Detection of viable fungal spores contaminant on documents and rapid control of the effectiveness of an ethylene oxide disinfection using ATP assay

Filamentous fungi are able to damage and even destroy archival and library materials. Nowadays the conventional method for detecting such micro‐organisms is to put them in cultures but such methods are laborious and time‐consuming. ATP methodology has been widely applied in other domains and its success on bacteria and yeast has been demonstrated. Several commercial reagent kits are available but they did not give satisfactory results on spores mould. We have elaborated new extraction strategies specific to fungi. A comparison of 42 extraction protocols of ATP from fungal spores was carried out. Extraction at 100°C with DMSO 90% in a Tris–acetate–EDTA buffer proved to be the best method. The viability of cells is estimated by the determination of adenylate energy charge (EC). We applied our method successfully on well‐known species such as Aspergillus flavus, A. niger, A. fumigatus, A. versicolor, Neosartorya fischeri, Eurotium chevalieri, Penicillium chrysogenum, Chaetomium globosum and Ulocladium spp. The results suggest that the ATP bioluminescence assay provides a sensitive and time‐saving method for detecting viable fungal spores. The validity of the procedure was also tested on spores killed by steam and on spores treated with ethylene oxide. We showed that EC determination could be used for a rapid control of the effectiveness of a disinfection process performed with ethylene oxide. Copyright © 2003 John Wiley & Sons, Ltd.     

ATP bioluminescence rapid detection of total viable count in soy sauce

The adenosine triphosphate (ATP) bioluminescence rapid determination method may be useful for enumerating the total viable count (TVC) in soy sauce, as it has been previously used in food and beverages for sanitation with good precision. However, many factors interfere with the correlation between total aerobic plate counts and ATP bioluminescence. This study investigated these interfering factors, including ingredients of soy sauce and bacteria at different physiological stages. Using the ATP bioluminescence method, TVC was obtained within 4 h, compared to 48 h required for the conventional aerobic plate count (APC) method. Our results also indicated a high correlation coefficient (r = 0.90) between total aerobic plate counts and ATP bioluminescence after filtration and resuscitation with special medium. The limit of quantification of the novel detection method is 100 CFU/mL; there is a good linear correlation between the bioluminescence intensity and TVC in soy sauce in the range 1 × 10²–3 × 10⁴ CFU/mL and even wider. The method employed a luminescence recorder (Tristar LB‐941) and 96‐well plates and could analyse 50–100 samples simultaneously at low cost. In this study, we evaluated and eliminated the interfering factors and made the ATP bioluminescence rapid method available for enumerating TVC in soy sauce. Copyright © 2011 John Wiley & Sons, Ltd.     

Detection of E. coli in beach water within 1 hour using immunomagnetic separation and ATP bioluminescence

The contamination of beach waters occurs from the discharge of storm water and sanitary sewer over?ows containing faecal material. Additional faecal material derives from discharge of animals and waterfowl. In order to protect public from exposure to faecal‐contaminated water, it is required to test enteric indicators in beach water. The problem is that the traditional culture‐based methods cannot meet this goal because it takes too long (>24 h), so the results are not available until a day later. A rapid method for testing beach water for Escherichia coli within 1 h has been developed. Immunomagnetic separation (IMS) and ATP bioluminescence were used for selective capture and quanti?cation, respectively. This rapid method was compared to the current method (m‐TEC) using beach water samples. The beach samples were pre?ltered with a 20 µm pore size ?lter in order to remove algae, plant debris and large particles. The results showed that the pre?ltration step did not trap the bacteria which were present in the original water samples. The pre?ltered water was then passed through a 0.45 µm pore size ?lter for concentration. The deposited bacteria were resuspended and then mixed with superparamagnetic polystyrene beads (diameter of 0.6 µm) that were coated with E. coli antibodies. After IMS, the quanti?cation of the E. coli was done by ATP bioluminescence. The results obtained with IMS‐ATP bioluminescence correlated well with the plate count method (Rsq = 0.93). The detection limit of the assay was about 20 CFU/100 mL, which is well below the US EPA limits for recreational water. The entire procedure can be completed in less than 1 hour. The necessary equipment is portable and was tested on‐site. Copyright © 2004 John Wiley & Sons, Ltd.     

Enumeration of phagocytosed Staphylococcus aureus inside cells of the mouse macrophage cell line J774 by bioluminescence,fluorescence and viable counting techniques

In order to quantify intracellular Staphylococcus aureus within a macrophage-like cell line by a bioluminescence technique, the mouse cell line J774 and opsonized Staphylococcus aureus were incubated together to allow phagocytosis to occur. Experiments using UV microscopy and fluorescent stained S. aureus were performed to determine an estimate of the mean intracellular bacterial numbers. For enumeration of intracellular bacteria by a bioluminescence technique, extracellular bacteria were removed by washing, the macrophages lysed mechanically and osmotically and treated with apyrase to remove somatic ATP. Bacterial cells were washed and the intracellular ATP measured by firefly luciferase bioluminescence in a luminometer. This new method of enumerating intracellular bacteria was compared to the conventional method of viable counts and found to correlate (r = 0.78). The bioluminescence assay developed was found to be a relatively rapid alternative method to the techniques currently used to enumerate intracellular bacteria and could prove advantageous in studies of intracellular killing and effects of antimicrobial agents on intracellular pathogens.     

Application of bioluminescence ATP measurement for evaluation of fungal viability of foxing spots on old documents

An adenosine triphosphate (ATP) bioluminescence‐based protocol was tested to assess the viability of fungal species in old documents damaged by foxing. Foxing appears as scattered yellow brownish‐red stains, damaging the aesthetics of documents and their long‐term readability. In the field of cultural heritage conservation, the debate over the mechanism of foxing is ongoing. Previous studies found evidence of mold‐like structures in some coloured areas; however, many species have not yet been identified and their role in the phenomenon is not understood. To better understand their involvement in this type of paper decay, we focused our attention first on their viability. We demonstrated the reliability and sensitivity of the ATP bioluminescence assay compared with conventional methods based on cultivation, which has rarely given rise to in vitro growth from foxed papers. From nine books dating back from the 19th and 20th centuries, the mean ATP amount of foxed spots ranged from 0.29 to 3.63 ng/cm², suggesting the presence of strains inside the brownish spots and providing evidence of their viability. Outside the spots, ATP content was considered negligible, with a mean ATP amount of 0 to 0.03 ng/cm². ATP assay appears to be a useful and robust method for the detection and quantification of viable elements in foxing spots. Copyright © 2012 John Wiley & Sons, Ltd.     

Susceptibility testing of dermatophytes

The dermatophytes are a specialized group of fungi that infect the keratinized tissues of humans (hair, nails, and skin) and cause superficial infections. Although several studies have been conducted to develop methods to determine the susceptibilities of yeast and filamentous fungi, similar studies for dermatophytes have only recently taken place. We review how susceptibility testing of dermatophytes was developed and how it has already been applied to clinical samples. We also review recent advances in the development of disk diffusion and colorimetric methodologies for determining the antifungal susceptibility of dermatophytes. With several agents now available for treating infections due to dermatophytes, susceptibility testing will serve as a valuable tool for clinicians as they choose the most appropriate treatment option. Studies are still needed to establish interpretive breakpoints for antifungal agents used in the treatment of superficial fungal infections.     

Disposable bioluminescence-based biosensor for detection of bacterial count in food

A biosensor for rapid detection of bacterial count based on adenosine 5′-triphosphate (ATP) bioluminescence has been developed. The biosensor is composed of a key sensitive element and a photomultiplier tube used as a detector element. The disposable sensitive element consists of a sampler, a cartridge where intracellular ATP is chemically extracted from bacteria, and a microtube where the extracted ATP reacts with the luciferin–luciferase reagent to produce bioluminescence. The bioluminescence signal is transformed into relevant electrical signal by the detector and further measured with a homemade luminometer. Parameters affecting the amount of the extracted ATP, including the types of ATP extractants, the concentrations of ATP extractant, and the relevant neutralizing reagent, were optimized. Under the optimal experimental conditions, the biosensor showed a linear response to standard bacteria in a concentration range from 10³ to 10⁸ colony-forming units (CFU) per milliliter with a correlation coefficient of 0.925 (n = 22) within 5 min. Moreover, the bacterial count of real food samples obtained by the biosensor correlated well with those by the conventional plate count method. The proposed biosensor, with characteristics of low cost, easy operation, and fast response, provides potential application to rapid evaluation of bacterial contamination in the food industry, environment monitoring, and other fields.     

COMPARISON OF MICROBIOLOGICAL METHODS FOR MONITORING CHICKEN CARCASS QUALITY

Many methods have been developed to determine microbial levels in food products and these include ATP bioluminescence, hydrophobic grid membrane filtration (HGMF), impediometry and turbidimetry. A comparison of these techniques for detecting microbial levels in chicken carcass rinses was conducted.
Only the ATP bioluminescence assay and the HGMF system showed a good correlation with plate counts (r = 0.82 and r = 0.83, respectively). The repeatability of these methods was acceptable. There was also a significant correlation between results obtained with two turbidimetric methods and HGMF as well as between HGMF and ATP bioluminescence data. However, only the ATP bioluminescence assay was able to provide results of microbial levels on a realtime basis (within 10 min). This would be beneficial for HACCP (Hazard Analysis of Critical Control Points) based programs.     

A new,rapid and sensitive bioluminescence assay for drug screening on Leishmania

We validated a new method, based on luciferine/luciferase bioluminescence, for drug screening on promastigotes of different Leishmania species. Results obtained with this new, rapid, reproducible, and reliable method are in good accordance with results obtained by the conventional MTT assay. This bioluminescence assay has a lower detection limit.     

Use of a newly developed rapid microbial ATP bioluminescence assay to detect microbial contamination on poultry carcasses

A newly developed rapid microbial ATP bioluminescence test (R-mATP) was shown to be an adequate means to assay the microbial load of poultry carcasses. This assay utilizes differential extraction and filtration to separate somatic from microbial ATP in a very rapid timeframe. The assay requires approximately 5 min to complete; approximately 3.5 min to sample and 90 s analytical time. Correlation coefficient (r) between aerobic colony counts and R-mATP test results (n=329) was 0.82. Post-test probabilities to correctly classify carcasses with different levels of microbial contamination were as high as 98% for samples of ≥3.5 log aerobic CFU per ml. Given the rapidity of this assay, the R-mATP holds potential for monitoring the microbial load of carcasses at poultry-processing critical control points. Other potential applications of this new version of the microbial ATP bioluminescence test are discussed.     

EVALUATION OF ADENOSINE TRIPHOSPHATE (ATP) BIOLUMINESCENCE FOR ESTIMATING BACTERIA ON SURFACES OF BEEF CARCASSES

A rapid (<15 min), inexpensive and simple method has been developed to estimate the concentration of bacteria on surfaces of beef carcasses using adenosine triphosphate (ATP) bioluminescence. Surfaces (5x5 cm²) of beef carcasses (n= 159) were collected by excision. An ATP assay and aerobic plate count were performed on each sample. A significant (p < 0.001) positive linear relationship (r = 0.83) between plate count and ATP assay was obtained for 159 beef carcass samples. When thresholds levels were set at 1 × 10⁴, 1 × 10⁵ and 1 × 10⁶ CFU/cm², there was moderate to good agreement between the ATP bioluminescence assay and the aerobic plate count as determined by the k-statistic. The application of this ATP bioluminescence test to HACCP systems for beef slaughter processes is discussed.     

ATP amplification for ultrasensitive bioluminescence assay: detection of a single bacterial cell

We developed an ultrasensitive bioluminescence assay of ATP by employing (i) adenylate kinase (ADK) for converting AMP + ATP to two molecules of ADP, (ii) polyphosphate (polyP) kinase (PPK) for converting ADP back to ATP (ATP amplification), and (iii) a commercially available firefly luciferase. A highly purified PPK-ADK fusion protein efficiently amplified ATP, resulting in high levels of bioluminescence in the firefly luciferase reaction. The present method, which was approximately 10,000-fold more sensitive to ATP than the conventional bioluminescence assay, allowed us to detect bacterial contamination as low as one colony-forming unit (CFU) of Escherichia coli per assay.     

In vitro susceptibility of dermatomycoses agents to six antifungal drugs and evaluation by fractional inhibitory concentration index of combined effects of amorolfine and itraconazole in dermatophytes

To investigate the antifungal drug susceptibility of fungi responsible for dermatomycoses, minimum inhibition concentration (MIC) tests were performed in 44 strains of dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton tonsurans, Microsporum canis, Microsporum gypseum and Epidermophyton floccosum, with six antifungal drugs (amorolfine, terbinafine, butenafine, ketoconazole, itraconazole and bifonazole) by broth microdilution assay according to Clinical Laboratory Standard Institute protocols. Six possible dermatomycosis‐causing non‐dermatophytic fungi were also tested. The two major causes of tinea, T. rubrum and T. mentagrophytes, showed significantly different sensitivities to ketoconazole and bifonazole. Clinically derived dermatophytes were sensitive to the six antifungal drugs tested. However, non‐dermatophytes, especially Fusarium spp., tended to be resistant to these antifungal drugs. In Trichophyton spp., the MICs of non‐azole drugs had narrower distributions than those of azoles. To evaluate the effects of antifungal drug combinations, the fractional inhibitory concentration index was calculated for the combination of amorolfine and itraconazole as representative external and internal drugs for dermatophytes. It was found that this combination had synergistic or additive effects on most dermatophytes, and had no antagonistic effects. The variation in susceptibility of clinically derived fungal isolates indicates that identification of causative fungi is indispensable for appropriately choosing effective antifungal drugs in the early stages of infection. The results of combination assay suggest that multiple drugs with different antifungal mechanisms against growth of dermatophytes should be used to treat refractory dermatomycoses, especially onychomycosis.     

Fast Monitoring of Indoor Bioaerosol Concentrations with ATP Bioluminescence Assay Using an Electrostatic Rod-Type Sampler

A culture-based colony counting method is the most widely used analytical technique for monitoring bioaerosols in both indoor and outdoor environments. However, this method requires several days for colony formation. In this study, our goal was fast monitoring (Sampling: 3 min, Detection: < 1 min) of indoor bioaerosol concentrations with ATP bioluminescence assay using a bioaerosol sampler. For this purpose, a novel hand-held electrostatic rod-type sampler (110 mm wide, 115 mm long, and 200 mm tall) was developed and used with a commercial luminometer, which employs the Adenosine triphosphate (ATP) bioluminescence method. The sampler consisted of a wire-rod type charger and a cylindrical collector, and was operated with an applied voltage of 4.5 kV and a sampling flow rate of 150.7 lpm. Its performance was tested using Staphylococcus epidermidis which was aerosolized with an atomizer. Bioaerosol concentrations were measured using ATP bioluminescence method with our sampler and compared with the culture-based method using Andersen cascade impactor under controlled laboratory conditions. Indoor bioaerosol concentrations were also measured using both methods in various indoor environments. A linear correlation was obtained between both methods in lab-tests and field-tests. Our proposed sampler with ATP bioluminescence method may be effective for fast monitoring of indoor bioaerosol concentrations.     

A study of the use of rapid methods for preservative efficacy testing of pharmaceuticals and cosmetics

Three rapid microbiological methods, impedance, the direct epifluorescence technique (DEFT-MEM) and ATP bioluminescence (ATP-B) were evaluated for their applicability to preservative efficacy testing (PET) of pharmaceuticals and cosmetics. A good correlation between rapid method response and total colony counts was obtained for untreated suspensions of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans with all three methods but, for Aspergillus niger , with impedance only. For chlorhexidine-treated suspensions of Staph. aureus and C. albicans , a good dose—response curve was obtained with impedance, but ATP-B and DEFT-MEM methods underestimated the kill by the order of 1–6 logs. From the results of this study it is concluded that impedance offers an alternative method to colony counting methods for PET but, at their present level of method development, neither DEFT-MEM nor ATP-B can be considered as satisfactory.     

Comparative evaluation of Trichosporon asahii susceptibility using ASTY colorimetric microdilution and CLSI M27‐A2 broth microdilution reference methods

The in vitro activity of AMPH‐B, 5‐FC, FLCZ, MCZ, ITCZ, and VRCZ against 50 isolates of T. asahii was determined using CLSI M27‐A2 microdilution and ASTY colorimetric methods. Observed agreement ranged from 96 to 100% according to the drug. Overall, the agreement between two methods was 97.7%. The ASTY colorimetric method was thus determined to be comparable to the CLSI reference method when testing the susceptibility of T. asahii to a variety of antifungal agents.     

Enumeration of bacterial cell numbers by amplified firefly bioluminescence without cultivation

We recently developed a novel bioluminescent enzymatic cycling assay for ATP and AMP with the concomitant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK), where AMP and pyrophosphate produced from ATP by firefly luciferase were converted back into ATP by PPDK. Background luminescence derived from contaminating ATP and AMP in the reagent was reduced using adenosine phosphate deaminase which degrades ATP, ADP, and AMP, resulting in constant and highly amplified bioluminescence with low background luminescence. To detect bacterial cells without cultivation, we applied the above bioluminescent enzymatic cycling reagent to rapid microbe detection system. ATP spots (0.31-5.0 amol/spot) at the level of a single bacterial cell were detected with 5 min signal integration, signifying that integrated luminescence was amplified 43 times in comparison to traditional ATP bioluminescence. Consequently, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Lactobacillus brevis in beer were detected without cultivation. Significant correlation was observed between the number of signal spots obtained using this novel system and the colony-forming units observed with the conventional colony-counting method (R(2)=0.973).     

The effect of selected human steroid hormones upon the growth of dermatophytes with different adaptation to man

The inhibitory effect exerted by steroid hormones on thein vitro growth characteristics of dermatophytes is poorly understood. As a hypothesis this inhibition could result from fungal adaptation to the human host. Therefore, in this study the susceptibility of representative anthropophilic, zoophilic and geophilic dermatophytes to hormonal inhibition was compared. As a result, in agar dilution assaysprogesterone,testosterone, andestradiol proved to reduce fungal growth, whereashydrocortisone had no such effect. In general, anthropophilic dermatophytes were shown to be more responsive to steroid hormones than geophilic species, suggesting a correlation of steroid susceptibility with adaptation to human skin. However, since fungal response to hormones consisted of growth inhibition and occurred only at steroid concentrations much higher than present in human skin, it cannot be assumed to contribute to this adaptation.     

实验动物皮肤病原真菌DNA快速少量提取法

PCR技术应用于实验动物皮肤病原真菌检测,方法简单、省时。但是,真菌的DNA提取较为困难。本文推荐一种既简单又经济快速的提取皮肤真菌DNA的方法,并能成功用于实验动物皮肤病原真菌质量检测研究。     

Application of a bioluminescence ATP assay in brewery wastewater treatment studies

The importance of having a rapid method for determining the viable biomass in activated-sludge wastewater treatment plants (WWTP) for process control and development is well recognized. The firefly bioluminescence ATP assay has been proposed for this purpose. Such an assay using partially purified firefly luciferase and synthetic firefly luciferin for the bioluminescence reaction, a liquid scintillation counter in the out-of-coincidence mode as luminescence detector, and a sludge ATP extraction technique involving dimethyl sulfoxide at room temperature is described. Experiments with several pure bacteria cultures were done to demonstrate the feasibility of applying this assay to activated sludge to activated sludge WWTP investigation and control. The ATP content of samples taken from various points in a 350000 gal/day brewery activated-sludge WWTP was monitored for 4.5 months. Good linear correlation between ATP and mixed-liquor suspended solids, return sludge suspended solids, and effluent suspended solids were observed. Percentage viabilities of the various sludge samples were derived from the ATP results.