Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1991-10-21
2002-11-05
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S320100, C435S252300, C435S252330, C435S455000, C435S007100, C435S029000, C435S325000, C435S235100, C435S254100, C536S023100, C536S023500, C536S024100
Reexamination Certificate
active
06475719
ABSTRACT:
Biotests are methods where one uses living cells or organisms as tools to detect different analytes. Many of those methods utilize bacterial or yeast cells. Procaryotic organisms and especially
Escherichia coli
bacterium are very well characterized. Yeast cells are eucaryotic organisms and grow as single cells. The cultivation of yeast is easier than the cultivation of higher eucaryotes. Yeast cells grow in simple cultivation media and they do not need addition of complicated growth factors. The knowledge of yeast is expanding rapidly and comprehensive maps of genes are known. Hundreds of specific mutations for both bacteria and yeast are known. With knowledge of specific mutations it is possible to study the activity of specific reactions and metabolic pathways. For instance with antibiotic sensitive bacterial mutants trace amounts of antibiotics cause changes in the metabolism or in the membranes. Using antibiotic sensitive bacterial mutants, one is able to develop very sensitive tests to measure residual antibiotics from biological material. Bacteria and yeast with mutations in their DNA repair mechanisms, or mutants whose cell membranes might be porous for different small molecular weight substances, e.g., antibiotics, are more sensitive to genotoxic substances than wild type. Using different mutant strains, one is able to measure for the presence of antibiotics and toxic or mutagenic agents. Genetic engineering techniques can be used to transfer new characteristics into bacteria or yeast cells. The new characteristics can be provided by proteins which are encoded by viruses. The protein do not exist naturally in the target organism. Use of genetic engineering techniques expands the applicability of bacteria and yeast cells for use in biotests.
The universal genetic code of DNA is similar in each organism. The relationship between carcinogenicity and mutagenicity is the basis for using tests for mutagenic agents as prescreening tests for carcinogenic agents. Testing for carcinogenicity in animals is extremely expensive and time consuming. Use of tests for mutagenic agents as a quick screening method for carcinogenicity has raised hope and interest. The quick screening method for carcinogenicity would decrease animal-based carcinogenicity testing.
The AMES-test (Ames, B. N., McCann, J. and Yamasaki, E. (1975) Mutat. Res. 31, 347) is the test used most often to screen for mutagenic agents. The AMES-test utilizes
Salmonella typhimurium
as a test organism. Utilizing the AMES-test one is able to detect the genotoxicity of most mycotoxins, aromatic amines and polycyclic hydrocarbons. However, the Ames-test is not able to detect the genotoxicity of carcinogenic metal salts or chlorinated hydrocarbons. The
S. typhimurium
strains used in the AMES-test contain point mutations in the biosynthetic route of the amino acid histidine. As the bacteria are exposed to the action of mutagenic substance, a reversion mutation occurs in the gene for histidine biosynthesis and the bacterium starts to produce histidine endogenously. Endogenous production of histidine gives the cell the ability to grow on minimal growth medium containing no added histidine. A pitfall in the AMES-test is poor sensitivity and slow performance. In the AMES-test all other genotoxic changes such as those acting on enzymes remain undetected. The test is also rather expensive for each particular compound tested.
A test for the detection of genotoxic substances based on bioluminescence is known (Ulizur, S., Weiser, I. and Yannai, S. (1980) Mut. Res., 74, 113-121). In this method, dark mutants of
Photobacterium leioanathi
and
P. fischeri
are used. In the presence of genotoxic substances, these strains start to emit light. The theoretical background of the method remains somewhat obscure. It has been speculated that the effect of removing a repressor or preventing its formation combined with a change in the chromosomal DNA of the bacterium might trigger the formation of light producing proteins. Different genotoxic substances act with different rates in this test due to the variety of different classes of substances. This test is faster than the Ames-test but is by no means easier to use. The bacteria used in the test should produce light during long and varying periods of time (30 min to 10 h) depending on the substance. The bacteria used are not capable of stably emitting light, which makes the method somewhat problematic. Due to these facts, the method is not easily automated for use in routine work when there are a lot of specimens to be analyzed. Additionally, being of marine origin, the cultivation temperature of the bacteria is rather low, 15° C.—it is not known how well the effect of genotoxic substances correlate to the effects on man whose body temperature is 37° C.
Antibiotics, used as medicines against microbial invasion, are detected from body fluids in order to study the dosage and penetration of the medicine. The effective therapeutic range of the antibiotic is often rather narrow and the risks due to overdosage might be large. It is also important to measure the presence/concentration of antibiotics in meat and cow milk due to symptoms in people with allergies to antibiotics. The cow milk used in cheese production should not contain antibiotics due to the fact that cheese making bacteria are not able to grow in antibiotic-contaminated milk. Common methods for detecting antimicrobial medicines are microbiological methods performed on agar. A direct method is to measure the inhibition of the growth of sensitive bacterial strains. One can also measure some metabolic parameters, such as acid production of a sensitive strain of bacteria, using proper color indicators.
Typical examples of agar diffusion tests are cylinder, hole or disk methods. The difference between these tests is in the way the sample is applied to the agar and also in the way the bacteria are utilized in the test.
Since microbiological methods utilize bacteria or their spores, the sensitivity of the test bacteria is of utmost importance. In the tests described above compromises had to be made in the choice of a suitable test strain since great sensitivity against antimicrobial agents and other characteristics needed for the test strain have not been found in the same strain of bacteria.
Major drawbacks when using microbes in antibiotic residue tests are slow speed and insensitive performance. In these methods one controls the growth of a test strain and thus the test cannot be performed in an hour. This is due to the fact that growth of microbes is a slow process even in its fastest mode. In addition, in many cases spores or freeze-dried microbes are used which make the tests even slower to perform.
Antibiotic detection methods based on bioluminescence measurement are known. Ulizur (1986, Methods Enzymol., 133, 275-284) describes three different ways to use bioluminescence for the detection of antimicrobial agents: a) lysis-test, b) induction test and c) bacteriophage test. In the first one, the lux-genes isolated from
Vibrio fischeri
produce luciferase protein which in the presence of substrates produces light. The genes have been cloned into a plasmid and transferred to
Bacillus subtilis
. The
B. subtilis
strain utilized is sensitive to antibiotics which affect bacterial membranes. Examples of such antibiotics are penicillins and cephalosporins. In the lysis-test, the lux-gene-containing
B. subtilis
is grown together with a test sample. If the test sample contains an antibiotic, the synthesis of cell wall components is prevented and the bacteria are lysed. Thus the culture yields lower light emission when compared to a culture lacking the test sample.
The induction test utilizes dim mutants of
P. phosphoreum
bacteria, which do not produce light. The induction test and other bioluminescence tests developed by Ulizur are based on exploitation of the chromosomal DNA of the target cell. Antibiotics affecting protein synthesis are detected in the induction test. When the bacteria are incubated together with compounds that bind
Karp Matti
Korpela Matti
Banner & Witcoff , Ltd.
Bio-Orbit Oy
Guzo David
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