozane-tazobactam by a flow cytometry assay directly from positive blood cultures.

The urgent need for rapid antimicrobial susceptibility is broadly apparent from government reports to the lay press. Accordingly, we developed a flow-cytometry assay (FCM) for evaluating ceftolozane-tazobactam (C/T) susceptibility directly on blood cultures (BC) requiring < 2 h from flag positivity to report. The protocol was optimized with C/T-susceptible and C/T-resistant gram-negative bacilli inoculated in BC aerobic bottles (Becton-Dickinson, USA), and afterward optimized for different C/T concentrations (1/4, 2/4, 4/4, and 8/4 mg/L) for 1 h incubation (37 °C), followed by FCM and software analysis. Fluorescent membrane permeability and membrane potential dyes were comparatively used to detect early cell lesions using the CytoFLEX cytometer (Beckman-Coulter, USA). Repeatability, reproducibility, and stability of the assay up to 48 h after BC positivity were determined. Internal validation was performed in spiked BC bottles with 130 Enterobacterales and 32 Pseudomonas aeruginosa isolates from Porto University (Portugal), including 13 ATCC isolates. Additionally, 64 gram-negative bacilli recovered from positive BC at Ramon y Cajal Hospital (Madrid, Spain) were tested. Categorical agreement (CA) and analytical errors were calculated comparing FCM with broth microdilution results. Only the membrane potential dyes clearly distinguished CT-susceptible and CT-resistant isolates. Excellent repeatability, reproducibility, and inter-method concordance was observed. Overall, CA was 99.1% using EUCAST criteria with 2 major errors and 98.7% with CLSI criteria with 2 major and 1 minor errors. A new, accurate, and ultra-rapid FCM (< 2 h) for testing C/T susceptibility gave accurate results and would expand current FCM antimicrobial susceptibility assay.

Microbes and Cancer: Friends or Faux?

Cancer is one of the most aggressive and deadly diseases in the world, representing the second leading cause of death. It is a multifactorial disease, in which genetic alterations play a key role, but several environmental factors also contribute to its development and progression. Infections induced by certain viruses, bacteria, fungi and parasites constitute risk factors for cancer, being chronic infection associated to the development of certain types of cancer. On the other hand, susceptibility to infectious diseases is higher in cancer patients. The state of the host immune system plays a crucial role in the susceptibility to both infection and cancer. Importantly, immunosuppressive cancer treatments increase the risk of infection, by decreasing the host defenses. Furthermore, alterations in the host microbiota is also a key factor in the susceptibility to develop cancer. More recently, the identification of a tumor microbiota, in which bacteria establish a symbiotic relationship with cancer cells, opened a new area of research. There is evidence demonstrating that the interaction between bacteria and cancer cells can modulate the anticancer drug response and toxicity. The present review focuses on the interaction between microbes and cancer, specifically aiming to: (1) review the main infectious agents associated with development of cancer and the role of microbiota in cancer susceptibility; (2) highlight the higher vulnerability of cancer patients to acquire infectious diseases; (3) document the relationship between cancer cells and tissue microbiota; (4) describe the role of intratumoral bacteria in the response and toxicity to cancer therapy.

Antibacterial Action Mechanisms of Honey: Physiological Effects of Avocado, Chestnut, and Polyfloral Honey upon Staphylococcus aureus and Escherichia coli.

Numerous studies have explored the antibacterial properties of different types of honey from all around the world. However, the data available describing how honey acts against bacteria are few. The aim of this study was to apply a flow cytometry (FC) protocol to examine and characterize the primary effects of three varieties of honey (avocado, chestnut and polyfloral) upon physiological status of Staphylococcus aureus and Escherichia coli cells to reveal their antibacterial action mechanisms. The effects of honey samples on membrane potential, membrane integrity, and metabolic activity were assessed using different fluorochromes, in a 180 min time course assay. Time-kill experiments were also carried out under similar conditions. Exposure of S. aureus and E. coli to the distinct honey samples resulted in physiological changes related to membrane polarization and membrane integrity. Moreover, honey induced a remarkable metabolic disruption as primary physiological effect upon S. aureus. The different honey samples induced quite similar effects on both bacteria. However, the depth of bacteria response throughout the treatment varied depending on the concentration tested and among honey varieties, probably due to compositional differences in the honey.

Hard-to-heal wounds, biofilm and wound healing: an intricate interrelationship.

Hard-to-heal wounds are a major public health problem that incur high economic costs. A major source of morbidity, they can have an overwhelming impact on patients, caregivers and society. In contrast to acute wound healing, which follows an 'orderly and timely reparative process', the healing of hard-to-heal wounds is delayed because the usual biological progression is interrupted. This article discusses hard-to-heal wounds, the impact they have on patients and healthcare systems, and how biofilms and other factors affect the wound-healing process. Controlling and preventing infection is of utmost importance for normal wound healing. Rational use of anti-infectious agents is crucial and is particularly relevant in the context of rising healthcare costs. Knowledge of the complex relationship between hard-to-heal wounds, biofilm formation and wound healing is vital for efficient management of hard-to-heal wounds.

Rapid Flow Cytometry Test for Identification of Different Carbapenemases in Enterobacteriaceae.

A flow cytometry test was developed to identify carbapenemase production by Enterobacteriaceae and to discriminate between the different types of carbapenemases (classes A, B, and D). It is based on the detection of meropenem activity against bacteria, coupled with different carbapenemase inhibitors, which is assessed by flow cytometry. It represents a convenient, fast, and reliable approach (100% sensitivity and 100% specificity) for the detection and characterization of different carbapenemases.

New Insights Regarding Yeast Survival following Exposure to Liposomal Amphotericin B.

In vitro resistance to amphotericin B is an extremely rare event among pathogenic yeasts. However, in vivo response is sometimes reduced, resulting in an unfavorable outcome. Such adverse outcomes might be related to subfungicidal plasma concentrations. We aimed to clarify the mechanisms of liposomal amphotericin B (AMB-L; AmBisome)-induced lesions and the mechanisms responsible for yeast cell recovery following exposure at plasma concentrations. The physiological statuses developing following exposure to AMB-L at simulated plasma concentrations (20 to 0.1 mg/liter) and at a constant concentration (3 mg/liter) were assessed in a 24-h time course assay. Time-kill experiments also were carried out under the same AMB-L treatment conditions. Our results suggest that yeast cells develop compensatory responses related to membrane polarization, metabolic activity, and reactive oxygen species (ROS) production after exposure to high plasma concentrations (20 to 5 mg/liter) during the first 6 h; in the remaining 18 h, when exposed to lower concentrations, cells reveal almost full recovery with no evidence of fungicidal activity. In contrast, whenever cells are exposed to a constant concentration above the MIC, despite initially exhibiting compensatory stress responses, soon afterwards they exhibit membrane depolarization, a decrease of metabolic activity, increasing ROS production, and lastly, programmed cell death and necrosis, resulting in succumbing to AMB-L fungicidal effects. This study may represent a step forward in the support of AMB-L use for clinical treatment of invasive fungal infections, since it demonstrates the importance of maintaining levels of AMB-L above the MIC in plasma and tissues to ensure it produces its fungicidal effects.

Fluconazole and Voriconazole Resistance in Candida parapsilosis Is Conferred by Gain-of-Function Mutations in MRR1 Transcription Factor Gene.

Candida parapsilosis is the second most prevalent fungal agent causing bloodstream infections. Nevertheless, there is little information about the molecular mechanisms underlying azole resistance in this species. Mutations (G1747A, A2619C, and A3191C) in the MRR1 transcription factor gene were identified in fluconazole- and voriconazole-resistant strains. Independent expression of MRR1 genes harboring these mutations showed that G1747A (G583R) and A2619C (K873N) are gain-of-function mutations responsible for azole resistance, the first described in C. parapsilosis.

Ibuprofen potentiates the in vivo antifungal activity of fluconazole against Candida albicans murine infection.

Candida albicans is the most prevalent cause of fungemia worldwide. Its ability to develop resistance in patients receiving azole antifungal therapy is well documented. In a murine model of systemic infection, we show that ibuprofen potentiates fluconazole antifungal activity against a fluconazole-resistant strain, drastically reducing the fungal burden and morbidity. The therapeutic combination of fluconazole with ibuprofen may constitute a new approach for the management of antifungal therapeutics to reverse the resistance conferred by efflux pump overexpression.

In vitro antifungal activity and in vivo antibiofilm activity of cerium nitrate against Candida species.

OBJECTIVES: The objective of this study was to clarify the antifungal properties of cerium, a lanthanide member, against Candida species. A comprehensive study with planktonic and sessile cells was performed. The ability of cerium nitrate (CN) to impair in vitro and in vivo biofilm formation was evaluated and its potential use in biofilm treatment was also evaluated. METHODS: Forty-eight clinical isolates of different Candida species and the type strain ATCC 90028 were tested according to the protocol M27-A3. The MICs and minimum lethal concentrations were determined. A time-kill assay was performed and a cytometric kinetic study was performed using live/dead markers. Biofilm inhibition and biofilm susceptibility in the presence of cerium was evaluated by quantification of the biofilm metabolic activity and total biomass with XTT and crystal violet assays, respectively. CN in vivo efficacy as a coating for medical indwelling devices was evaluated for the first time for Candida parapsilosis, using a mouse subcutaneous foreign body model using polyurethane catheter segments. Scanning electron microscopy was used to assess biofilm architecture after CN treatment. RESULTS: The MICs for planktonic cells correlated with severe cellular metabolic activity impairment and membrane damage after 3 h of incubation. Moreover, CN efficiently prevented biofilm formation both in vitro and in vivo in segments of polyurethane catheters. At higher concentrations, it was also able to disorganize and almost eradicate preformed biofilms. CONCLUSIONS: Our results strongly suggest that CN application in the clinical setting might be effective in preventing the formation of biofilm-associated infections, namely through catheter coating and ultimately as an antimicrobial lock therapy.

Antibiofilm and Antimicrobial Activity of Polyethylenimine: An Interesting Compound for Endodontic Treatment.

AIM: Bacteria levels of necrotic teeth are greatly reduced after endodontic treatment procedures but the presence of persisting microorganisms leads to continuous efforts to develop materials with antimicrobial properties. The purpose of the study was to determine the antimicrobial activity of polyethylenimine (PEI) against common bacteria and yeasts, regarding planktonic cells and biofilm, and to clarify its antimicrobial mechanism of action through flow cytometry. MATERIALS AND METHODS: The antibiofilm and antimicrobial effect of PEI was determined against Enterococcus faecalis, Staphylococcus aureus, Escherichia coli and Candida albicans strains using reference protocols. The effect of PEI was evaluated regarding adhesion, biofilm formation and biofilm disaggregation. In order to understand PEI cellular effects flow cytometric analysis was performed with different fluorescent markers. RESULTS: It was verified that minimal inhibitory concentrations (MIC) values and minimal lethal concentrations (MLC) obtained for PEI were similar and ranged between 50 and 400 mg/l, proving the microbicidal and fungicidal activity of this compound. Antibiofilm activity was also proved for all the microorganisms. Severe lesion of the membrane and cell depolarization was demonstrated. CONCLUSION: Polyethylenimine showed antimicrobial and antibiofilm activity against microorganisms often associated with apical periodontitis. CLINICAL SIGNIFICANCE: Theoretically, prolonging the antibacterial effects of materials used in endodontics may be interesting to help prevent reinfection and possibly to affect residual bacteria that survived the treatment procedures.

In vivo and in vitro acquisition of resistance to voriconazole by Candida krusei.

Candida krusei is an important agent of opportunistic infections that often displays resistance to several antifungals. We describe here the in vivo acquisition of resistance to voriconazole (VRC) by C. krusei isolates recovered from a leukemia patient during a long period of VRC therapy. In order to mimic the in vivo development of VRC resistance, a susceptible C. krusei isolate was exposed daily to 1 µg/ml of VRC in vitro. Interestingly, after 5 days of exposure to VRC, a MIC of 4 µg/ml was achieved; this value remained constant after 25 additional days of treatment with VRC and also after 30 consecutive days of incubation in VRC-free medium. Our objective was to determine the associated molecular resistance mechanisms, such as expression of efflux pump genes and ERG11 gene mutations, among the resistant strains. Synergistic effects between the efflux blocker tacrolimus (FK506) and VRC were found in all of the resistant strains. Moreover, ABC1 gene expression increased over time in both the in vivo- and in vitro-induced resistant strains, in contrast to the ABC2 and ERG11 genes, whose expression was invariably lower and constant. ERG11 gene sequencing showed two different types of mutations, i.e., heterozygosity at T1389T/C, corresponding to synonymous mutations, in C. krusei strains and a missense mutation at position T418C, resulting in a change from Tyr to His, among resistant C. krusei clinical isolates. This study highlights the relevance of ATP-dependent efflux pump (namely, Abc1p) activity in VRC resistance and describes new mutations in the ERG11 gene among resistant C. krusei clinical isolates.

Development of cross-resistance by Aspergillus fumigatus to clinical azoles following exposure to prochloraz, an agricultural azole.

BACKGROUND: The purpose of this study was to unveil whether azole antifungals used in agriculture, similar to the clinical azoles used in humans, can evoke resistance among relevant human pathogens like Aspergillus fumigatus, an ubiquitous agent in nature. Additionally, cross-resistance with clinical azoles was investigated. Antifungal susceptibility testing of environmental and clinical isolates of A. fumigatus was performed according to the CLSI M38-A2 protocol. In vitro induction assays were conducted involving daily incubation of susceptible A. fumigatus isolates, at 35°C and 180 rpm, in fresh GYEP broth medium supplemented with Prochloraz (PCZ), a potent agricultural antifungal, for a period of 30 days. Minimal inhibitory concentrations (MIC) of PCZ and clinical azoles were monitored every ten days. In order to assess the stability of the developed MIC, the strains were afterwards sub-cultured for an additional 30 days in the absence of antifungal. Along the in vitro induction process, microscopic and macroscopic cultural observations were registered. RESULTS: MIC of PCZ increased 256 times after the initial exposure; cross-resistance to all tested clinical azoles was observed. The new MIC value of agricultural and of clinical azoles maintained stable in the absence of the selective PCZ pressure. PCZ exposure was also associated to morphological colony changes: macroscopically the colonies became mostly white, losing the typical pigmentation; microscopic examination revealed the absence of conidiation. CONCLUSIONS: PCZ exposure induced Aspergillus fumigatus morphological changes and an evident increase of MIC value to PCZ as well as the development of cross-resistance with posaconazole, itraconazole and voriconazole.

Environmental azole fungicide, prochloraz, can induce cross-resistance to medical triazoles in Candida glabrata.

Acquisition of azole resistance by clinically relevant yeasts in nature may result in a significant, yet undetermined, impact in human health. The main goal of this study was to assess the development of cross-resistance between agricultural and clinical azoles by Candida spp. An in vitro induction assay was performed, for a period of 90 days, with prochloraz (PCZ) - an agricultural antifungal. Afterward, the induced molecular resistance mechanisms were unveiled. MIC value of PCZ increased significantly in all Candida spp. isolates. However, only C. glabrata developed cross-resistance to fluconazole and posaconazole. The increased MIC values were stable. Candida glabrata azole resistance acquisition triggered by PCZ exposure involved the upregulation of the ATP binding cassette multidrug transporter genes and the transcription factor, PDR1. Single mutation previously implicated in azole resistance was found in PDR1 while ERG11 showed several synonymous single nucleotide polymorphisms. These results might explain why C. glabrata is so commonly less susceptible to clinical azoles, suggesting that its exposure to agricultural azole antifungals may be associated to the emergence of cross-resistance. Such studies forward potential explanations for the worldwide increasing clinical prevalence of C. glabrata and the associated worse prognosis of an infection by this species.

Evaluation of Giardia duodenalis viability after metronidazole treatment by flow cytometry.

Giardia duodenalis (syn. lamblia; syn. intestinalis) susceptibility testing is not routinely performed because the classical culture methods are very time-consuming and laborious. We developed a novel flow cytometry (FC) assay to evaluate the susceptibility of G. duodenalis trophozoites to metronidazole (MTZ). Different concentrations of MTZ were added to cultures of trophozoites (10 5 /mL) and the cultures were incubated for different periods. The 50% inhibitory concentration was calculated and propidium iodide (PI) was used to quantify the number of dead cells. After treatment, PI-positive trophozoites increased with increasing drug concentration and exposure time. An excellent correlation was found between FC and the classical method. A novel, accurate and reliable method is now available to evaluate G. duodenalis viability.

Epidemiological cutoff values for fluconazole, itraconazole, posaconazole, and voriconazole for six Candida species as determined by the colorimetric Sensititre YeastOne method.

In the absence of clinical breakpoints (CBP), epidemiological cutoff values (ECVs) are useful to separate wild-type (WT) isolates (without mechanisms of resistance) from non-WT isolates (those that can harbor some resistance mechanisms), which is the goal of susceptibility tests. Sensititre YeastOne (SYO) is a widely used method to determine susceptibility of Candida spp. to antifungal agents. The CLSI CBP have been established, but not for the SYO method. The ECVs for four azoles, obtained using MIC distributions determined by the SYO method, were calculated via five methods (three statistical methods and based on the MIC50 and modal MIC). Respectively, the median ECVs (in mg/liter) of the five methods for fluconazole, itraconazole, posaconazole, and voriconazole (in parentheses: the percentage of isolates inhibited by MICs equal to or less than the ECVs; the number of isolates tested) were as follows: 2 (94.4%; 944), 0.5 (96.7%; 942), 0.25 (97.6%; 673), and 0.06 (96.7%; 849) for Candida albicans; 4 (86.1%; 642), 0.5 (99.4%; 642), 0.12 (93.9%; 392), and 0.06 (86.9%; 559) for C. parapsilosis; 8 (94.9%; 175), 1 (93.7%; 175), 2 (93.6%; 125), and 0.25 (90.4%; 167) for C. tropicalis; 128 (98.6%; 212), 4 (95.8%; 212), 4 (96.0%; 173), and 2 (98.5; 205) for C. glabrata; 256 (100%; 53), 1 (98.1%; 53), 1 (100%; 33), and 1 (97.9%; 48) for C. krusei; 4 (89.2%; 93), 0.5 (100%; 93), 0.25 (100%; 33), and 0.06 (87.7%; 73) for C. orthopsilosis. All methods included ≥94% of isolates and yielded similar ECVs (within 1 dilution). These ECVs would be suitable for monitoring emergence of isolates with reduced susceptibility by using the SYO method.

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.