CA2186728A1 - A rapid microbiological test for the detection of antibacterial compounds - Google Patents
A rapid microbiological test for the detection of antibacterial compoundsInfo
- Publication number
- CA2186728A1 CA2186728A1 CA002186728A CA2186728A CA2186728A1 CA 2186728 A1 CA2186728 A1 CA 2186728A1 CA 002186728 A CA002186728 A CA 002186728A CA 2186728 A CA2186728 A CA 2186728A CA 2186728 A1 CA2186728 A1 CA 2186728A1
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- Prior art keywords
- test
- agar medium
- growth
- sample
- organism
- Prior art date
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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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
Abstract
The present invention relates to a microbiological test method for detecting antibacterial compounds in liquid samples such as milk, meat juice, serum and urine. The method employs an acid-base or redox indicator to detect growth of a test organism and is characterised in that a thermophilic strain of Bacillus or Streptococcus, e.g. Bacillus stearothermophilus var. calidolactis C953, is employed at a concentration of above 107 CFU/ml, preferably between 2 x 107 and 3 x 108 CFU/ml.
Description
W0 96/23901 r .,~ ~ -2 ~ 8672~
.
A RAPID MICROBIOLOGICAL TEST FOR THE DETECTION OF
ANTIBACTERIAL COMPOUNDS
FIELD OF THE INVENTION
This invention relates to a noYel ~ 0 ~ test method for the rapid d~L~Illilldtio~l of the presence or absence of dllLi~a~;Le:lidl compounds in liquids such as milk, meat juice, serum and urine. The invention also relates toa unit for the detection of residues of antibacterial compounds in liquid samples and the use of the unit.
DE~CR~ ION OF THE PRIOR ART
to I~ jOl~Y;~l test methods for the d~L~ dLiul~ of dl ~LiL~al;L~:lidl compounds, particularly residues of dllLiL iulics and ch~llluLll~ld,u~tics such as sulphas, in liquid samples such as milk, meat juice, serum and urine have been known for a long time. Examples of such tests have been described in GB-A-1467439, EP 0005891. DE 3613794, CA 2056581 and EP 0285792. These des~,,it,Liu":, all deal with ready to use tests that make use of a test organism and will give a result generally within 21/2 to 41/2, hours by the change of colour of an acid-base or redox indicator added to the test system. The principle is that when an dl ,Li,a,L~,ial compound is present in the sample in a Cûl~Ctn~LIdLion sufficient to inhibit the growth of the test organism the colour of the indicator will stay the same, while when no inhibition occurs the growth of the test organism is accc""~anied by the formation of acid or reduced ", ~ that will change the colour of the indicator.
The known test units ",~"Lio,1ed above may include an agar medium inoculated with a suitable test organism, preferably a strain of Bacillus or W0 96123901 I ~
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Streptncocc~ls and an acid-base indicator or a redox indicator. The suitable test organism and the indicator are introduced into an optionally buffered agar solution, optionally nutrients are added to the solution and optionally substances to change the sensitivity to certain d";i",iu,ul,ial compounds in a 5 positive or a negative way are added to the solution. Finally, the agar solution is allowed to solidify to form the agar medium in such a way that the test olyarliall~a stay alive but cannot multiply because of lack of nutrients andlor low temperature. Optionally compounds may be added to the agar medium, e.g.
the nutrients or the i, Idi~d~u~(s) as a separate source, e.g. as a tablet or a paper disc.
A preferred species of Streptococcus for use in conventional I l liul ~Jbi~lQyi~ tests for ~ ,li~auLtn idl compounds is Streptf-coccl~s therrrlophilus. A preferred species of B2cillus for use in such tests is Bacillus ~a~ erlllûp~ . This species may be employed for this purpose as spores.
In most documents cu"c~",i"g ~k;l~ l tests for antibacterial compounds, a very broad cu, ~ut~ ..t~an of the test organism of 105 to 10~ - 1 09 colony forming units (CFU) per ml is ",e"~iu"ed. Examples of such documents are GB-A-1467439, EP 000581, EP 0611001, DE 3429823, US 3941658, US
4946777 and EP 0285792. HoweYer, none of these documents has an example 20 specifying a UUl~Ctlll~ldtiul) of test organism higher than 10' CFU/ml. This reflects the fact that in practice Illiul~ o~J;~Al tests for d"~iL,ait~:,ial compounds have preYiously routinely been carried out with a test organism CO~Ctll Itld~iUI~ below 10' CFUlml. In CA 2056581 (page 11, line 27), it is -r r ~ ~Iy stated that the cu"c~, Itl dtiOI I of test organism - in this particular 25 case the spores of Bacillus ~,ull,e""~pl.:~s - may vary, but may never exceed a cullc~llLIdLioll of 10' CFU per ml agar medium.
Thus, up till now, it has been commonly accepted that a test organism cùl~ctll~ an of 10' CFU per ml is the maximum ~UII~ LId~iUII that could be used in the type of l"i~l~Jl~i~Jlo~J;~l tests described in this document.
30 Up till now, It has been commonly thought that use of a higher Illi~ilUUlydlli ~ WO9rjl239o~ ,6,'l ~t 3- 2 ~ 867~8 cùnc~nl, dliùr, in such a test will result in loss of sensitiYity for relevant ~,LiL,a~,lc,ial con~poi nds.
Of course, a suitable rapid test for dl ,liL,a.,~Lcrial compounds should among others fulfil the following requirement:
-a high sensitivity for a wide range of a~ ,~iL,a~,lc,ial compounds used in practice.
A test with a bacterium CUIl.icll~dliUll of 10~ to 107 CFU per ml generally gives a result between 2l/2 to 41/2 hours or even after a longer time.Although a test duration of minimal l1/2 hours s~,,,cLi,,,c~ is mentioned (for 0 example EP 000581, GB-A-1467439, US 3941658, US 4946777) such atest has never been dclllulls~,dLcd in an example or anywhere else.
Up till now, it was not co, Isid~, cd possible to carry out a Illi~,lu~ioloyicdl test for rmtibacterial compounds with a test duration less than 2t/2 hours by using a col1~cl lll dLi~7n of test organism higher than 107 per ml1S while retaining good sensitivity for a wide range of dl llibd~.1Lclie~l compounds.
~he present :".e~f,cd'L;r,n now teaches that, contrary to expectation, such ",j~"~.hj~7~ tests are feasible.
DESCRIPTION OF THE INVENTION
In one aspect, the present invention provides a method for detecting one ot more ~ a,,Lcridl compounds in a liquid sample, such as milk, meat juice, serum or urine, which ~,ull,~.,iae~ the steps of:
(i) ~01 lldl_;lil l9 said sample with therein a test mic, uu, ydl li:~l l l selected 2S from Illcllllopl ' strains of B2cillus and Streptococcus, prior to addition of said sample growth of said test Illi~:lUOlydlli~lll being prevented;
(ii) providing conditions for said test ",i~,uorya~ "~ whereby in the absence of dllli~aul~lidl compounds growth of said test ",;~,uùryal~ia"~ can occur and can be detected; and (iii~ detecting the growth of said ",i,,,uu,ydllia", after a time suf~icient fordetection of test ",i.,,uu,ya~ l growth in the absence of antibacterial WO 96/23901 1 ~
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compounds, but during which period d~ le test microorganism grov~th is I l Idil l~dil ,ed by a known amount of an ~ ,li~auL~, ial compound in a test sample;
clldlault:lisad in that said test Illiuluulydr,i~", is present at above lû7 CFU/ml.
Such a test method provides a marked reduction in test duration S CC.lll,udll::d with conventional Illiull,i ~lo.J; ~I tests for d"Liua, L~,ial compounds.
Whi~e conventional Illi~iluhiol~ .l tests for antibacterial compounds generally sive a result between 2t/2 and 41/2 hours, a method described in this document gives a result in less than 21/2 hours, e.g 70 minutes or possibly less e.g. 60 minutes or less. This is of extreme il~Ipul ~ e to the user because the quallty of the sample liquid is known more quickly thus allowing an earlier delivery or pruCeSai"g, Moreover, surprisingly, by employing a ~ ub;uloyical test of the invention with a test organism ~ c~"L, dLi.oll of more than 1 07 CFU per ml, a result can be obtained in less than 150 minutes without losing sensitivity. Thus, it has been found possible using a Il~ ioloy;~ test of the invention to detect in well below 21/2 hours, e.g. as little as 70 minutes, Penicillin G at 0.003 lU/ml in a milk sample (see Examples).
The test Illi-;lUOlydlli~lll present in the test is preserved under such cur,diLiu,-s that they are activated, even after at least one year, when contacted with the test sample.
The test " ,i~, uu, ydl lial 1 l may be preserved in dispersed form in a solidi~led medium e.g. solidified agar medium.
According to another ~",L,odi",~"L the test Illi~lUolydl,is", is preserved in co~)c~"l~dL~d form e.g. in dried form. In case the Illk;luoryd"i~", is dispersed in solidified medium, the amount or presence of the Illiuluolydllialll is defined as CFU of said Illiuluulydl~ialll per ml of said medium (CFU/ml). In case the Illiuluulydllialll iS present in other forms (e.g. freeze-dried), the amount or presence of the Illi-;luulydllialll iS defined as CFU's of said Illi.;luuly~llialll per ml test sample (e.g. milk) added during the method of detection (CFU/ml).
A"LiL,a~,la~idl compounds which can be detected in acculddllut: with the present invention are antibiotics and ClltnllOLlle:ld,u.3Utics like sulpha WO 96/23901 P.~
.
compounds. A test method of the invention may be used for the detection of dll~iba~t~lidl compounds in a wide range of liquid samples, for example milk, mi~k products, meat juices, serum, urine, eggs, honey, water and extracts of meat, eggs or honey.
In a further aspect, the invention provides a test unit for carrying out a method of the invention cU",~ i"g a container holding a solidified agar medium having dispersed therein a test organism selected from ~ o~l, 'ic strains of Bacillus and Streptncocc~s at above 10' CFU/ml, said agar medium optionally addi~ion..:'y containing nutrients for growth of said test organism 10 and/or one or more components for detectlon of said growth. Examples of CUl ~dil lt~ useful for the purpose of the invention are tubes or blocks with tubular i, Id~ d~iUI l~ for example, lldll:l~)Cllt71 I~ tubes, single or in a set, or combined in the fomm of a block of translucent material having a number of holes shaped therein, e.g. microtiter plates.
The agar medium in such a unit may be an optionally buffered agar medium to which is added a test organism, a nutrient source cc""~ ,isi"g sufficient nutrients to support growth of the test organism, an acid-base indicator or one or more redox indicators and optionally substances to change the sensitivity to certain d"t;",i~,ubial compounds in a positive or a negative 20 way. All the test cu",p~l~e"t~ may be incorporated into the initial agar solution used for p, ~:pdl dliol~ of the test unit or at least some may be added separately to the agar medium.
Thus, optionally at least part of the nutrients may be c~ ~hse~ ntly il ICul l~UI dL~:d into the agar medium in the test unit from a separate applied2~ source, for example a tablet or paper disc, placed on the agar medium before carrying out the test. At least one indicator component may also be added separately to the agar medium, e.g. by inclusion in a separate nutrient source.
Also a buffer and the test organism may be added as a separate source. The nutrient source and / or one or more indicator components, e.g. redox 30 indicators, may be added to the test sample.
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When nutrients and / or indicator (s) and / or the test organism are added to the agar medium or test sample from a separate applied source, e.g.
a tablet, measures are preferably taken to avoid moisture transport from the agar medium into the applied additive source. When a tablet is employed for s this purpose, this may be done by coating the tablet with a moisture resistant layer, for example a wax, which coating must degrade or melt during the test procedure. A wax having a meiting temperature of 35 to 55C, preferably 40 to 45C is suitable.
The nutrients are added to enable the m~ of the test organism in the absence of ~"LiL,a~,idl compounds. Suitable nutrients are for example as:,i",ild~le carbon sources (e.g. Iactose, glucose or dextrose), as~i",ildble nitrogen sources (e.g. peptone) and sources of growth factors and minerals (e.g. yeast extract).
The growth of the test Illi~lUUlydlli~lll can be detected by colour change of the agar medium or the test sample resulting from the presence of an acid base or redox indicator.
If the indicator used is an acid-base indicator, an indicator for a pH of about 5.5 is suitable, preferably b,u,,,o~,~sul~urple or phenolred. Other acid-base indicators may however be used as well.
If it is desired to use a redox indicator or a c~l,lbil,dtiu,1 of two or more redox indicators, suitable indicators are e.g. Brilliant Black, Methylene Blue, Toluidine Blue, Nile Blue A, 2,3,5-triphenyll~L, " ~rn, Safranine û, Indigo Carmin, Thionin, Gallocyanine, Brilliant Crocein MOO, Acid Yellow 38, Acid Orange 51, Acid Blue 120, Basic Blue 3, Azure A, Azure B, Congo Red, 1-10 PII~ Llll~ " ,e, Janus Green B, Brilliant Cresyl Blue. Other redox indicators (redox mediators, redox catalysts and electron carriers) may be used as well.
Preferred redox indicators are Briliiant Black, Methylene Blue, Toluidine Blue and Nile Blue A or ~ulllLJil ~s thereof, e.g. a c~lllL,illaliull of Brilliant Black and Toluidine Blue.
~ W096123901 1~,1/~,6. -: - 7 - 2 1 8 67 2~
, A suitable test organism for a method of the invention is Streptococcus thermophilus, preferably strept~cocclls thermophilus J101 (DSM 4022, deposited on March 3, 1987).
Bacillus al~rllulller~ philus may alternatively be employed. Particulariy preferred for use in a method of the invention is Bacillus ~e&luU~ opl7;lus var.~.~'c~ rt;~ C963. Strain C953 of Bacillus stearothermophilus var. cAI;~lAr~tis was deposited w'lth the Laboratory of l\l:~lubioloyy of the Technical University of Delft under the accession number LMD 74.1 in 1974 and with the Centraal Bureau voor Schimmelcultures (CBS), Baarn under the accession number CBS
o 760.83 in 1983 where the strain is available to the public.
Bacillus stearothermophilus var. cAliCIo/~ C953 and St,~t~coccus thermophilus T101 are very sensitive to dl ILiba~iLelidl compounds, especially dllLiL,iuLi~s such as peniciliins and ~:llell,uLl,e,df.eutics such as sulpha compounds. These Illi.;lUOlydllisllls are fast growing and have the additional advantage that their optimal growing temperature is high (Bacil/us s~,ull,e""olphilus: between 50C and 70C; Sllt~ cocc~l~ thermophilus:
between 35C and 45C). This makes these Illi~;lUUlydlli~ very suitable for a test according to the invention as they cannot grow at storage temperature (e.g.room temperature) and there is little possibility that Olyalli "":, which are possibly present in the test liquid or which have otherwise been included in thetest system would affect the result of the test.
When the test organism is a Bacillus strain, it is preferably i, Ico",o, dLed into the agar medium in the form of a spore suspension which may be prepared and illCol~oldled into the agar medium prior to ~ by known methods (see, for example, GB-A-1467439). AlL~Ill.. ~;~cly, as hereinbefore indicated, the test organism may be added as a separate source, for example by a~ of a tablet or paper disc to the agar medium.
When the test organism is a St,~ ~coc~ strain, the bacteria are preferably in~u~ul dLed into the agar medium in the form of bacterial cells which may be prepared according to known methods (EP 0 28~ 792).
WO 96/239~
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When the test ~ uu~ydllialll is in col~ct"L,dl~d form (e.g. freeze-dried form, tablet, paper disc, etc.) the concentrate is measured into a vessel which may be a conventional ampoule, a sealable test tube, a sample bottle, a hole of a block such as a microtiter plate and the like (see for example EP-û285792).
s The cul lc~ dLioll of test organism in the agar medium is suitably above 10' CFUlml, but desirably does not exceed 10' CFU/ml, e.g. a test organism CUl~Ct~ ldLiUI~ between 2 x 107 and 109 CFU/ml may be employed, preferably 2 x 10' to 3 x 10~ CFU/ml. Most preferably, the test organism Cul~C~ ld~iul~ in the is between 3 x 10' and 2 x 10~ CFU/ml.
According to a preferred embodiment of the present application, the sensitivity of the test organism is a~ lct~hle. The sensitivity may be altered by various means, for example by adding certain substances, by changing the test cùndiliolls such as the pH or concentration of buffering substances or agar or by varying the ratio of the volumes of agar and test liquid.
rj Examples of substances that may be added to the test system to change sensitivity are n~lrle~sid~c such as aclc,llosicle and antifolates such as trimethoprim, ormethoprim and ~LIuxuulilll which improve the sensitivity of the test organism to sulpha compounds. Salts of oxalic acid or hydrofluoric acid may be added to improve the sensitivity to tetracyclines. Cysteine may be added ~o diminish the sensitivity to penicillins.
I Ir . ~:il Id~ l the invention will be discussed in more detall for a test C~l I l,ur iSil Ig the agar medium, however, the skilled in the art will d~prl:CidL~ that these teachings can also be applied for tests without an agar medium.
As hereinbefore indicated, components needed or optionally desirable in 2s the agar medium, including optionally substances to change the sensitivity to certain dl,Li~a.,L~,ial compounds, may be added to an optionally buffered agar solution. The agar solution is allowed to solidify in the test units to form theagar medium in such a way that the test organism stays alive but cannot multiply because of lack of nutrients and / or low temperature. The needed compounds which have not been added to the agar solution prior to ~ WO 96t23901 l .,~
9 2 1 8 6 7 2 ~
s ' ' ~ ~ 1 have to be added from a separate source, for example a tablet or paper disc.
Needed ~"~pO~ "ts are the test organism, one or more indicators and the nutrients. Other compounds, e.g. substances to improve or diminish the sensitivity to certain a~ a~,t~lidl compounds and buffer solutions, are optional.
It is preferred to carry out the process of the present invention in such a way that the test organism does not grow or multiply in the agar medium in the presence of a known minimum amount of an antibacterial compound to be detected. Also the test units, e.g test tubes co, ILdil lil ,g the agar medium, have to be stored in such a way that the test organism, does not multiply in the agar medium. This is generally achieved by depriving the organism of nutrients until the test is carried out or by " Idil ILclil lil l9 the culture (during the production process) and the test units (during storage) at a sufficiently low temperature, preferably between 4C and 30C, more preferably between 4C and 15C, or 5 both.
The test units are preferably closed air-bght during storage in which condibon they may be stored for at least several months.
The optionally buffered agar medium co, lldil lil l9 the test organism and /
or the i, I~ dL~ ~ (S) and / or the nutrients and / or certain substances to alter the 20 sensitivity of the test is allowed to solidify in upright test units e.g. tubes.
The unlts preferably have d~l~"),i"ed sizes. This is because of the reliability of the test. The height of the agar medium in the test units is of great i""~..,Ld,~;e. If an antibiotic is present in the test sample it will diffuse into the agar. The sensitivity of the test is therefore preferably asc~, Ldil leCI with variation 25 of the height of the solidified agar in the test unit.
Preferably, the height of the agar medium in the test unit is selected in such a way that the indicator changes its colour when the antibacterial compound to be detected is present in a con~ n below a certain value, but does not change its colour when the co"c~"~, dliun is above this value.
30 Preferably, the test is arranged to be a test from which one can read whether a certain conc~, ItldLi-,ll of an al ,tiL,a. L~, idl compound is present or not. For this, , . , , , _ _ _ _ _ _ _ ,, _,,, ,,, , . , ,, . ,, . , ,, .. ,, ,,, ,, _ .. , _ . _ WO96/2390~ ~ s.~ I ~
~J ~ 3 ` -10 - 2 1 8 6 7 2~
the height of the test unit will preferably be high enough to contain an amount of agar medium and sample corresponding to a height of 3-30 mm,more preferably 6-15 mm. The internal cross-sectional .li,,,~,,siu,~ of the test units is preferably 1-20 mm, more preferably 1-14 mm.
The volume of the agar medium in the test unit is determined by the height of the test unit, the internal cross-sectional dillldl~io,1 of the test unit and the p~ l lldg~ of the volume of the test unit which is filled with the agar medium. The volume of the agar medium is preferably 10 ~l - 3 ml, more preferably 15 ~l -1 ml and most preferably 20 ~l - 500 ~
A suitable liquid test sample Yolume is for example 0,01 - 0,5 ml. Thus, for example, a test unit of the invention formed by solidifying 0.3 ml agar solution in an upright sterile test tube of internal diameter 9 mm has been found suitable for detecting 0.003 lU/ml Penicillin G in a milk sample.
When spores of 3acill~1s stearothermophilus var. c~lidol~tis are used, suitable incubation temperatures are between 55C and 70C, more preferably between 60C and 66C. Incubation can be carried out in any conventional form of incubator, e.g. a waterbath, a block heater, a stove or a dry incubator.
When cells of S~ o~,o~ s thermophil~s are used, suitable incubation temperatures are between 36 and 44C, more preferably between 38 and 42C.
As hereinbefore indicated, an incubation period of less than 150 minutes is feasible, e.g. 70 minutes, possibly as little as 60 minutes or even 30 minutes.
Such an incubation period is markedly shorter than the incubation period of other conventional I~ ,lobi~lo~J~r~l test sytems for antibacterial compounds employing less than 10' CFU's/ml.
A test of the invention is verY simple to carry out, so that persons who perform the test do not have to be specially educated. The result is very simpleto determine. After the incubation period, the colour of the agar medium cu, ,' ;. ,i"g the indicator shows if test organism growth did occur or not.
All documents mentioned in this r, ~',~ ~ 1 are herein incorporated by reference to the same extent as if each individual '~Pt '' " .1 or patent was -r~ lly and individually indicated to be ill~UI~JUldL~d by reference.
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Leaend to the Fiaures Fig. 1: shows the test duration as a function of the spore concentration for test units of type A employed as in Example IV.
Fig.2: shows the test duration as a function of the spore concentration for test units of type B employed as in Example V.
EXAMPLE I
Fl~;JdldLiUn of test tubes of tvpe A to detect antibiotics Test tubes of type A contained an agar medium prepared as follows:
A solubon was made of 12 9 agar, 9 9 sodium chloride and 50 ml of a 0,1 M L,i~Ll,d"old",i"e buffer solution (pH = 8.0) in 1000 ml distilled water. An amount of a solution of L~,u,,,o.;,~sull.urple to give a final cu, ICt" ~I~dtiùn of 20 mg per 1000 ml was also added. The final solution was sterilized for 20 minutes at 121C and cooled to about 60C.
To this solution different amounts of a suspension of Bacillus alullle~ 7philus VAr, c~ i~lcllacli~ C953 spores in distilled water were added to give final cu~1C~ dLiùns between 10~ and 10' spores per ml.
Sterile tubes with a diameter of about 9 mm were filled with 0,3 ml of the agar solution under aseptic ~u"ditiol~s and i,,l,,,ediclt,ly sealed, e.g. with aluminium foil. The agar solutlon in the test tubes was allowed to solidify in the test tubes while the test tubes were held in an upright position.
The tubes were stored at a temperature between 5C and 1 5C.
EXAMPLE ll r,~d,dLiùl~ of test tubes of tvr~e B to detect antibiotics Test tubes of type B contained an agar medium prepared as follows:
A solution was made of 10 9 agar, 9 9 sodium chloride, 2 9 glucose, 2 g peptone 2 9 tryptone 4 9 yedst extract and 50 m~ of a 0.1 M llitLlldll~ lille WO96/23901 ~ /r.l,r~
buffer solution (pH = 8.0) in 1000 ml distilled water. The final solution was sterilized for 20 minutes at 121C and cooled to about 60C.
An amount of a solution of Brilliant Black to give a final concentration of 80 mg per 1000 ml and an amount of a solution of Toluidine Blue to give a final 5 CUllCellLldi;UI) of 3 mg per 1000 ml were also added.
To this solution different amounts of a suspension of Bacillus .3. ulller~ p~ ' rs var. ' _'c ' C953 spores in distilled water were added to give final conct:"~, ~ 1S between 10 and 10' spores per ml.
Sterile tubes with a diameter of about 9 mm were filled with 0,3 ml of the 10 agar solution under aseptic col1diLiulls and immediately sealed e.g. with an aluminium foil. The agar solution in the test tubes was allowed to solidify in the test tubes while the test tubes were held in an upright position.
The tubes were stored at a temperature between 5C and 15C.
EXAMPLE lll r, t:,lJdl dLiol~ of nutrient tablets A mixture was made of 100 9 dextrose, 160 9 Avicel PH101, 50 9 tryptose, 14 9 yeast extract and 15 9 precirol. This mixture was sufficient to prepare about 18000 tablets with a diameter of d~pru,~ ldl~ly 3 mm and a thickness of a~p,uAi,,,dL~ly 1.9 mm.
EXAMPLE IV
Carrvinc~ out the test tvoe A
One of each of the test tubes of type A with different COIlCt:llLldLiol1s of Bacillus ~alulllellllophilus var. c~ tis spores as described in Example I
were opened by removing the seals. A nutrient tablet as described in Example 30 lll was added to each of the test tubes. Then 0.1 ml of a fresh cow's milk =
~ W096/2390~ CK
` ', ,.; ` -13- 21 ~6728 sample was added to each of the test tubes and the test tubes were didL~ :ly placed in a waterbath kept at 64C.
Observations were made after 30 minutes to 2 hours and 30 minutes.
- The time at which thè colour of the agar medium became yellow was d~L~I ",i"ed. As is shown in Figure 1, the test duration correlated with the number of spores added to the agar medium.
If at this specific time, the colour of the agar medium was blue, the sample was known to contain a d~ le amount of an dllLiLJd,~lidl compound (e.g. 0.003 I.U. per ml or more penicillin G). Figure 1 shows that a test duration reduction to at least 60 minutes is realizable.
EXAMPLE V
Carryina out the test tvpe B
The procedure described in example IV was followed except that test tubes of type B were used and no tablet was added to the test tube.
hgure 2 shows that a test duration reduction to at least 70 minutes is realizable.
2c EXAMPLE Vl Carrvina out the test tvr e B
An amount of penicillin G of 0.003 I.U. per ml was added to fresh cow's milk. The control sample contained no penicillin G.
Two of each of the test tubes of type B with cc,llc~:llLIdLions of Bacillus ~u,~,ll.~r",~phi~us var. ~- ~ ' spores as described in Example 11 were opened by removing the seal. Then 0,1 ml of each of the two milk samples was added to each of the test tubes and the test tubes were illllll~didL~ly placed in a waterbath kept at 64C.
wo s6n3so~
1 4 - 2 ~ 8 6 7 2 8 Observations were made after 30 minutes to 3 hours. The time at which the colour of the agar medium became yellow was determined. As is shown in Table 1, the test duration correlated with the amount of spores added to the agar medium without influencing the sensitivity of the test in a negative way.
Table 1 shows a decreasing test duration down to at least 70 minutes.
Even at a test duration of 70 minutes, a cul~c~l ILI d[iOIl of 0.003 I.U. per mlpenicillin G can be detected. Of course also higher CullC~ dLiulls of penicillinG can be detected.
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C~ X +
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~,7 E o C~ ;
o ¢,7 ~ ~ +
,~¦ a) ~ E E E o a~
U) o ,J ~ ~ U ~ 16 -EXANIPLE Vll Detection of antibiQtics and sulPha compounds in ",i~:, uLil~ dL~ (test tvpe C) s For the ~ Jdl dLiul ~ of the test plates (test type C) the procedure descri-bed in Example I was followed except that the nutrients described in Example lll(dextrose, tryptose and yeast extract) were not added separately but were added to the initial agar medium in the same Cvl~Ct:l ,i,dLiùn as described in Example 1. Also together with the buffer solution an amount of trimethoprim solution was added to give afinal CvllCt:lltldLiOIl of 60 ~9 per litre.
To the wells of sterile ,,,ki,uLiLt:,ulates 20 ~l of the agar solution was added under asceptic cu"di~i.."s. The plates were i"""edidL~ly sealed with aluminium foil.
The plates were stored at a temperature between 5C and 15C.
An amount of penicillin G of 3 ppb was added to fresh cow s milk.
Also a milk sample was prepared which contains an amount of sul~ adia~ of 10û ppb.
The control sample contained no penicillin G or s~l,ulladid~;, ,e.
The Illi._luLiLt:l~JlaL~ were opened by removing the seal. Then 20 ~l of 20 each of the three milk samples was added to each of the test plates (duplo) co"Ldi"i"g different cul~ccnlLIaLiùlls of ~acillus ~ .,v~ ophilus var.
c~ spores C9~3. The plates were i"""eclidL~:ly placed in a waterbath kept at 64C.
Observations were made a~ter 30 minutes to 150 minutes. The time at 25 which the colour of the agar medium became yellow was dt:L~:""i"ed. As is shown in table 2 the test duration correlated with the amount of spores added to the agar medium without influencing the sensitivity of the test in a negativeway.
Even at a test duration of 90 minutes both penicillin G and 30 SUI~ ddiaL,~ 'e can be detected at conc~"L dLions of respectively 3 ppb penicillin WO 96/23901 r~ c -17- 2-~ 8672~
~ . ~
G and 100 ppb sulfadiazine. Penicillin G (3 ppb) can even be detected at a test duration of 80 minutes.
Of course also higher con~"i,dlions of both penicillin G and sLl~ a.lid~; ,e can be detected.
Table 2: Sensitivitv of test tvpe C at different spore co, Ic~"~ s.
Test duration 140 t23 lOS 90 80 (minutes) 10spores/ml medium 1.3 x 107 2.5 x 107 5.1 x 107 1.0 X 10~ 2.0 x 10' milk without antibio tica milk with 3 ppb peni-cillin G + + + + +
15milk with 100 ppb + + + +
EX~MPLE Vlll 20 Detection of dl b . in milk (test tvDe D) In this ~A~.e,i",e"I S~ Vlv~ op~ T101 (DSM 4022) was used for the ck ~ . " ,i, , of aJ l~ibi. ;_s in milk.
.Si 7~ OGC~l~ Ulv~ opl ' l~ T101 was grown using methods known per se.
An amount of a solution of L, u" ,o~, ~sc l,uurple to give a final ~o". ~"I, dliUI I of 4 mg per 1000 ml was added to fresh cow s milk.
To part of the fresh cow s milk also an amount of penicillin G of 4 ppb was added.
The control sample contained no penicillin G.
SUBSTITUTE SHEET (RULE 26) wo 96/2390~
To the milk samples different amounts of Streptococcus ll,e""o~ ;lus T1ûl were added to give final conct~, ILldtiUI 15 between 10' and 1 09 CFU per ml.
Illlllledial~ly after pl~drdLi~n of the samples sterile tubes with a diameter of about 9 mm were filled with 0.3 ml of each milk sample (duplo).
5 The tubes were i~ l,edidlely placed in a waterbath kept at 42C.
Observations were made after 30 minutes to 2 hours and 30 minutes.
The time at which the colour of the agar medium became yellow was cl~lt:l 11 ~il ,ed. As is shown in table 3, the test duration correlated with the amount of cells added to the milk without influencing the sensitivity of the test in a negative way.
Even at a test duration of 80 minutes penicillin G can be detected at a cùnc~ ILl..t~k~l I of 4 ppb or higher.
Table 3: Sensitivity of test type D at different cell conc~, ILIdLiul~s.
Test duration 145 125 103 80 (minutes) cellslml 3.0 x 10' 6.0 x 10' 1,3 x 10~ 2,5 x 10 medium 20milk without Li~iuli~a milk with + + + +
4ppb penicillin G
.
A RAPID MICROBIOLOGICAL TEST FOR THE DETECTION OF
ANTIBACTERIAL COMPOUNDS
FIELD OF THE INVENTION
This invention relates to a noYel ~ 0 ~ test method for the rapid d~L~Illilldtio~l of the presence or absence of dllLi~a~;Le:lidl compounds in liquids such as milk, meat juice, serum and urine. The invention also relates toa unit for the detection of residues of antibacterial compounds in liquid samples and the use of the unit.
DE~CR~ ION OF THE PRIOR ART
to I~ jOl~Y;~l test methods for the d~L~ dLiul~ of dl ~LiL~al;L~:lidl compounds, particularly residues of dllLiL iulics and ch~llluLll~ld,u~tics such as sulphas, in liquid samples such as milk, meat juice, serum and urine have been known for a long time. Examples of such tests have been described in GB-A-1467439, EP 0005891. DE 3613794, CA 2056581 and EP 0285792. These des~,,it,Liu":, all deal with ready to use tests that make use of a test organism and will give a result generally within 21/2 to 41/2, hours by the change of colour of an acid-base or redox indicator added to the test system. The principle is that when an dl ,Li,a,L~,ial compound is present in the sample in a Cûl~Ctn~LIdLion sufficient to inhibit the growth of the test organism the colour of the indicator will stay the same, while when no inhibition occurs the growth of the test organism is accc""~anied by the formation of acid or reduced ", ~ that will change the colour of the indicator.
The known test units ",~"Lio,1ed above may include an agar medium inoculated with a suitable test organism, preferably a strain of Bacillus or W0 96123901 I ~
; -2- ~ 1 8672&
Streptncocc~ls and an acid-base indicator or a redox indicator. The suitable test organism and the indicator are introduced into an optionally buffered agar solution, optionally nutrients are added to the solution and optionally substances to change the sensitivity to certain d";i",iu,ul,ial compounds in a 5 positive or a negative way are added to the solution. Finally, the agar solution is allowed to solidify to form the agar medium in such a way that the test olyarliall~a stay alive but cannot multiply because of lack of nutrients andlor low temperature. Optionally compounds may be added to the agar medium, e.g.
the nutrients or the i, Idi~d~u~(s) as a separate source, e.g. as a tablet or a paper disc.
A preferred species of Streptococcus for use in conventional I l liul ~Jbi~lQyi~ tests for ~ ,li~auLtn idl compounds is Streptf-coccl~s therrrlophilus. A preferred species of B2cillus for use in such tests is Bacillus ~a~ erlllûp~ . This species may be employed for this purpose as spores.
In most documents cu"c~",i"g ~k;l~ l tests for antibacterial compounds, a very broad cu, ~ut~ ..t~an of the test organism of 105 to 10~ - 1 09 colony forming units (CFU) per ml is ",e"~iu"ed. Examples of such documents are GB-A-1467439, EP 000581, EP 0611001, DE 3429823, US 3941658, US
4946777 and EP 0285792. HoweYer, none of these documents has an example 20 specifying a UUl~Ctlll~ldtiul) of test organism higher than 10' CFU/ml. This reflects the fact that in practice Illiul~ o~J;~Al tests for d"~iL,ait~:,ial compounds have preYiously routinely been carried out with a test organism CO~Ctll Itld~iUI~ below 10' CFUlml. In CA 2056581 (page 11, line 27), it is -r r ~ ~Iy stated that the cu"c~, Itl dtiOI I of test organism - in this particular 25 case the spores of Bacillus ~,ull,e""~pl.:~s - may vary, but may never exceed a cullc~llLIdLioll of 10' CFU per ml agar medium.
Thus, up till now, it has been commonly accepted that a test organism cùl~ctll~ an of 10' CFU per ml is the maximum ~UII~ LId~iUII that could be used in the type of l"i~l~Jl~i~Jlo~J;~l tests described in this document.
30 Up till now, It has been commonly thought that use of a higher Illi~ilUUlydlli ~ WO9rjl239o~ ,6,'l ~t 3- 2 ~ 867~8 cùnc~nl, dliùr, in such a test will result in loss of sensitiYity for relevant ~,LiL,a~,lc,ial con~poi nds.
Of course, a suitable rapid test for dl ,liL,a.,~Lcrial compounds should among others fulfil the following requirement:
-a high sensitivity for a wide range of a~ ,~iL,a~,lc,ial compounds used in practice.
A test with a bacterium CUIl.icll~dliUll of 10~ to 107 CFU per ml generally gives a result between 2l/2 to 41/2 hours or even after a longer time.Although a test duration of minimal l1/2 hours s~,,,cLi,,,c~ is mentioned (for 0 example EP 000581, GB-A-1467439, US 3941658, US 4946777) such atest has never been dclllulls~,dLcd in an example or anywhere else.
Up till now, it was not co, Isid~, cd possible to carry out a Illi~,lu~ioloyicdl test for rmtibacterial compounds with a test duration less than 2t/2 hours by using a col1~cl lll dLi~7n of test organism higher than 107 per ml1S while retaining good sensitivity for a wide range of dl llibd~.1Lclie~l compounds.
~he present :".e~f,cd'L;r,n now teaches that, contrary to expectation, such ",j~"~.hj~7~ tests are feasible.
DESCRIPTION OF THE INVENTION
In one aspect, the present invention provides a method for detecting one ot more ~ a,,Lcridl compounds in a liquid sample, such as milk, meat juice, serum or urine, which ~,ull,~.,iae~ the steps of:
(i) ~01 lldl_;lil l9 said sample with therein a test mic, uu, ydl li:~l l l selected 2S from Illcllllopl ' strains of B2cillus and Streptococcus, prior to addition of said sample growth of said test Illi~:lUOlydlli~lll being prevented;
(ii) providing conditions for said test ",i~,uorya~ "~ whereby in the absence of dllli~aul~lidl compounds growth of said test ",;~,uùryal~ia"~ can occur and can be detected; and (iii~ detecting the growth of said ",i,,,uu,ydllia", after a time suf~icient fordetection of test ",i.,,uu,ya~ l growth in the absence of antibacterial WO 96/23901 1 ~
`."; ~J3 ~ 5 -4- ;~ 1 8672~
compounds, but during which period d~ le test microorganism grov~th is I l Idil l~dil ,ed by a known amount of an ~ ,li~auL~, ial compound in a test sample;
clldlault:lisad in that said test Illiuluulydr,i~", is present at above lû7 CFU/ml.
Such a test method provides a marked reduction in test duration S CC.lll,udll::d with conventional Illiull,i ~lo.J; ~I tests for d"Liua, L~,ial compounds.
Whi~e conventional Illi~iluhiol~ .l tests for antibacterial compounds generally sive a result between 2t/2 and 41/2 hours, a method described in this document gives a result in less than 21/2 hours, e.g 70 minutes or possibly less e.g. 60 minutes or less. This is of extreme il~Ipul ~ e to the user because the quallty of the sample liquid is known more quickly thus allowing an earlier delivery or pruCeSai"g, Moreover, surprisingly, by employing a ~ ub;uloyical test of the invention with a test organism ~ c~"L, dLi.oll of more than 1 07 CFU per ml, a result can be obtained in less than 150 minutes without losing sensitivity. Thus, it has been found possible using a Il~ ioloy;~ test of the invention to detect in well below 21/2 hours, e.g. as little as 70 minutes, Penicillin G at 0.003 lU/ml in a milk sample (see Examples).
The test Illi-;lUOlydlli~lll present in the test is preserved under such cur,diLiu,-s that they are activated, even after at least one year, when contacted with the test sample.
The test " ,i~, uu, ydl lial 1 l may be preserved in dispersed form in a solidi~led medium e.g. solidified agar medium.
According to another ~",L,odi",~"L the test Illi~lUolydl,is", is preserved in co~)c~"l~dL~d form e.g. in dried form. In case the Illk;luoryd"i~", is dispersed in solidified medium, the amount or presence of the Illiuluolydllialll is defined as CFU of said Illiuluulydl~ialll per ml of said medium (CFU/ml). In case the Illiuluulydllialll iS present in other forms (e.g. freeze-dried), the amount or presence of the Illi-;luulydllialll iS defined as CFU's of said Illi.;luuly~llialll per ml test sample (e.g. milk) added during the method of detection (CFU/ml).
A"LiL,a~,la~idl compounds which can be detected in acculddllut: with the present invention are antibiotics and ClltnllOLlle:ld,u.3Utics like sulpha WO 96/23901 P.~
.
compounds. A test method of the invention may be used for the detection of dll~iba~t~lidl compounds in a wide range of liquid samples, for example milk, mi~k products, meat juices, serum, urine, eggs, honey, water and extracts of meat, eggs or honey.
In a further aspect, the invention provides a test unit for carrying out a method of the invention cU",~ i"g a container holding a solidified agar medium having dispersed therein a test organism selected from ~ o~l, 'ic strains of Bacillus and Streptncocc~s at above 10' CFU/ml, said agar medium optionally addi~ion..:'y containing nutrients for growth of said test organism 10 and/or one or more components for detectlon of said growth. Examples of CUl ~dil lt~ useful for the purpose of the invention are tubes or blocks with tubular i, Id~ d~iUI l~ for example, lldll:l~)Cllt71 I~ tubes, single or in a set, or combined in the fomm of a block of translucent material having a number of holes shaped therein, e.g. microtiter plates.
The agar medium in such a unit may be an optionally buffered agar medium to which is added a test organism, a nutrient source cc""~ ,isi"g sufficient nutrients to support growth of the test organism, an acid-base indicator or one or more redox indicators and optionally substances to change the sensitivity to certain d"t;",i~,ubial compounds in a positive or a negative 20 way. All the test cu",p~l~e"t~ may be incorporated into the initial agar solution used for p, ~:pdl dliol~ of the test unit or at least some may be added separately to the agar medium.
Thus, optionally at least part of the nutrients may be c~ ~hse~ ntly il ICul l~UI dL~:d into the agar medium in the test unit from a separate applied2~ source, for example a tablet or paper disc, placed on the agar medium before carrying out the test. At least one indicator component may also be added separately to the agar medium, e.g. by inclusion in a separate nutrient source.
Also a buffer and the test organism may be added as a separate source. The nutrient source and / or one or more indicator components, e.g. redox 30 indicators, may be added to the test sample.
wo 96/2390~
J,~ 6 - 2 1 8672~
When nutrients and / or indicator (s) and / or the test organism are added to the agar medium or test sample from a separate applied source, e.g.
a tablet, measures are preferably taken to avoid moisture transport from the agar medium into the applied additive source. When a tablet is employed for s this purpose, this may be done by coating the tablet with a moisture resistant layer, for example a wax, which coating must degrade or melt during the test procedure. A wax having a meiting temperature of 35 to 55C, preferably 40 to 45C is suitable.
The nutrients are added to enable the m~ of the test organism in the absence of ~"LiL,a~,idl compounds. Suitable nutrients are for example as:,i",ild~le carbon sources (e.g. Iactose, glucose or dextrose), as~i",ildble nitrogen sources (e.g. peptone) and sources of growth factors and minerals (e.g. yeast extract).
The growth of the test Illi~lUUlydlli~lll can be detected by colour change of the agar medium or the test sample resulting from the presence of an acid base or redox indicator.
If the indicator used is an acid-base indicator, an indicator for a pH of about 5.5 is suitable, preferably b,u,,,o~,~sul~urple or phenolred. Other acid-base indicators may however be used as well.
If it is desired to use a redox indicator or a c~l,lbil,dtiu,1 of two or more redox indicators, suitable indicators are e.g. Brilliant Black, Methylene Blue, Toluidine Blue, Nile Blue A, 2,3,5-triphenyll~L, " ~rn, Safranine û, Indigo Carmin, Thionin, Gallocyanine, Brilliant Crocein MOO, Acid Yellow 38, Acid Orange 51, Acid Blue 120, Basic Blue 3, Azure A, Azure B, Congo Red, 1-10 PII~ Llll~ " ,e, Janus Green B, Brilliant Cresyl Blue. Other redox indicators (redox mediators, redox catalysts and electron carriers) may be used as well.
Preferred redox indicators are Briliiant Black, Methylene Blue, Toluidine Blue and Nile Blue A or ~ulllLJil ~s thereof, e.g. a c~lllL,illaliull of Brilliant Black and Toluidine Blue.
~ W096123901 1~,1/~,6. -: - 7 - 2 1 8 67 2~
, A suitable test organism for a method of the invention is Streptococcus thermophilus, preferably strept~cocclls thermophilus J101 (DSM 4022, deposited on March 3, 1987).
Bacillus al~rllulller~ philus may alternatively be employed. Particulariy preferred for use in a method of the invention is Bacillus ~e&luU~ opl7;lus var.~.~'c~ rt;~ C963. Strain C953 of Bacillus stearothermophilus var. cAI;~lAr~tis was deposited w'lth the Laboratory of l\l:~lubioloyy of the Technical University of Delft under the accession number LMD 74.1 in 1974 and with the Centraal Bureau voor Schimmelcultures (CBS), Baarn under the accession number CBS
o 760.83 in 1983 where the strain is available to the public.
Bacillus stearothermophilus var. cAliCIo/~ C953 and St,~t~coccus thermophilus T101 are very sensitive to dl ILiba~iLelidl compounds, especially dllLiL,iuLi~s such as peniciliins and ~:llell,uLl,e,df.eutics such as sulpha compounds. These Illi.;lUOlydllisllls are fast growing and have the additional advantage that their optimal growing temperature is high (Bacil/us s~,ull,e""olphilus: between 50C and 70C; Sllt~ cocc~l~ thermophilus:
between 35C and 45C). This makes these Illi~;lUUlydlli~ very suitable for a test according to the invention as they cannot grow at storage temperature (e.g.room temperature) and there is little possibility that Olyalli "":, which are possibly present in the test liquid or which have otherwise been included in thetest system would affect the result of the test.
When the test organism is a Bacillus strain, it is preferably i, Ico",o, dLed into the agar medium in the form of a spore suspension which may be prepared and illCol~oldled into the agar medium prior to ~ by known methods (see, for example, GB-A-1467439). AlL~Ill.. ~;~cly, as hereinbefore indicated, the test organism may be added as a separate source, for example by a~ of a tablet or paper disc to the agar medium.
When the test organism is a St,~ ~coc~ strain, the bacteria are preferably in~u~ul dLed into the agar medium in the form of bacterial cells which may be prepared according to known methods (EP 0 28~ 792).
WO 96/239~
2 ~ 8672~
When the test ~ uu~ydllialll is in col~ct"L,dl~d form (e.g. freeze-dried form, tablet, paper disc, etc.) the concentrate is measured into a vessel which may be a conventional ampoule, a sealable test tube, a sample bottle, a hole of a block such as a microtiter plate and the like (see for example EP-û285792).
s The cul lc~ dLioll of test organism in the agar medium is suitably above 10' CFUlml, but desirably does not exceed 10' CFU/ml, e.g. a test organism CUl~Ct~ ldLiUI~ between 2 x 107 and 109 CFU/ml may be employed, preferably 2 x 10' to 3 x 10~ CFU/ml. Most preferably, the test organism Cul~C~ ld~iul~ in the is between 3 x 10' and 2 x 10~ CFU/ml.
According to a preferred embodiment of the present application, the sensitivity of the test organism is a~ lct~hle. The sensitivity may be altered by various means, for example by adding certain substances, by changing the test cùndiliolls such as the pH or concentration of buffering substances or agar or by varying the ratio of the volumes of agar and test liquid.
rj Examples of substances that may be added to the test system to change sensitivity are n~lrle~sid~c such as aclc,llosicle and antifolates such as trimethoprim, ormethoprim and ~LIuxuulilll which improve the sensitivity of the test organism to sulpha compounds. Salts of oxalic acid or hydrofluoric acid may be added to improve the sensitivity to tetracyclines. Cysteine may be added ~o diminish the sensitivity to penicillins.
I Ir . ~:il Id~ l the invention will be discussed in more detall for a test C~l I l,ur iSil Ig the agar medium, however, the skilled in the art will d~prl:CidL~ that these teachings can also be applied for tests without an agar medium.
As hereinbefore indicated, components needed or optionally desirable in 2s the agar medium, including optionally substances to change the sensitivity to certain dl,Li~a.,L~,ial compounds, may be added to an optionally buffered agar solution. The agar solution is allowed to solidify in the test units to form theagar medium in such a way that the test organism stays alive but cannot multiply because of lack of nutrients and / or low temperature. The needed compounds which have not been added to the agar solution prior to ~ WO 96t23901 l .,~
9 2 1 8 6 7 2 ~
s ' ' ~ ~ 1 have to be added from a separate source, for example a tablet or paper disc.
Needed ~"~pO~ "ts are the test organism, one or more indicators and the nutrients. Other compounds, e.g. substances to improve or diminish the sensitivity to certain a~ a~,t~lidl compounds and buffer solutions, are optional.
It is preferred to carry out the process of the present invention in such a way that the test organism does not grow or multiply in the agar medium in the presence of a known minimum amount of an antibacterial compound to be detected. Also the test units, e.g test tubes co, ILdil lil ,g the agar medium, have to be stored in such a way that the test organism, does not multiply in the agar medium. This is generally achieved by depriving the organism of nutrients until the test is carried out or by " Idil ILclil lil l9 the culture (during the production process) and the test units (during storage) at a sufficiently low temperature, preferably between 4C and 30C, more preferably between 4C and 15C, or 5 both.
The test units are preferably closed air-bght during storage in which condibon they may be stored for at least several months.
The optionally buffered agar medium co, lldil lil l9 the test organism and /
or the i, I~ dL~ ~ (S) and / or the nutrients and / or certain substances to alter the 20 sensitivity of the test is allowed to solidify in upright test units e.g. tubes.
The unlts preferably have d~l~"),i"ed sizes. This is because of the reliability of the test. The height of the agar medium in the test units is of great i""~..,Ld,~;e. If an antibiotic is present in the test sample it will diffuse into the agar. The sensitivity of the test is therefore preferably asc~, Ldil leCI with variation 25 of the height of the solidified agar in the test unit.
Preferably, the height of the agar medium in the test unit is selected in such a way that the indicator changes its colour when the antibacterial compound to be detected is present in a con~ n below a certain value, but does not change its colour when the co"c~"~, dliun is above this value.
30 Preferably, the test is arranged to be a test from which one can read whether a certain conc~, ItldLi-,ll of an al ,tiL,a. L~, idl compound is present or not. For this, , . , , , _ _ _ _ _ _ _ ,, _,,, ,,, , . , ,, . ,, . , ,, .. ,, ,,, ,, _ .. , _ . _ WO96/2390~ ~ s.~ I ~
~J ~ 3 ` -10 - 2 1 8 6 7 2~
the height of the test unit will preferably be high enough to contain an amount of agar medium and sample corresponding to a height of 3-30 mm,more preferably 6-15 mm. The internal cross-sectional .li,,,~,,siu,~ of the test units is preferably 1-20 mm, more preferably 1-14 mm.
The volume of the agar medium in the test unit is determined by the height of the test unit, the internal cross-sectional dillldl~io,1 of the test unit and the p~ l lldg~ of the volume of the test unit which is filled with the agar medium. The volume of the agar medium is preferably 10 ~l - 3 ml, more preferably 15 ~l -1 ml and most preferably 20 ~l - 500 ~
A suitable liquid test sample Yolume is for example 0,01 - 0,5 ml. Thus, for example, a test unit of the invention formed by solidifying 0.3 ml agar solution in an upright sterile test tube of internal diameter 9 mm has been found suitable for detecting 0.003 lU/ml Penicillin G in a milk sample.
When spores of 3acill~1s stearothermophilus var. c~lidol~tis are used, suitable incubation temperatures are between 55C and 70C, more preferably between 60C and 66C. Incubation can be carried out in any conventional form of incubator, e.g. a waterbath, a block heater, a stove or a dry incubator.
When cells of S~ o~,o~ s thermophil~s are used, suitable incubation temperatures are between 36 and 44C, more preferably between 38 and 42C.
As hereinbefore indicated, an incubation period of less than 150 minutes is feasible, e.g. 70 minutes, possibly as little as 60 minutes or even 30 minutes.
Such an incubation period is markedly shorter than the incubation period of other conventional I~ ,lobi~lo~J~r~l test sytems for antibacterial compounds employing less than 10' CFU's/ml.
A test of the invention is verY simple to carry out, so that persons who perform the test do not have to be specially educated. The result is very simpleto determine. After the incubation period, the colour of the agar medium cu, ,' ;. ,i"g the indicator shows if test organism growth did occur or not.
All documents mentioned in this r, ~',~ ~ 1 are herein incorporated by reference to the same extent as if each individual '~Pt '' " .1 or patent was -r~ lly and individually indicated to be ill~UI~JUldL~d by reference.
~ W0 96/239ol 1 ~
-11 - 2 ~ 86728 ,, , . ~ ~
Leaend to the Fiaures Fig. 1: shows the test duration as a function of the spore concentration for test units of type A employed as in Example IV.
Fig.2: shows the test duration as a function of the spore concentration for test units of type B employed as in Example V.
EXAMPLE I
Fl~;JdldLiUn of test tubes of tvpe A to detect antibiotics Test tubes of type A contained an agar medium prepared as follows:
A solubon was made of 12 9 agar, 9 9 sodium chloride and 50 ml of a 0,1 M L,i~Ll,d"old",i"e buffer solution (pH = 8.0) in 1000 ml distilled water. An amount of a solution of L~,u,,,o.;,~sull.urple to give a final cu, ICt" ~I~dtiùn of 20 mg per 1000 ml was also added. The final solution was sterilized for 20 minutes at 121C and cooled to about 60C.
To this solution different amounts of a suspension of Bacillus alullle~ 7philus VAr, c~ i~lcllacli~ C953 spores in distilled water were added to give final cu~1C~ dLiùns between 10~ and 10' spores per ml.
Sterile tubes with a diameter of about 9 mm were filled with 0,3 ml of the agar solution under aseptic ~u"ditiol~s and i,,l,,,ediclt,ly sealed, e.g. with aluminium foil. The agar solutlon in the test tubes was allowed to solidify in the test tubes while the test tubes were held in an upright position.
The tubes were stored at a temperature between 5C and 1 5C.
EXAMPLE ll r,~d,dLiùl~ of test tubes of tvr~e B to detect antibiotics Test tubes of type B contained an agar medium prepared as follows:
A solution was made of 10 9 agar, 9 9 sodium chloride, 2 9 glucose, 2 g peptone 2 9 tryptone 4 9 yedst extract and 50 m~ of a 0.1 M llitLlldll~ lille WO96/23901 ~ /r.l,r~
buffer solution (pH = 8.0) in 1000 ml distilled water. The final solution was sterilized for 20 minutes at 121C and cooled to about 60C.
An amount of a solution of Brilliant Black to give a final concentration of 80 mg per 1000 ml and an amount of a solution of Toluidine Blue to give a final 5 CUllCellLldi;UI) of 3 mg per 1000 ml were also added.
To this solution different amounts of a suspension of Bacillus .3. ulller~ p~ ' rs var. ' _'c ' C953 spores in distilled water were added to give final conct:"~, ~ 1S between 10 and 10' spores per ml.
Sterile tubes with a diameter of about 9 mm were filled with 0,3 ml of the 10 agar solution under aseptic col1diLiulls and immediately sealed e.g. with an aluminium foil. The agar solution in the test tubes was allowed to solidify in the test tubes while the test tubes were held in an upright position.
The tubes were stored at a temperature between 5C and 15C.
EXAMPLE lll r, t:,lJdl dLiol~ of nutrient tablets A mixture was made of 100 9 dextrose, 160 9 Avicel PH101, 50 9 tryptose, 14 9 yeast extract and 15 9 precirol. This mixture was sufficient to prepare about 18000 tablets with a diameter of d~pru,~ ldl~ly 3 mm and a thickness of a~p,uAi,,,dL~ly 1.9 mm.
EXAMPLE IV
Carrvinc~ out the test tvoe A
One of each of the test tubes of type A with different COIlCt:llLldLiol1s of Bacillus ~alulllellllophilus var. c~ tis spores as described in Example I
were opened by removing the seals. A nutrient tablet as described in Example 30 lll was added to each of the test tubes. Then 0.1 ml of a fresh cow's milk =
~ W096/2390~ CK
` ', ,.; ` -13- 21 ~6728 sample was added to each of the test tubes and the test tubes were didL~ :ly placed in a waterbath kept at 64C.
Observations were made after 30 minutes to 2 hours and 30 minutes.
- The time at which thè colour of the agar medium became yellow was d~L~I ",i"ed. As is shown in Figure 1, the test duration correlated with the number of spores added to the agar medium.
If at this specific time, the colour of the agar medium was blue, the sample was known to contain a d~ le amount of an dllLiLJd,~lidl compound (e.g. 0.003 I.U. per ml or more penicillin G). Figure 1 shows that a test duration reduction to at least 60 minutes is realizable.
EXAMPLE V
Carryina out the test tvpe B
The procedure described in example IV was followed except that test tubes of type B were used and no tablet was added to the test tube.
hgure 2 shows that a test duration reduction to at least 70 minutes is realizable.
2c EXAMPLE Vl Carrvina out the test tvr e B
An amount of penicillin G of 0.003 I.U. per ml was added to fresh cow's milk. The control sample contained no penicillin G.
Two of each of the test tubes of type B with cc,llc~:llLIdLions of Bacillus ~u,~,ll.~r",~phi~us var. ~- ~ ' spores as described in Example 11 were opened by removing the seal. Then 0,1 ml of each of the two milk samples was added to each of the test tubes and the test tubes were illllll~didL~ly placed in a waterbath kept at 64C.
wo s6n3so~
1 4 - 2 ~ 8 6 7 2 8 Observations were made after 30 minutes to 3 hours. The time at which the colour of the agar medium became yellow was determined. As is shown in Table 1, the test duration correlated with the amount of spores added to the agar medium without influencing the sensitivity of the test in a negative way.
Table 1 shows a decreasing test duration down to at least 70 minutes.
Even at a test duration of 70 minutes, a cul~c~l ILI d[iOIl of 0.003 I.U. per mlpenicillin G can be detected. Of course also higher CullC~ dLiulls of penicillinG can be detected.
~ WO 96/23901 I _1/~ 5,~::
-15- 2~86728 O
-U~ , +
o X +
o O X +
,.. ~
C~ X +
o o ~ o . +
~,7 E o C~ ;
o ¢,7 ~ ~ +
,~¦ a) ~ E E E o a~
U) o ,J ~ ~ U ~ 16 -EXANIPLE Vll Detection of antibiQtics and sulPha compounds in ",i~:, uLil~ dL~ (test tvpe C) s For the ~ Jdl dLiul ~ of the test plates (test type C) the procedure descri-bed in Example I was followed except that the nutrients described in Example lll(dextrose, tryptose and yeast extract) were not added separately but were added to the initial agar medium in the same Cvl~Ct:l ,i,dLiùn as described in Example 1. Also together with the buffer solution an amount of trimethoprim solution was added to give afinal CvllCt:lltldLiOIl of 60 ~9 per litre.
To the wells of sterile ,,,ki,uLiLt:,ulates 20 ~l of the agar solution was added under asceptic cu"di~i.."s. The plates were i"""edidL~ly sealed with aluminium foil.
The plates were stored at a temperature between 5C and 15C.
An amount of penicillin G of 3 ppb was added to fresh cow s milk.
Also a milk sample was prepared which contains an amount of sul~ adia~ of 10û ppb.
The control sample contained no penicillin G or s~l,ulladid~;, ,e.
The Illi._luLiLt:l~JlaL~ were opened by removing the seal. Then 20 ~l of 20 each of the three milk samples was added to each of the test plates (duplo) co"Ldi"i"g different cul~ccnlLIaLiùlls of ~acillus ~ .,v~ ophilus var.
c~ spores C9~3. The plates were i"""eclidL~:ly placed in a waterbath kept at 64C.
Observations were made a~ter 30 minutes to 150 minutes. The time at 25 which the colour of the agar medium became yellow was dt:L~:""i"ed. As is shown in table 2 the test duration correlated with the amount of spores added to the agar medium without influencing the sensitivity of the test in a negativeway.
Even at a test duration of 90 minutes both penicillin G and 30 SUI~ ddiaL,~ 'e can be detected at conc~"L dLions of respectively 3 ppb penicillin WO 96/23901 r~ c -17- 2-~ 8672~
~ . ~
G and 100 ppb sulfadiazine. Penicillin G (3 ppb) can even be detected at a test duration of 80 minutes.
Of course also higher con~"i,dlions of both penicillin G and sLl~ a.lid~; ,e can be detected.
Table 2: Sensitivitv of test tvpe C at different spore co, Ic~"~ s.
Test duration 140 t23 lOS 90 80 (minutes) 10spores/ml medium 1.3 x 107 2.5 x 107 5.1 x 107 1.0 X 10~ 2.0 x 10' milk without antibio tica milk with 3 ppb peni-cillin G + + + + +
15milk with 100 ppb + + + +
EX~MPLE Vlll 20 Detection of dl b . in milk (test tvDe D) In this ~A~.e,i",e"I S~ Vlv~ op~ T101 (DSM 4022) was used for the ck ~ . " ,i, , of aJ l~ibi. ;_s in milk.
.Si 7~ OGC~l~ Ulv~ opl ' l~ T101 was grown using methods known per se.
An amount of a solution of L, u" ,o~, ~sc l,uurple to give a final ~o". ~"I, dliUI I of 4 mg per 1000 ml was added to fresh cow s milk.
To part of the fresh cow s milk also an amount of penicillin G of 4 ppb was added.
The control sample contained no penicillin G.
SUBSTITUTE SHEET (RULE 26) wo 96/2390~
To the milk samples different amounts of Streptococcus ll,e""o~ ;lus T1ûl were added to give final conct~, ILldtiUI 15 between 10' and 1 09 CFU per ml.
Illlllledial~ly after pl~drdLi~n of the samples sterile tubes with a diameter of about 9 mm were filled with 0.3 ml of each milk sample (duplo).
5 The tubes were i~ l,edidlely placed in a waterbath kept at 42C.
Observations were made after 30 minutes to 2 hours and 30 minutes.
The time at which the colour of the agar medium became yellow was cl~lt:l 11 ~il ,ed. As is shown in table 3, the test duration correlated with the amount of cells added to the milk without influencing the sensitivity of the test in a negative way.
Even at a test duration of 80 minutes penicillin G can be detected at a cùnc~ ILl..t~k~l I of 4 ppb or higher.
Table 3: Sensitivity of test type D at different cell conc~, ILIdLiul~s.
Test duration 145 125 103 80 (minutes) cellslml 3.0 x 10' 6.0 x 10' 1,3 x 10~ 2,5 x 10 medium 20milk without Li~iuli~a milk with + + + +
4ppb penicillin G
Claims (13)
1. A method for detecting one or more antibactirial compounds in a liquid sample which comprises the steps of:
(i) contacting said sample with a test microorganism selected from thermophilic strains of Bacillus and Streptococcus, prior to addition of said sample growth of said test organism prevented;
(ii) providing conditions for said test microorganism whereby in the absence of antibacterial compounds growth of said test microorganism can occur and can be detected; and (iii) detecting the growth of said test microorganism after a time sufficient for detection of test microorganism growth in the absence of antibacterial compounds, but during which period detectable inhibition of test microorganism growth is maintained by a known amount of an antibacterial compound in a test sample;
whereby said test organism is present at above 107 colony-forming units (CFU)/ml.
(i) contacting said sample with a test microorganism selected from thermophilic strains of Bacillus and Streptococcus, prior to addition of said sample growth of said test organism prevented;
(ii) providing conditions for said test microorganism whereby in the absence of antibacterial compounds growth of said test microorganism can occur and can be detected; and (iii) detecting the growth of said test microorganism after a time sufficient for detection of test microorganism growth in the absence of antibacterial compounds, but during which period detectable inhibition of test microorganism growth is maintained by a known amount of an antibacterial compound in a test sample;
whereby said test organism is present at above 107 colony-forming units (CFU)/ml.
2. A method according to claim 1 wherein the test microorganism is dispersed in a solidified medium preferably in a solidified agar medium.
3. A method according to claim 1 wherein the test microorganism prior to addition of the test sample, is in concentrated form preferably in dried form.
4. A method according to any one of claims 1-3 suitable for detecting in less than 2.5 hours Penicillin G at 0.003 I.U. per ml.
5. A method according to any one of claims 14 wherein prior to addition of the sample, said test microorganism is present in said agar medium at 2 x 107 to 3 x 108 CFU/ml, or in case the test microorganism is not present in said medium, the test microorganism is after the addition of the sample present at 2 x 107 to 3 x 108 CFU/ml sample.
6. A method according to claim 1 whereby the growth of said test microorganism is detected by colour change of the agar medium resulting from presence of an acid-base or redox indicator.
7. A method according to any one of claims 1 to 6 wherein the test microorganism employed is a strain of Bacillus Stearothermophilus, preferably wherein Bacillus Stearothermophilus var. calidolactis C953 is employed, or wherein the test organism employed is a strain of Streptococcus thermophilus, preferably wherein Streptococcus thermophilus T101 is employed.
8. A method according to claim 2 wherein said agar medium has been formed from an agar solution containing sufficient nutrients to support growth of said test organism.
9. A method according to claim 2 wherein the nutrient source for said test organism, or part thereof, is applied to the solidified agar medium.
10. A method according to claim 2 wherein said agar medium contains at least one substance to change the sensitivity of the test organism to antibacterial compounds.
11. A method according to claim 2 wherein 10 µl - 3 ml agar medium is used.
12. A test unit for carrying out a method according to claim 2 comprising a container holding a solidified agar medium having dispersed therein a test organism selected from thermophilic strains of Bacillus and Streptococcus at above 107 CFU/ml, said agar medium optionally additionally containing nutrients for growth of said test organism and/or one or more components for detection of said growth.
13. A test unit as claimed in claim 12 wherein said container is a tube or a block with tubular indentations.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP95200243 | 1995-02-01 | ||
| EP95200243.4 | 1995-02-01 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2186728A1 true CA2186728A1 (en) | 1996-08-08 |
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ID=8219988
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002186728A Abandoned CA2186728A1 (en) | 1995-02-01 | 1996-02-01 | A rapid microbiological test for the detection of antibacterial compounds |
Country Status (18)
| Country | Link |
|---|---|
| US (1) | US6867015B1 (en) |
| EP (1) | EP0755456B1 (en) |
| JP (1) | JPH09511150A (en) |
| AT (1) | ATE197477T1 (en) |
| AU (1) | AU693993B2 (en) |
| BG (1) | BG100882A (en) |
| BR (1) | BR9603963A (en) |
| CA (1) | CA2186728A1 (en) |
| CZ (1) | CZ284196A3 (en) |
| DE (1) | DE69610879T2 (en) |
| ES (1) | ES2153562T3 (en) |
| HU (1) | HUP9602995A2 (en) |
| NO (1) | NO964142L (en) |
| NZ (1) | NZ301691A (en) |
| PL (1) | PL184460B1 (en) |
| SK (1) | SK123496A3 (en) |
| WO (1) | WO1996023901A1 (en) |
| ZA (1) | ZA96786B (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| ATE197477T1 (en) | 1995-02-01 | 2000-11-11 | Dsm Nv | A RAPID MICROBIOLOGICAL TEST FOR DETECTING ANTIBACTERIAL COMPOUNDS |
| EP1639123A1 (en) * | 2003-07-02 | 2006-03-29 | DSM IP Assets B.V. | Improved method for the determination of the presence of an antibiotic in a fluid |
| PL1639122T3 (en) * | 2003-07-02 | 2009-07-31 | Dsm Ip Assets Bv | Improved test system for the determination of the presence of an antibiotic in a fluid |
| ATE414784T1 (en) * | 2003-09-11 | 2008-12-15 | Dsm Ip Assets Bv | BLOOD AND URINE TEST |
| WO2005049809A1 (en) | 2003-11-18 | 2005-06-02 | Charm Sciences, Inc. | Inhibition assay method and device for detection of antibiotics |
| WO2005118837A1 (en) | 2004-06-02 | 2005-12-15 | Dsm Ip Assets B.V. | High ph test system for the determination of the presence of an antibiotic in a fluid |
| ES2263361B1 (en) * | 2004-11-30 | 2007-11-01 | Universidad De Zaragoza | METHOD OF DETECTION OF ANTIBIOTIC RESIDUES AND OTHER ANTIBACTERIAL COMPOUNDS. |
| US8476064B2 (en) * | 2006-05-09 | 2013-07-02 | Charm Sciences, Inc. | Inhibition assay method and device for detection of antibiotics |
| CA2701170A1 (en) | 2007-09-29 | 2009-08-27 | Timothy James Williams | Composition and method to modify sperm function and increase male gender ratio in mammals |
| JP5774586B2 (en) * | 2009-07-01 | 2015-09-09 | ビオメリュー・インコーポレイテッド | Method for neutralizing antibiotics in culture media |
| PL2756306T3 (en) | 2011-09-16 | 2020-03-31 | Dsm Ip Assets B.V. | Immunoassay for detecting antibiotics |
| WO2013037886A1 (en) | 2011-09-16 | 2013-03-21 | Dsm Ip Assets B.V. | Immunoassay for detecting antibiotics |
| WO2013037887A1 (en) | 2011-09-16 | 2013-03-21 | Dsm Ip Assets B.V. | Competitive immunoassay for the detection of antibiotics |
| WO2013037888A1 (en) | 2011-09-16 | 2013-03-21 | Dsm Ip Assets B.V. | Immunoassay for detecting antibiotics |
| WO2013057182A1 (en) | 2011-10-18 | 2013-04-25 | Dsm Ip Assets B.V. | Method for the determination of the presence of an antibiotic in a fluid |
| WO2014065191A1 (en) * | 2012-10-25 | 2014-05-01 | 学校法人 福岡大学 | Infection-evaluation-system model |
| CN104293661B (en) * | 2014-10-22 | 2016-08-24 | 哈德逊（天津）生物技术有限责任公司 | A kind of quick antibacterial test method and kit |
| CN106554987A (en) * | 2015-09-29 | 2017-04-05 | 北京华益精点生物技术有限公司 | The test kit and its detection method of antibiotic in a kind of detection food-borne animal tissue |
| CN105524975A (en) * | 2015-12-24 | 2016-04-27 | 丽珠集团新北江制药股份有限公司 | Detection medium for determining microbiological potency of enramycin |
| CN108463726B (en) | 2016-01-15 | 2021-06-08 | 帝斯曼知识产权资产管理有限公司 | Method for detecting an analyte |
| ES2910202T3 (en) | 2017-06-02 | 2022-05-11 | Fastinov S A | Therapeutic drug monitoring |
| CN114323876B (en) * | 2022-03-14 | 2022-05-13 | 广东江门中医药职业学院 | Method for detecting cloxacillin drug residue in aquatic product |
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| DE755456C (en) | 1938-10-05 | 1955-05-05 | Schaeffer & Budenberg G M B H | Safety valve with an elliptical link for vertical guidance of the valve body |
| GB1467439A (en) * | 1973-05-31 | 1977-03-16 | Gist Brocades Nv | Method for determination of the presence of antibiotics |
| NL7806086A (en) | 1978-06-05 | 1979-12-07 | Gist Brocades Nv | ANALYSIS UNIT. |
| DE3429823A1 (en) | 1984-08-14 | 1986-02-27 | Merck Patent Gmbh, 6100 Darmstadt | MEANS AND METHOD FOR DETECTING ANTIMICROBIALLY EFFECTIVE SUBSTANCES |
| FI75865C (en) * | 1987-04-07 | 1988-08-08 | Valio Meijerien | TESTANORDNING OCH FOERFARANDE FOER BESTAEMNING AV ANTIBIOTIKA I MJOELK SAMT I DESSA ANVAENDBAR NY STREPTOCOCCUS THERMOPHILUS-STAM. |
| US5354663A (en) * | 1988-05-04 | 1994-10-11 | Charm Sciences, Inc. | Microbial inhibition test kit and method |
| US5008255A (en) | 1989-01-12 | 1991-04-16 | Alcon Laboratories, Inc. | Method for enhancing solubility of trimethoprim with sodium sulfacetamide, and synergistic pharmaceutical compositions derived therefrom |
| CA2056581A1 (en) * | 1991-11-12 | 1993-05-13 | Stanley E. Charm | Microbial inhibition test kit and method |
| ES2176220T3 (en) * | 1993-02-11 | 2002-12-01 | Dsm Nv | PROCEDURE FOR THE DETECTION OF ANTIBACTERIAL COMPOUNDS IN TEST SAMPLES AND UNIT FOR THEIR REALIZATION. |
| DE4432771A1 (en) * | 1994-09-14 | 1996-03-21 | Landesvereinigung Der Bayerisc | Test medium for a microbiological inhibitor test and method for carrying it out |
| ATE197477T1 (en) | 1995-02-01 | 2000-11-11 | Dsm Nv | A RAPID MICROBIOLOGICAL TEST FOR DETECTING ANTIBACTERIAL COMPOUNDS |
-
1996
- 1996-02-01 AT AT96902983T patent/ATE197477T1/en not_active IP Right Cessation
- 1996-02-01 JP JP8523277A patent/JPH09511150A/en active Pending
- 1996-02-01 ES ES96902983T patent/ES2153562T3/en not_active Expired - Lifetime
- 1996-02-01 NZ NZ301691A patent/NZ301691A/en unknown
- 1996-02-01 HU HU9602995A patent/HUP9602995A2/en unknown
- 1996-02-01 EP EP96902983A patent/EP0755456B1/en not_active Revoked
- 1996-02-01 US US08/704,747 patent/US6867015B1/en not_active Expired - Fee Related
- 1996-02-01 PL PL96316582A patent/PL184460B1/en not_active IP Right Cessation
- 1996-02-01 ZA ZA96786A patent/ZA96786B/en unknown
- 1996-02-01 CZ CZ962841A patent/CZ284196A3/en unknown
- 1996-02-01 BR BR9603963A patent/BR9603963A/en not_active Application Discontinuation
- 1996-02-01 SK SK1234-96A patent/SK123496A3/en unknown
- 1996-02-01 CA CA002186728A patent/CA2186728A1/en not_active Abandoned
- 1996-02-01 AU AU47180/96A patent/AU693993B2/en not_active Ceased
- 1996-02-01 DE DE69610879T patent/DE69610879T2/en not_active Revoked
- 1996-02-01 WO PCT/EP1996/000488 patent/WO1996023901A1/en not_active Application Discontinuation
- 1996-09-30 NO NO964142A patent/NO964142L/en unknown
- 1996-10-01 BG BG100882A patent/BG100882A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| DE69610879T2 (en) | 2001-05-17 |
| SK123496A3 (en) | 1997-07-09 |
| ATE197477T1 (en) | 2000-11-11 |
| AU693993B2 (en) | 1998-07-09 |
| NO964142D0 (en) | 1996-09-30 |
| EP0755456A1 (en) | 1997-01-29 |
| PL316582A1 (en) | 1997-01-20 |
| AU4718096A (en) | 1996-08-21 |
| EP0755456B1 (en) | 2000-11-08 |
| ES2153562T3 (en) | 2001-03-01 |
| NZ301691A (en) | 1997-03-24 |
| HUP9602995A2 (en) | 1997-05-28 |
| BG100882A (en) | 1997-08-29 |
| ZA96786B (en) | 1996-08-12 |
| WO1996023901A1 (en) | 1996-08-08 |
| BR9603963A (en) | 1997-10-07 |
| NO964142L (en) | 1996-11-20 |
| HU9602995D0 (en) | 1997-01-28 |
| PL184460B1 (en) | 2002-10-31 |
| CZ284196A3 (en) | 1997-03-12 |
| MX9604494A (en) | 1997-11-29 |
| JPH09511150A (en) | 1997-11-11 |
| US6867015B1 (en) | 2005-03-15 |
| DE69610879D1 (en) | 2000-12-14 |
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