Abstract: The bioluminescence method is based on measurements of adenosine triphosphate (ATP) which is the principal energy carrier in all living organisms, including microorganisms. ATP reacts with luciferin in the presence of the catalyst - luciferase enzyme, and the effect of this oxidation reaction is emission of light, recorded by a luminometer. In hygiene monitoring, it is assumed that the amount of the microbial biomass is directly proportional to the amount of ATP in the sample. Detection of microbial contamination with the bioluminescence technique is often applied in clinical and environmental studies. The bioluminescence method of ATP investigation on solid surfaces has become well-established in food processing industry, particularly as part of the general
hazard analysis and critical control point (HACCP) measurements. It is a crucial alternative to time-consuming and labour-intensive traditional microbiological tests. This method provides an actual estimation of total surface cleanliness, which includes the presence of organic debris and microbial contamination. The limitation of this technique is the impossibility to directly convert the luminometric results to the number of microorganisms. Therefore, it is necessary to establish acceptable limits before the ATP test is applied. This method is commonly used to test if cleaning requirements are achieved satisfactorily. In environmental studies, the ATP-based method is developed and implemented for the assessment of the occurrence of biofilm on plumbing materials that have contact with drinking water or for evaluation of vitality and adhesion of bacteria on the surface of bioactive polymers. This method was also evaluated as an objective technique that allows
to assess the efficiency of cleaning in healthcare institutions and as a rapid method that detects the presence of pathogens responsible for healthcare associated infections (HAIs). Many of these studies indicate that intracellular ATP levels differ between microbial taxa so much that the ATP test should not be interpreted as an indicator of the presence of microbial pathogens.
Keywords: hygiene control, microorganisms, bioluminescence method, ATP assay
Introduction
The bioluminescence method uses the capacity of organic matter to release adenosine triphosphate (ATP), which is the principal energy source in all living organisms. This capacity is characteristic of bacteria, fungi and other microbes as well as food and food debris, including that on disinfected surfaces. The principle of the assay is based on the following enzymatic reaction:
ATP + luciferin/luciferase → AMP + PP + light
Luciferase catalyses the oxidation reaction of luciferin to the form of higher energy state. The reaction can proceed properly only when energy carried by ATP is delivered. ATP breaks down to adenosine monophosphate (AMP) and phosphoric residues (PP). The oxidized form of luciferin returns to its primary energy state by emitting light with the wave of 562 nm, the precise measurement of which enables indirect assessment of ATP concentration. The amount of light emitted is directly proportional to the concentration of ATP. Thus, it is assumed that the amount of
ATP in the sample is directly proportional to the amount of the microbial biomass. The amount of light emission, which results from the luminescence reaction, is measured with the use of a luminometer. This device contains a measuring chamber isolated from external light sources and a detector that processes the optic signal to the electrical one, which is expressed in relative light units (RLU). Detection of microbial contamination with ATP assay is applied in clinical trials, environmental examinations (of water from bathing sites or ground waters) and food industry. In practice, the material for investigations is collected from solid samples, such as meat, and fluid ones: water, milk or wastewater [Deininger and Lee, 2001, Samkutty et al., 2001]. However, it must be noted that food products contain large amounts of ATP which may considerably exceed the signal obtained from microbes when contamination is not high. In 1 g of meat or orange juice there
is 10-7 g of ATP whereas a single bacterial cell usually contains 10-15 g of this compound. The amount of released ATP depends on the type and physiological condition of microbes. It is assumed that Gram-positive bacteria contain more ATP than Gram-negative ones, and the presence of ATP on spores is nearly undetectable [Kręgiel, 2012].
It should be considered a rapid method in comparison with the other techniques used to assess the number of microbes, since the time of measurement is several seconds. In commonly applied culture-based methods, one has to wait for microbes to grow for a long time. Molecular techniques based on DNA, despite their sensitivity and reliability in terms of both quantity and quality, require time and are expensive due to the reagents used. Microscopic methods, in turn, determine the total number of microbes (living or dead) and require their high concentrations [Squirrell et al., 2002, Seshardi, 2009].
ATP test in hygiene monitoring in food industry
ATP bioluminescence assay may be used for hygiene monitoring in food industry. The microbiological methods assessing cleanliness of the surfaces in contact with food, such as swabbing and contact plate method, are broadly used, but time-consuming due to the culturing stage. In manufacturing conditions, a method for rapid evaluation of microbiological cleanliness is sought for. This is associated with the requirements of the systems that assure safety of food production, which consist in rapid and efficient hazard detection and estimation in the control points of the HACCP system [Champiat et al., 2001]. The technique of bioluminescence is applied in sectors producing animal products, in particular meat, and in farms with dairy and slaughter animals as well as in dairy industry [Finger and Sischo, 2001]. Not only does this technique allow microbial ATP to be measured, but also indicates the ATP level of food debris and other organic contaminants, which might become a culture medium for microbes. This enables to detect incorrectness in the process of cleaning and disinfection, but it requires establishing certain critical limit s. Moreover, this method may be used on a continuous basis during the production process, which, if incorrectness has been detected, enables to undertake immediate action so as to make corrections and
establish hazards in real-time [Aycicek et al., 2006, Kręgiel, 2012].
A practical nuisance of the method is the fact that it is not easy to recount the luminometric results to the number of microbes. To interpret the luminometric results obtained at individual control points in a given industry or factory, it is necessary to establish the acceptable level of ATP. This depends of various factors, such as type of resources, production and processing procedures, materials which the analysed surfaces are made of and risk associated with the product. Therefore, the limits for values cannot be unequivocally standardised. However, the manufacturers of luminometers indicate approximate limits for the main groups of food products. The most frequently proposed idea is to accept the median value (based on numerous measurements conducted at one point) as the acceptable limit, in which case the inadmissible limit would be the acceptable value multiplied by three. The introduction of limits to the software of the device helps to accelerate
the measurements and work in the pass/fail system, where all values above “fail” attest to incorrect cleaning and disinfecting process and indicate the necessity to repeat it. The level “pass” indicates a satisfactory sanitary level [Hy-Lite Merck information materials].
Luminometric analyses conducted in dairy industry are primarily associated with investigating the surfaces of instruments: milking equipment, milk containers, pipelines, milk receivers etc., Vilar et al. (2008), assessed the hygienic effect after the application of various automatic and manual cleaning practices. The highest ATP level (100 000 RLU) was observed on surfaces cleaned with cool
water without any cleaning agents. Low ATP levels were found in closed systems, particularly those cleaned automatically, and in large containers that are easy to clean. The most discrepant results (ranging from 9 to 38 570 RLU) were obtained in animal farms in which non-chlorinated water was used for cleaning. Low pressure and too low temperature were the reasons for the lack of adequate hygiene. These studies revealed that the luminometric method is a fast and easy tool that may help breeders to control the effectiveness of cleaning processes.
The ATP-based method was also used to compare the level of hygiene in fish-processing factories where the HACCP system has been implemented and in those operating without such a system. Hwang et al. (2011), used this technique in the critical control points on individual production stages in order to evaluate the hygiene level on the surfaces that have direct contact with food (transporters, baskets, knives or tables for cutting). The outcomes were compared with the results of swabbing tests performed on the surfaces of 10 cm2. The level of ATP on the surfaces ranged from 460-85 000 RLU/10 cm2, which corresponded to 1.0 – 3.5 log CFU/10 cm2 of bacteria cultured. Despite certain discrepancies between the results, it was agreed that the tested method enables to rapidly determine statistically significant differences in hygiene levels in factories with and without HACCP system.
Research on improving the luminometric method to detect microbial contamination in food is underway. Luo et al. (2009), obtained a very good correlation of the measurements with the culture-based method by using an additional stage of extracting intracellular ATP from bacteria. There are various methods of ATP extraction from cells based on ultrasounds: microwaves, organic
solvents, strong acids and surfactants. It was shown that complete lysis may be achieved with the use of trichloroacetic acid (TCA) and cetyltrimethylammonium bromide (CTAB). Partial lysis, in turn, may be achieved using sodium dodecyl sulphate (SDS). The level of ATP detected in suspensions of bacterial cells lysed this way was considerably higher, which increases the reliability of assays when non-bacterial ATP is present in food samples. However, the chemical extraction method may negatively affect enzymatic activity of reaction-inducing luciferase. The residue of CTAB and TCA in the mixtures extracted, if not neutralized, may inhibit luciferase activity and decrease the level of detectable ATP. On the other hand, alkaline substances, used as neutralizers, may also decrease activity of this enzyme. Again, the authors emphasise that the usage of this method to directly determine the presence of bacteria in food is limited due to a high non-microbial
ATP levels.
Bioluminescence in environmental studies The bioluminescence method is also applied in checking and validating devices used for environmental measurements. Seshadri et al. (2009), tested the
ATP-based method for rapid assessment of bioaerosol samples collected by impaction in air microbiological tests. The number of RLU units that corresponded to the number of bacteria was verified by direct count of acridine orange-stained cells under the fluorescence microscope. The study revealed high correlation between the methods for determining the microbial count for the two tested species, Pseudomonas fluorescens and vegetative cells of Bacillus subtilis, in filter extracts on which air samples were aspirated in different sampling flow rates. It was concluded that the bioluminescence method gives better outcomes in assessing efficacy and bacterial bioaerosol sampling than the microscopy and culture-based methods since it enables to localise and quantitatively assess losses in bacterial recovery resulting from their depositing on the sampler’s elements. It was indicated that the method could be applied in the validation procedures of samplers used for collecting air for microbial testing. Moreover, when determining the ATP level that agreed with the number of Pseudomonas fluorescens and Bacillus subtilis bacteria in reference suspensions as calculated under the fluorescence microscope, the differences between these two species were identified. In suspensions of the same density with 104 – 107 cells, the content of ATP was higher for B. subtilis than for P. fluorescens. This confirmed the necessity to draw up separate reference curves for each microbial species tested. In both cases, this method enabled to detect bacteria with the concentration of 105 – 109 CFU/ml. It gave representative outcomes with high concentrations of bioaerosols which are seen in highly contaminated environments.
The bioluminescence method is also recognised in hygiene monitoring of materials used in installations for conditioning and distributing drinking water. Apart from technical properties, the materials used to build water supply systems, predominantly plastic ones, must be corrosion-resistant. In accordance with the EU Directive 98/83/EC, the substances and contaminants which accompany these materials must not be present in water intended for human consumption in concentrations which may be noxious for health. Apart from testing migration of hazardous chemical substances, microbiological testing concerning the susceptibility of material surfaces to form biofilms, is also required. A given material or product may be used in contact with water intended for human consumption only if it has the authorisation of the National Sanitary Inspectorate based on the hygiene certificate issued by the National Institute of Public Health - National Institute of Hygiene. The research method used in this institution consists in bioluminescence assay of swabs from materials following sample exposition to a dynamic water flow for 8-10 weeks. These studies are conducted with control plates: positive ones - paraffin-coated glass plates, and negative ones - stainless steel plates. The examined material is approved when the bioluminescence level measured on its surface after a given time period is not greater than its tenfold value for the negative control. The fact that the bioluminescence level tends to change during tests and the size of the surface which will remain in contact with water intended for human consumption are also taken into account when issuing the opinion. It is not easy to interpret the results. The analyses demonstrate numerous fluctuations of bioluminescence on various polyethylene and polypropylene materials, particularly in the first weeks of testing [Szczotko and Krogulski, 2010]. In other countries, the phenomenon of bioluminescence is also adapted to such testing. In the Netherlands, the BPP test (Biomass Production Potential) is conducted. It is used to measure ATP in static conditions without water flow [van der Kooij et al., 2001].
The usefulness of ATP assay in food assessment and the capacity of bacteria to adhere to bioactive polymers were analysed by Gutarowska et al. (2012). They demonstrated that it is possible to obtain well-correlated results concerning antimicrobial activity of polymers with the analyses performed using fluorescence microscopy. This method was considered a good marker of viability
of cells in which, according to the literature, we observe their atrophy and inhibition of ATP synthesis as well as decomposition of this compound by ATPases and phosphatases. For certain species of bacteria, this was observed as soon as after several or several dozen hours of culturing on a bioactive polymer. In these studies, ATP measurement also demonstrated differences in viability and adhesion between the strains examined.
Bioluminescence in hygiene monitoring in healthcare facilities The ATP-based method is not widely acknowledged in the assessment of cleanliness of surfaces in healthcare institutions. This is because monitoring usually concerns frequently cleaned and disinfected surfaces, and the expected number of microbes on these surfaces is low, which, in turn, translates into a weak
bioluminescence signal, and is a limitation of this method. Different studies reveal numerous discrepancies concerning the accuracy of measurements resulting, among other things, from using various types of luminometers or manners of sample preparation [Davidson et al., 1999, Larson et al., 2003, Boyce et al., 2009, Aiken et al., 2011]. For instance, during the studies reported by Brown et al. (2010), one device showed the value of 3 352 RLU whereas the other displayed the value of 163 RLU for the same fluid. Moreover, of six measurements performed, one was false positive. Discrepancies were also noted in reproducibility of assays using the same device. In many cases, such inaccuracy of measurements may be explained by the usage of various cleaning detergents and chemical washing agents which may affect luminometric readings. However, the validation of the measuring procedure and establishing critical limits seems to be impossible in such a situation. Shama and Malik (2013) emphasise that the ATP-based method is not appropriate for monitoring microbial pathogens, particularly those with low infective doses. They also mention difficulties associated with material sampling from uneven or coarse surfaces in hospital settings, such as fabrics covered with biofilm enclosed by polysaccharide matrix. The authors draw attention to significant differences in the degree of intracellular ATP release depending on the species and physiological condition of a microbe. Such differences may even amount to 250% in the cases analysed by these authors. In the studies of Willis et al. (2007), the attempts to correlate the results of bioluminometric tests and culturebased analyses, conducted in hospital settings on various surfaces, such as floors, tables, windows or bathrooms, produced discrepant outcomes. The correlation coefficient indicated was very low and equalled 0.078 which is typical of this type of environment. It was, however, possible to find more contaminated surfaces, which was the floor under patients’ beds, and less contaminated ones, such as personal belongings.
Bioluminescence method in textile hygiene monitoring It was attempted to use the ATP-based method to detect microbial contamination on textiles. A common method to dispose of microbes from textiles is washing them. Hence, in the analysed case, the presence of bacteria was investigated on highly contaminated materials washed prior to the measurements. It was also checked whether bacteria can be detected on fabrics which might become contaminated while being washed together with other contaminated fabrics. Moreover, the presence of microbes was also investigated in the washing bath and water after rinsing. A suspension with Escherichia coli with the concentration of 6 x 108 CFU/ml was placed on two different textile types (100% cotton fabric - warp and
weft and blend fabric: warp - 100% cotton and weft - 100% polyester). Both contaminated fabrics and non-contaminated samples were washed with the use of a standard washing agent in the temperature of 40 ± 2ºC. Subsequently, the samples underwent extraction in order to recover the microbes. The count of living bacteria in the samples washed was conducted with the culture-based method using selective medium. The outcomes were compared with luminometric signals recorded for the corresponding samples. Prior to conducting tests on fabrics that did not undergo extraction, a sample material was taken with a swab free from ATP, which was then placed in a reaction mixture, and bioluminescence tests were conducted with the use of a HY-LiTE®2 device (Merck). The average results from three measurements for each of the methods, including standard deviation values, are presented in table 1.
Table 1. The number of E.coli bacteria in extracts of fabrics washed together with textiles contaminated with a suspension with E.coli measured using culture-based method, and a corresponding level of ATP signal
Samples of textiles and
water after washing
Bacteria count in textile
extracts
[CFU/ml]
ATP level in textile
samples after washing
[RLU/25cm2]
contaminated textiles, without washing
cotton fabric
13.33 x 106 ± 7.57 x 106
3 267 ± 1 358
blend fabric
8.67 x 106 ± 5.18 x 106
9 867 ± 6 493
contaminated textiles, after washing
cotton fabric
absent1
85 ± 12
blend fabric
absent
187 ± 38
non-contaminated textiles, after washing with contaminated cotton fabrics
cotton fabric
40 ± 30
44 ± 8
blend fabric
absent
31 ± 7
washing bath
absent
1 433 ± 58
water after the last rinsing
70 ± 18
61 ± 3
non-contaminated textiles, after washing with contaminated blend fabrics
cotton fabric
83 ± 77
43 ± 3
blend fabric
absent
38 ± 18
washing bath
absent
850 ± 132
water after the last rinsing
115 ± 21
51 ± 14
1absent - bacteria undetected by the culture-based method
Source: own research
The outcomes presented in table 1 indicate that when textiles are highly contaminated a reliable bioluminescence signal may be obtained - at the level of several thousand units. It corresponds to several million CFU per 1 ml of textile extracts analysed with the culture-based method on microbiological medium. Following washing, the bioluminescence signal decreases considerably in the case of previously contaminated samples and those that could become contaminated in these washing process. Nevertheless, it indicates the presence of microbes in the fabrics whereas the culture-based method failed to detect bacteria in extracts from numerous textile samples. Bacteria were present in washed fabrics, which is demonstrated by their growth on Endo media plates (fig. 1). The bioluminescence signal was also registered to a satisfactory degree in fluids after washing, particularly in washing baths. Therefore, it may be concluded that this methods
enables to indicate the path of textile contamination during washing.
Conclusions
The bioluminescence method is being acknowledged as a tool to monitor the efficacy of cleaning and disinfecting practices in both food industry and health care. However, microbial contamination monitoring by ATP measurement cannot be commonly applied due to considerable limitations of this method. Among others, these limitations result from: low sensitivity of commercially available
luminometers used for detection of microbes, poor reproducibility of results, particularly between surfaces with various properties and purposes, as well as unfavourable influence of environmental factors on the measurement outcomes. Nevertheless, it must be emphasised that the bioluminescence method may serve as a good educational and informational tool which is capable of indicating sites of higher susceptibility to microbial contamination in an easy and rapid way.
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