CHAPTER 2: REVIEW OF ENDOSPORE DETECTION TECHNOLOGY
2.5 Metabolism
All living cells respire and produce metabolites, each possessing unique intrinsic fluorescence property. Although endospores elicit a non detectable level of metabolism, metabolites such as reduced pyridine nucleotides, oxidized flavoproteins and ATP can readily be detected during the later phase of germination and outgrowth. ATP can be detected using a firefly luciferin/luciferase bioluminescence system. Other metabolites can be detected based on their intrinsic fluorescence using microspectrofluorometry, epifluorescence techniques. Multi wavelength intrinsic fluorescence detection enables pattern recognition algorithms for microbial fingerprinting. Intrinsic fluorescence markers
2.5.1 Impedance measurement
When bacteria grow, they produce metabolites which alter the conductivity of the medium, a property first observed in 1898. With the use of modern electrical measuring equipment, this nineteenth-century observation has been put to use as a rapid impedance microbiology technique52. However, use of charged antimicrobials, such as silver ions or cationic surfactant biocides, can lead to interpretive problems.
2.5.2 Microcalorimetry
Microcalorimetry, as a rapid analytical method for biocide testing, is based upon the principle that bacteria and other microorganisms produce heat when they metabolize.
Microcalorimeters can detect the small amount of heat produced53. Any surviving cells, following an inimical treatment, will, during subsequent incubation, metabolize and produce heat. Morgan et al. used microcalorimetry to examine the effect of antomicrobials on Streptococcus mutans and suggested that the data obtained by this technique gave a “better indication of antimicrobial efficacy than merely determining concentrations at which an antimicrobial agent is bacteriostatic or bactericidal”54.
The overall metabolic capacity, as measured by the heat released by cells, the number of cells of a sample is dependent on the number of cells present and can be measured using a microcalorimeter such as the Thermal Activity Monitor. Spores, after induced to germinate, start actively metabolizing and produce heat which can be measured using microcalorimetry.
2.5.3 ATP firefly luciferin-luciferase assay
Adenosine 5’triphosphate (ATP) is the primary source of chemical energy and a ubiquitous energy currency in all living organisms. The use of a firefly (Photuris pyralis) enzyme to quantify ATP in biological systems was first proposed by McElroy and Strehler in the 1940s55,56. The detection is based on the conversion of chemical energy to light energy during the breakdown of ATP. Firefly luciferase catalyzes the ATP- dependent oxidative decarboxylation of luciferin in the presence of oxygen and
magnesium ions into AMP and light. One photon of light is produced per molecule of ATP hydrolyzed when ATP is the limiting component in the reaction57. Measurement of ATP is a direct indication of cellular metabolism and is often reckoned as a metric for viability58,59.
First, nonmicrobial ATP is eliminated from the sample using a somatic cell releasing agent and a subsequent incubation in apyrase or ATPase. Bacterial cells are then disrupted using chemicals such as benzalkonium chloride. The ATP released is quantified using the luciferin-luciferase reaction. A differential filtration procedure is also reported to separate somatic from microbial cells60. Several different ATP reagents are available commercially and the protocol has been optimized over the years. For instance, a mutant luciferase resistant to benzalkonium chloride has been isolated to achieve maximum extraction of intracellular ATP from microbes and inactivation of the ATP- eliminating enzymes for removal of extracellular ATP61. Hattori et al. achieved a detection limit of 7.7 cfu mL-1 using vegetative cells of B. subtilis, while Promega Corporation reports a detection limit of 10 cfu mL-1 of vegetative cells of B. cereus.
Challenges are encountered in the detection of endospores using the luciferin/luciferase system. While a vegetative bacterium contains approximately 10-17 mole of ATP per cell62, dormant spores of a number of Bacillus species have no detectable biosynthetic or metabolic activity and contain low levels of ATP63-65. Kodaka et al. reported that an endospore contains about 10-21 mole ATP per cell, four orders of magnitude lower than that of a vegetative bacterium66. In addition, ATP cannot be sufficiently extracted from endospores, unlike their vegetative counterparts, due to a nonporous and hardy proteinaceous spore coat67-69. Theoretically speaking, if the current
luminometers can detect approximately ten vegetative bacilli cells per milliliter, the limit of detection for endospores will be 105 endospores mL-1.
There are several approaches for endospore detection using the firefly luciferase assay. One method is the screening for endospores by heat shock at 80°C for 15 minutes allowing only spores to survive, followed by incubation and detection of ATP in the subsequent outgrowth of vegetative cells. In this manner, bacterial outgrowth from endospores on test strips was measured after five hours of incubation to validate the sterilization efficiency of autoclaves70. This method is relatively faster than the traditional cultivation method, but can only provide semi-quantitative counts of the original endospore population.
Other approaches take advantage of the production of ATP from endospores during germination. Less than 1% of the adenine nucleotide pool in spores is ATP, but it accounts for 80% in vegetative cells. Most of the nucleotides in endospores are stored in the form of 3-phosphoglyceric acid64. Nevertheless, within the first minute of germination, the large depot of 3-phosphoglyceric acid is catabolized into ATP71. In addition, coat porosity increases after the onset of germination, which permits easy extraction of intracellular ATP72. Fujunami et al. measured a large increase in light intensity of B. subtilis spores after 30 minutes of incubation in nutrient broth supplemented with L-alanine in the presence of various white powders73. Rapid accumulation of ATP upon nutrient induced germination also held true for anaerobic Clostridium spores74.
Pressured-induced germination of B. subtilis at 100 MPa resulted in a rapid production of ATP, but no ATP was formed during germination at 600 MPa75, while
hydrogen peroxide-treated spores can germinate, but accumulate very little ATP. The mechanism of ATP accumulation during germination is still not very clear. Further study may shed light on the subject of endospore viability and the phenomenon of germinable- but-not-culturable.
Direct extraction of ATP from endospores is also another feasible method for detection. Venkateswaran used ATP as a biomarker of viable microorganisms in clean- room facilities. The use of benzalkonium chloride (Kikkoman International, Inc.) completely lyzed vegetative cells and endospores in surface swab samples to release ATP for detection. A low ATP-CFU ratio has been associated with endospores, ranging from 10-18 to 10-20 moles ATP per CFU76.
Compared with other endospore detection techniques, ATP bioluminescence measurements offer many advantages, such as high sensitivity, large dynamic range, high specificity, and rapidity. The luciferase assay can easily be automated for high throughput processing. The instruments tend to be inexpensive, portable and can detect viable bacteria in relatively complex media, such as powder and milk. False positives are rare because the enzyme is highly specific for ATP, and ATP is lost rapidly upon cell death.
Nevertheless, the assay suffers from drawbacks such as a low level of ATP in endospores and interference from extracellular and somatic ATP. Also the assay is not species- specific and some food samples have been shown to contain inhibitory substances that interfere with luciferase activity77.
At present, the ATP assay is mainly used to detect and enumerate vegetative cells of pathogenic bacteria for food quality control and hygiene testing78. It has been used to detect as few as 104 cfu mL-1 of bacteria in milk in five to ten minutes79, a bacterial
population of 105 cfu mL-1 in fruit juice80 and 5 × 104 cfu g-1 in meat81. The application of the ATP assay on endospore detection in foodstuffs is still in its initial phase. Because endospores are the likely candidates for surviving pasteurization, steaming, and vacuum processes, the ATP assay could be expected to play a bigger role in the detection of pathogenic foodborne endospores in the future.
More recently, the ATP luciferase assay has shown promising results in detecting anthrax spores. The lysin plyG, isolated from a phage that infects B. anthracis was used to specifically target and lyse germinating B. anthracis spores causing a pronounced release of intracellular ATP. ATP could be detected within 60 minutes of the addition of germinants from as few as 100 spores using a phage sensitive B. cereus as a surrogate for B. anthracis82. The ATP assay was also used to detect airborne bacterial spores. A detection limit of 105 cfu mL-1 was reported using aerosolized B. globigii spores—a surrogate for B. anthracis83. In a nutshell, the luciferin-luciferase reaction could potentially be used for the detection of anthrax spores and validation of decontamination regimes after an anthrax attack.