Enhanced microbial biomass assay using mutant luciferase resistant to benzalkonium chloride.
In a biomass assay based on adenosine 5(')-triphosphate (ATP) bioluminescence, extracellular ATP is removed; then intracellular ATP is extracted from the microorganism by an ATP extractant and subsequently reacted with luciferase. To provide a highly sensitive assay, the concentration of benzalkonium chloride (BAC) in the ATP extractant was optimized by using a mutant luciferase resistant to BAC. The use of 0.2% BAC, which was acceptable for the luciferase, simultaneously achieved the maximum extraction of intracellular ATP from microorganisms and the inactivation of the ATP-eliminating enzymes for removal of extracellular ATP. The detection limit (blank+3 SD) for ATP was 1.8x10(-14)M (1.8x10(-18)mol/assay) in the presence of the ATP extractant with coefficients of variation of 0.7 to 6.3%. The reagent system coupled with the ATP-eliminating enzymes allowed for the detection of 93 colony-forming units (CFU)/ml of Escherichia coli ATCC 25922, 170CFU/ml of Pseudomonas aeruginosa ATCC 27853, 170CFU/ml of Proteus mirabilis ATCC 29906, 68CFU/ml of Staphylococcus aureus ATCC 25923, and 7.7CFU/ml of Bacillus subtilis ATCC 6051. The yeast cell of Saccharomyces cerevisiae IFO 10217 could be detected at 1CFU/ml. With 54 kinds of microorganisms, the average ATP extraction efficiency compared to the trichloroacetic acid extraction method was 81.0% in 24 strains among gram-negative bacteria, 99.4% in 13 strains among gram-positive bacteria, and 97.0% in 17 strains among yeast. The ATP contents of the gram-negative bacteria, gram-positive bacteria, and yeasts ranged from 0.40 to 2.70x10(-18)mol/CFU (mean=1.5x10(-18)mol/CFU), from 0.41 to 16.7x10(-18)mol/CFU (mean=5.5x10(-18)mol/CFU), and from 0.714 to 54.6x10(-16)mol/CFU (mean=8.00x10(-16)mol/CFU), respectively.
Hattori
Noriaki
N
Research and Development Division, Kikkoman Corp., Noda City, Chiba pref., Japan. 8345@mail.kikkoman.co.jp
Sakakibara
Tatsuya
T
Kajiyama
Naoki
N
Igarashi
Toshinori
T
Maeda
Masako
M
Murakami
Seiji
S
D000255:Adenosine Triphosphate / Q000032:analysis* / Q000378:metabolism; D001548:Benzalkonium Compounds / Q000737:chemistry* / Q000494:pharmacology; D018533:Biomass; D015169:Colony Count, Microbial; D024881:Drug Resistance, Bacterial; D006090:Gram-Negative Bacteria / Q000254:growth & development / Q000378:metabolism; D006094:Gram-Positive Bacteria / Q000254:growth & development / Q000378:metabolism; D007700:Kinetics; D008156:Luciferases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements; D014238:Trichloroacetic Acid / Q000737:chemistry
D001548:Benzalkonium Compounds; D014238:Trichloroacetic Acid; D000255:Adenosine Triphosphate; D008156:Luciferases