SE424090B - Method for determining the concentration of an antibiotic - Google Patents
Method for determining the concentration of an antibioticInfo
- Publication number
- SE424090B SE424090B SE8104898A SE8104898A SE424090B SE 424090 B SE424090 B SE 424090B SE 8104898 A SE8104898 A SE 8104898A SE 8104898 A SE8104898 A SE 8104898A SE 424090 B SE424090 B SE 424090B
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- antibiotic
- concentration
- phage
- luminescent
- antibiotics
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- 230000003115 biocidal effect Effects 0.000 title claims abstract description 28
- 239000003242 anti bacterial agent Substances 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title claims abstract description 13
- 241000894006 Bacteria Species 0.000 claims abstract description 23
- 241001515965 unidentified phage Species 0.000 claims abstract description 13
- 230000000694 effects Effects 0.000 claims abstract description 5
- 238000011534 incubation Methods 0.000 claims abstract description 3
- 230000001419 dependent effect Effects 0.000 claims abstract 2
- 229940088710 antibiotic agent Drugs 0.000 claims description 15
- 210000004027 cell Anatomy 0.000 description 15
- 238000004020 luminiscence type Methods 0.000 description 10
- 238000001727 in vivo Methods 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 5
- 230000015556 catabolic process Effects 0.000 description 4
- 238000006731 degradation reaction Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 230000006820 DNA synthesis Effects 0.000 description 3
- 229940123573 Protein synthesis inhibitor Drugs 0.000 description 3
- 230000006819 RNA synthesis Effects 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000002458 infectious effect Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000003865 nucleic acid synthesis inhibitor Substances 0.000 description 3
- 238000001243 protein synthesis Methods 0.000 description 3
- 239000000007 protein synthesis inhibitor Substances 0.000 description 3
- 230000014616 translation Effects 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 229930189077 Rifamycin Natural products 0.000 description 2
- 230000000845 anti-microbial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 229960005091 chloramphenicol Drugs 0.000 description 2
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 229960000210 nalidixic acid Drugs 0.000 description 2
- MHWLWQUZZRMNGJ-UHFFFAOYSA-N nalidixic acid Chemical compound C1=C(C)N=C2N(CC)C=C(C(O)=O)C(=O)C2=C1 MHWLWQUZZRMNGJ-UHFFFAOYSA-N 0.000 description 2
- 229960003292 rifamycin Drugs 0.000 description 2
- HJYYPODYNSCCOU-ODRIEIDWSA-N rifamycin SV Chemical compound OC1=C(C(O)=C2C)C3=C(O)C=C1NC(=O)\C(C)=C/C=C/[C@H](C)[C@H](O)[C@@H](C)[C@@H](O)[C@@H](C)[C@H](OC(C)=O)[C@H](C)[C@@H](OC)\C=C\O[C@@]1(C)OC2=C3C1=O HJYYPODYNSCCOU-ODRIEIDWSA-N 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- WZEJGSYLAFXHCK-YQVWRLOYSA-N (2s)-2-amino-n-[2-[[(1r,2r)-1,3-dihydroxy-1-(4-nitrophenyl)propan-2-yl]amino]acetyl]-3-phenylpropanamide Chemical compound C([C@H](N)C(=O)NC(=O)CN[C@H](CO)[C@H](O)C=1C=CC(=CC=1)[N+]([O-])=O)C1=CC=CC=C1 WZEJGSYLAFXHCK-YQVWRLOYSA-N 0.000 description 1
- 238000009636 ATP test Methods 0.000 description 1
- 229940122029 DNA synthesis inhibitor Drugs 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000701533 Escherichia virus T4 Species 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- YJQPYGGHQPGBLI-UHFFFAOYSA-N Novobiocin Natural products O1C(C)(C)C(OC)C(OC(N)=O)C(O)C1OC1=CC=C(C(O)=C(NC(=O)C=2C=C(CC=C(C)C)C(O)=CC=2)C(=O)O2)C2=C1C YJQPYGGHQPGBLI-UHFFFAOYSA-N 0.000 description 1
- 229940123752 RNA synthesis inhibitor Drugs 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 241000607618 Vibrio harveyi Species 0.000 description 1
- 241000607365 Vibrio natriegens Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000012984 antibiotic solution Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 238000005415 bioluminescence Methods 0.000 description 1
- 230000029918 bioluminescence Effects 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 229960002950 novobiocin Drugs 0.000 description 1
- YJQPYGGHQPGBLI-KGSXXDOSSA-N novobiocin Chemical compound O1C(C)(C)[C@H](OC)[C@@H](OC(N)=O)[C@@H](O)[C@@H]1OC1=CC=C(C(O)=C(NC(=O)C=2C=C(CC=C(C)C)C(O)=CC=2)C(=O)O2)C2=C1C YJQPYGGHQPGBLI-KGSXXDOSSA-N 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000011533 pre-incubation Methods 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229940073490 sodium glutamate Drugs 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
_Hs1o4s9s-5 2 10 15 20 25 30 35 Antimicrobial Ag. Chemotherapy lá (1979) 43-45). Den andra metoden utnyttjar syraproduktionen från laktos (Ericsson, H.M. och Sherris, J.C., Acta pathol. et Miorobiol. Scan.Sect. B Suppl. gll 1971). Ett kortare test (90-120 minuter) har fram- tagits av Nilsson (Nilsson, L., Antimicrobial Ag. Chemotheraphy lg (1978) 812-816) och av andra (Whitehead, T.P., L.J. Kricka, T.J.N. Carter och C.H.G. Thorpe, Clin.Chem. gå (1979) 1531-1546) och utgår från det faktum att utomcellulärt ATP hos den bakterie- kultur som behandlas med antibiotikumet ökar med en ökande antibiotikumkoncentration. De minsta koncentrationer av antibio- z tikumet som bestämdes med dessa test var 2_pg kloramfenikol per ml med hjälp av Beneckea natriegens testet och llng gentamicin per ml med användning av ATP-testet. Användningen av luminösa (eller luminiscenta) bakterier för bestämning av antibiotika föreslogs ursprungligen av Rake, McKee och Jones (Proc. Soc. _Hs1o4s9s-5 2 10 15 20 25 30 35 Antimicrobial Ag. Chemotherapy (1979) 43-45). The second method utilizes the acid production from lactose (Ericsson, H.M. and Sherris, J.C., Acta pathol. Et Miorobiol. Scan.Sect. B Suppl. Gll 1971). A shorter test (90-120 minutes) has been developed by Nilsson (Nilsson, L., Antimicrobial Ag. Chemotherapy (1978) 812-816) and by others (Whitehead, TP, LJ Kricka, TJN Carter and CHG Thorpe, Clin.Chem. Go (1979) 1531-1546) and is based on the fact that extracellular ATP in the bacterial culture treated with the antibiotic increases with an increasing antibiotic concentration. The minimum concentrations of the antibiotic determined by these tests were 2_pg chloramphenicol per ml using the Beneckea natriegens test and llng gentamicin per ml using the ATP test. The use of luminous (or luminescent) bacteria for the determination of antibiotics was originally suggested by Rake, McKee and Jones (Proc. Soc.
Exper.Biol. & Med., ål (1942) 273-274).Exper.Biol. & Med., Eel (1942) 273-274).
Avsikten med föreliggande uppfinning är att åstadkomma ett förfarande för momentan bestämning av ett antibiotikum i av- sevärt lägre koncentrationer än de som bestämts med de ovan beskrivna metoderna, varvid förfarandet erfordrar en betydligt kortare tid. Uppfinningens kännetecken framgår av de efter beskrivningen följande patentkraven.The object of the present invention is to provide a method for instantaneous determination of an antibiotic in considerably lower concentrations than those determined by the methods described above, the method requiring a considerably shorter time. The features of the invention appear from the claims following the description.
Uppfinningen kommer nu att förklaras i detalj med hänvisning till.bifogade ritning på vilken: Fig. l - 3 visar diagram illustrerande användningen av upp- finningen för tre olika antibiotika.The invention will now be explained in detail with reference to the accompanying drawing, in which: Figs. 1-3 show diagrams illustrating the use of the invention for three different antibiotics.
Enligt föreliggande uppfinning mätes kocnentrationen av ett antibiotikum genom utnyttjande.av ett biologiskt kopplat system vid vilket starkt luminiscenta bakterier blandas med en specifik bakteriofag. I frånvaro av antibiotikumet pacificeras de lumini- scenta bakterierna och deras in vivo luminiscens går ner nästan 14.111 noll .According to the present invention, the concentration of an antibiotic is measured by using a biologically coupled system in which highly luminescent bacteria are mixed with a specific bacteriophage. In the absence of the antibiotic, the luminescent bacteria are pacified and their in vivo luminescence decreases by almost 14,111 zeros.
Bakterier är värdorganismer för en speciell grupp av virus som kallas bakteriofager. Bakteriofagadsorptionens kinetik och de intracellulära stegen hos fagutvecklingen är väl beskrivna.Bacteria are host organisms for a special group of viruses called bacteriophages. The kinetics of bacteriophage adsorption and the intracellular stages of phage development are well described.
Under optimala villkor adsorberas fagen på värdcellen varefter 10 15 20 25 30 35 3 8104898-5 dess nukleinsyror införes i denna. Under ett första skede (eklipsskedet) styr fagen syntesen av nya enzymerrhos värdcellen.Under optimal conditions, the phage are adsorbed on the host cell, after which its nucleic acids are introduced into it. During a first stage (eclipse stage), the phage controls the synthesis of new enzymes in the host cell.
Dessa nya proteiner (tidiga proteiner) omfattar enzymer som är nödvändiga för syntes av fag-DMA. Senare under eklipsperioden uppträder "sena proteiner" vilka omfattar underenheter hos fagens ändar liksom ett lysozym som bryter ner värdcellens cell- vägg. Det sista skedet innebär att cellen brister och de nybildade infektuösa fagerna frigöres.These new proteins (early proteins) include enzymes necessary for the synthesis of phage DMA. Later in the eclipse period, "late proteins" appear which comprise subunits at the ends of the phage as well as a lysozyme which breaks down the cell wall of the host cell. The last stage means that the cell breaks down and the newly formed infectious phages are released.
Luminiscenta bakterier är liksom andra prokarioter angripbara av vissa bakteriofager. Den lytiska cykeln hos dessa fager är i princip densamma som den hos den mest kända fagen T4 hos E.coli.Luminescent bacteria, like other prokaryotes, are attackable by certain bacteriophages. The lytic cycle of these phages is basically the same as that of the most well-known phage T4 in E.coli.
Då starkt luminiscenta bakteriekulturer infekteras av sina specifika bakteriofager kan inga speciella förändringar observeras under "eklipsperioden“ som normalt varar cirka 30-45 minuter.As highly luminescent bacterial cultures are infected by their specific bacteriophages, no special changes can be observed during the "eclipse period" which normally lasts about 30-45 minutes.
Nedgången i in vivo luminiscensen uppträder samtidigt som den infektuösa fagen uppträder i mediet. Således representerar nivån av in vivo luminiscensen hos den infekterade cellen produktionen av den bakteriofag som är en följd av nedbrytningen av de luminösa bakterierna. Den fullständiga syntesen av en infektuös fag erfordrar en komplett serie av DNA, RNA och protein- synteser. Antibiotika som blockerar åtminstone någon av dessa synteser blockerar även produktionen av fagen och förhindrar därigenom nedbrytningen av de luminiscenta bakterierna.The decrease in in vivo luminescence occurs at the same time as the infectious phage appears in the medium. Thus, the level of in vivo luminescence of the infected cell represents the production of the bacteriophage resulting from the degradation of the luminous bacteria. The complete synthesis of an infectious phage requires a complete series of DNA, RNA and protein syntheses. Antibiotics that block at least one of these syntheses also block the production of the phage, thereby preventing the degradation of the luminescent bacteria.
Föreliggande uppfinning hänför sig till en metod som utnyttjar detta kopplade biologiska system för bestämning av DNA, RNA och proteinsynteshämmande antibiotika.The present invention relates to a method utilizing this coupled biological system for the determination of DNA, RNA and protein synthesis inhibiting antibiotics.
Avsikten med uppfinningen är att åstadkomma en metod för snabb bestämning av DNA, RNA och proteinsynteshämmare. Det beskrivna testet erfordrar en tid av endast 60-90 minuter och kan automati- seras i det närmaste fullständigt.The object of the invention is to provide a method for rapid determination of DNA, RNA and protein synthesis inhibitors. The test described requires a time of only 60-90 minutes and can be almost completely automated.
Det nya testet baserar sig på ett biologiskt kopplat system vid vilket starkt lysande bakterier blandas med en specifik bakteriofag. I frånvaro av ett antibiotikum bryts bakterierna ner inom 45-60 minuter och deras in vivo luminiscens reduceras med en faktor 100-2002 Sådana antibiotika som skyddar de lumini- scenta bakterierna från nedbrytning av bakteriofagen är av tre typer: -8104898-5 4 10 15 20 _25 30 '35 (a) Antibiotika som hämmar DNA-syntesen såsom novobiocin eller nalidixicsyra, (b) Antibiotika som hämmar RNA-syntesen såsom rifamycin, (c) Antibiotika som hämmar proteinsyntesen såsom exempelvis kloramfenikol.The new test is based on a biologically coupled system in which strongly luminous bacteria are mixed with a specific bacteriophage. In the absence of an antibiotic, the bacteria degrade within 45-60 minutes and their in vivo luminescence is reduced by a factor of 100-2002 Such antibiotics that protect the luminescent bacteria from degradation of the bacteriophage are of three types: -8104898-5 4 10 15 (A) Antibiotics that inhibit DNA synthesis such as novobiocin or nalidixic acid, (b) Antibiotics that inhibit RNA synthesis such as rifamycin, (c) Antibiotics that inhibit protein synthesis such as chloramphenicol.
Samtliga dessa antibiotika påverkar ej in vivo luminiscensen hos de luminiscenta bakterierna under inkubationstiden vid testet. Om man önskar specifikt detektera verkningssättet hos det aktuella antibiotikumet (om det verkar som DNA-syntes hämmare eller som proteinsynteshämmare) skall det testade antibiotikumet tillföras under den sista tredjedelen av den latenta faginfektions- perioden, Under denna del av den intercellulära fagutvecklingen kommer endast proteinsynteshämmare att mera påtagligt skydda de luminiscenta bakterierna. DNA-synteshämmare är aktiva endast under begynnelseskedet av infektionscykeln, d.v.s. 10-20 minuter efter infektering.All of these antibiotics do not affect the in vivo luminescence of the luminescent bacteria during the incubation period of the test. If it is desired to specifically detect the mode of action of the antibiotic in question (whether it acts as a DNA synthesis inhibitor or as a protein synthesis inhibitor), the tested antibiotic should be administered during the last third of the latent phage infection period. During this part of the intercellular phage development, only protein synthesis inhibitors will more significantly protect the luminescent bacteria. DNA synthesis inhibitors are active only during the initial stage of the infection cycle, i.e. 10-20 minutes after infection.
En ytterligare fördel med användning av fagtest för antibiotika är möjligheten att använda faginfekterade frystorkade lumini- scenta bakterier. Därvid nedfryses och torkas de luminiscenta bakterierna kort efter det att de infekterats av fagerna och fagens DNA har trängt in i cellen (en förinkubering av under 10 minuter vid 30°C). Sådana infekterade frystorkade celler kan vid lagring vid 4-6°C återfå sin luminiscens i full utsträckning vid vibrering i lämplig buffert. Emellertid kommer dessa celler att brytas sönder efter cirka 60-90 minuter på grund av den intercellulära fagutvecklingen. Antibiotika som tillföras vid hydreringsskedet hämmar fagutvecklingen och cellnedbrytningen.An additional advantage of using phage tests for antibiotics is the possibility of using phage-infected freeze-dried luminescent bacteria. The luminescent bacteria are frozen and dried shortly after they have been infected by the phage and the phage's DNA has entered the cell (a pre-incubation of less than 10 minutes at 30 ° C). Such infected lyophilized cells, when stored at 4-6 ° C, can fully recover their luminescence upon vibration in the appropriate buffer. However, these cells will disintegrate after about 60-90 minutes due to the intercellular phage development. Antibiotics administered during the hydration phase inhibit phage development and cell degradation.
Användning av frystorkade system kräver i princip endast två steg för antibiotikabestämning: 1. Alikvoter av de hydrerade faginfekterade luminiscenta bakterierna tillföres såväl provet som en känd standard av det testade antibiotikumet, 2. Proven inkuberas under 90 minuter och nivån hos in vivo luminiscensen bestäms.The use of lyophilized systems basically requires only two steps for antibiotic determination: 1. Aliquots of the hydrated phage-infected luminescent bacteria are added to both the sample and a known standard of the tested antibiotic, 2. The samples are incubated for 90 minutes and the level of in vivo luminescence is determined.
Förfarandet enligt uppfinningen kommer nu att beskrivas i detalj med hjälp av ett exempel: 10 15 20 25 30 35 8104898-5 5 Exempel (l) Luminiscenta bakterier av stammen Beneckea Harveyi (2) (3) (4) (5) (6) (7) (8) (9) (10) (ll) får växa vid 30°C i ett flytande komplex ASWRP(l) medium till en slutlig eelitärher ev s - 108 en luminiscens av l-2 ' 1012 kvanta per sekund och ml. celler per ml och Den luminiscenta kulturen infekteras med bakteriofagen "hv-3" i förhållande 10 PFU per luminiscent bakterie.The process according to the invention will now be described in detail by means of an example: Example (1) Luminous bacteria of the strain Beneckea Harveyi (2) (3) (4) (5) (6) (7) (8) (9) (10) (ll) may grow at 30 ° C in a liquid complex ASWRP (l) medium to a final elitist or s - 108 a luminescence of l-2 '1012 quantities per second and ml. cells per ml and The luminescent culture is infected with the bacteriophage "hv-3" in a ratio of 10 PFU per luminescent bacterium.
Blandningen inkuberas under 10-15 minuter vid 30°C så att fagen adsorberas och dess DNA tränger in i cellen.The mixture is incubated for 10-15 minutes at 30 ° C so that the phage is adsorbed and its DNA enters the cell.
Den infekterade kulturen nedkyles snabbt i is och de infekterade cellerna centrifugeras vid Oo. _Cellkulorna nedsänkes-i en blandning bestående av 5% havsvatten innehållande 0,15 N natriumglutamat och 0,25 N sykros vid pH 7,0. Den slutliga cellkoncentrationen är ej kritisk men ett lämpligt värde är 109 celler per mi. 0,5 ml av den sålunda behandlade kulturen fylles på lämp- liga flaskor och kulturerna frystorkas under standardbe- tingelser.The infected culture is rapidly cooled in ice and the infected cells are centrifuged at 0 ° C. The cell beads are immersed in a mixture consisting of 5% seawater containing 0.15 N sodium glutamate and 0.25 N sucrose at pH 7.0. The final cell concentration is not critical but a suitable value is 109 cells per ml. 0.5 ml of the culture thus treated is filled into suitable bottles and the cultures are lyophilized under standard conditions.
De frystorkade kulturerna kan förvaras vid 4-9°C under åtminstone 4 månader utan att deras aktivitet går förlorad.The lyophilized cultures can be stored at 4-9 ° C for at least 4 months without losing their activity.
Före rester riilföree s mi kallt Aswnr (1) medium till den frystorkade kulturen.Before residues riilföree s mi cold Aswnr (1) medium to the freeze-dried culture.
Den testade antibiotikalösningen placeras i l ml ASWRP medium eller tillföres NaCl så att en slutlig Nacl kon- centration av 3 % erhålles. Lösningens pH-värde bör ligga mellan 6 och 8,5 (lämpligen 7,2). 0,1 ml av den hydrerade faginfekterade kulturen tillföres testlösningen och ett antal kända antibiotikakoncentrationer liksom även ett prov utan antibiotikum.The tested antibiotic solution is placed in 1 ml of ASWRP medium or added to NaCl so that a final Nacl concentration of 3% is obtained. The pH of the solution should be between 6 and 8.5 (preferably 7.2). 0.1 ml of the hydrated phage-infected culture is added to the test solution and a number of known antibiotic concentrations as well as a sample without antibiotic.
Blandningen inkuberas under lätt skakning vid 30-37°C under 60-90 minuter eller till dess att luminiscensen icke ytterligare sjunker i provet utan antibiotikum.The mixture is incubated with slight shaking at 30-37 ° C for 60-90 minutes or until the luminescence does not further drop in the sample without antibiotic.
Luminiscensen hos samtliga prov bestämmes vid samma tid- punkt (inom 5 minuter) och koncentrationen hos det aktuella provet bestämmes med hjälp av en kalibreringskurva som er- hålles från standardproverna. . 10 15 20 D 25 8104898-5 6 I figurerna l - 3 visas kurvor för tre olika antibiotika vars koncentrationer bestämts enligt den ovan beskrivna metoden. Fig. l visar därvid de kurvor som erhålles för antibiotikumet nalidixicsyra som hämmar DNA-syntesen. Fig. 2 visar motsvarande kurvor för rifamycin som hämmar RNA-syntesen och Fig. 3 visar de kurvor som erhålles för kloramfenicol som hämmar proteinsyntesen. På abskissan angives därvid tiden i sekunder, på ordinatan antalet ljuspulser per sekund och ml lösning samt till höger i figuren de använda mängderna ifug/ml.The luminescence of all samples is determined at the same time (within 5 minutes) and the concentration of the sample in question is determined using a calibration curve obtained from the standard samples. . Figures 15 to 310 show curves for three different antibiotics whose concentrations have been determined according to the method described above. Fig. 1 shows the curves obtained for the antibiotic nalidixic acid which inhibits DNA synthesis. Fig. 2 shows the corresponding curves for rifamycin which inhibits RNA synthesis and Fig. 3 shows the curves obtained for chloramphenicol which inhibits protein synthesis. The abscissa indicates the time in seconds, the ordinate the number of light pulses per second and ml of solution and to the right of the figure the amounts of ifug / ml used.
Den ovan beskrivna metoden kan även användas då mer än ett anti- biotikum finnes i provet, varvid man kan använda ett antal olika antibiotika resistenta mutanter av luminiscenta bakterier för att bestämma verkningssättet hos detaktuella antibiotikumet.The method described above can also be used when more than one antibiotic is present in the sample, whereby a number of different antibiotic resistant mutants of luminescent bacteria can be used to determine the mode of action of the antibiotic in question.
De väsentliga fördelarna vid det nya bioluminiscenstestet är följande; ' (1) Testerna är mycket snabba och känsliga. (2) Testerna är specifika och bestämmer specifikt aktiviteten hos det testade antibiotikumet i dess naturliga tillstånd. (3) Testerna är ekonomiska, enkla och kan utföras automatiskt. (4) Den minsta volymen som erfordras för dessa test är 0,1 ml eller eventuellt mindre. Därför erfordras endast l-5 pl av provet. (5) Det är möjligt att utnyttja en bakteriestam som är resistent mot vissa antibiotika för att specifikt bestämma verkan av andra antibiotika i antibiotikablandningar.The essential benefits of the new bioluminescence test are as follows; '(1) The tests are very fast and sensitive. (2) The tests are specific and specifically determine the activity of the tested antibiotic in its natural state. (3) The tests are economical, simple and can be performed automatically. (4) The minimum volume required for these tests is 0,1 ml or possibly less. Therefore, only 1-5 μl of the sample is required. (5) It is possible to use a bacterial strain resistant to certain antibiotics to specifically determine the effect of other antibiotics in antibiotic mixtures.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE8104898A SE424090B (en) | 1981-08-18 | 1981-08-18 | Method for determining the concentration of an antibiotic |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE8104898A SE424090B (en) | 1981-08-18 | 1981-08-18 | Method for determining the concentration of an antibiotic |
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| SE8104898L SE8104898L (en) | 1982-02-26 |
| SE424090B true SE424090B (en) | 1982-06-28 |
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| SE8104898A SE424090B (en) | 1981-08-18 | 1981-08-18 | Method for determining the concentration of an antibiotic |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990012887A1 (en) * | 1989-04-20 | 1990-11-01 | Bio-Orbit Oy | Determination of factors affecting gene regulation and/or gene replication |
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1981
- 1981-08-18 SE SE8104898A patent/SE424090B/en unknown
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990012887A1 (en) * | 1989-04-20 | 1990-11-01 | Bio-Orbit Oy | Determination of factors affecting gene regulation and/or gene replication |
| US6475719B1 (en) | 1989-04-20 | 2002-11-05 | Bio-Orbit Oy | Determination of factors affecting gene regulation and/or gene replication |
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| Publication number | Publication date |
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| SE8104898L (en) | 1982-02-26 |
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