WO1994016097A1 - Antibiotic assay and kits for the use thereof - Google Patents
Antibiotic assay and kits for the use thereof Download PDFInfo
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- WO1994016097A1 WO1994016097A1 PCT/IT1994/000001 IT9400001W WO9416097A1 WO 1994016097 A1 WO1994016097 A1 WO 1994016097A1 IT 9400001 W IT9400001 W IT 9400001W WO 9416097 A1 WO9416097 A1 WO 9416097A1
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- 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
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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- C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
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- This invention relates to an antibiotic assay and to kits for the use thereof.
- this invention relates to a method to detect the growth of bacterial strains, which are resistant to one or more antibiotics, by means of pH variation sensitive colorimetric indicators, on samples from biological fluids, waters, emissions, etc.
- This assay type shows some advantages, when compared with the conventional Kirby and Bauer's method; but it is also involving art-skilled operators and expensive equipment; furthermore the method does not allow a clear and univocal result evaluation, as it often shows intermediate values.
- the authors of the present invention developed a system to detect bacterial growth, or an inhibition of bacterial growth, in the presence of an effective amount of antibiotics, as function of pH changes in the culture medium. "Effective amount” here means a concen- tration, that is able to inhibit the growth of a microorganism strain, which is sensitive to such given antibiotic. pH changes, depending from the metabolism of sugars into the culture medium, are detected by means of color indicators.
- pH changes are identified by a pH color indicator contained into said culture medium.
- It is a further object of the invention a method to detect the microorganism growth from a sample comprising the steps, as follows: to add a microorganism suspension to a culture medium suitable to grow said microorganism and containing a pH color indicator, preferably phenolsulfonphtalein (red phenol), to get a mixture;
- a pH color indicator preferably phenolsulfonphtalein (red phenol)
- said culture medium for said microorganism comprises:
- Said containers may comprise test tubes, trays, microtitration plates, wherein the antibiotic was previously added steadily.
- microtitration trays comprise different wells, each one containing a different amount of different antibiotics, in order to enable to assay various antibiotics at the same time.
- a kit to detect growth a microorganism from a sample in the presence of an effective amount of an antibiotics comprising:
- a sterile microtitration plate comprising different wells, each one containing an antibiotic effective amount in a stabilized state; said plate at least containing two different antibiotics in two different wells and at least one antibiotic-free well;
- a sterile container containing an adequate amount of a culture medium suitable to promote the growth of said microorganism as well as a pH indicator, preferably a red phenol.
- said antibiotic is contained at least into a pair of wells, at the minimum effective concentration in the former and at a higher concentration in the latter.
- the plate is prepared by dissolving the antibiotic as instructed by the Supplier, diluting properly the obtained solution, filling wells at the same time with 50 ⁇ l of said solution, by using an automatic multichannel dosing device, and finally dehydrating said wells until the solvent is full evaporated, at a temperature not able to deactivate said antibiotics.
- Sterile plates could be maintained up to one year at a temperature ranging from 4°C up to 8°C.
- the system consists of 17 dried antibiotics into various wells of a microtitration tray at single specific concentrations, allowing to assay the
- sensitivity to antibiotics of the most common bacteria that could be collected from an urine culture. Bacteria are resuspended in a culture medium, containing a growth indicator, and then loaded in each system wells. After having been incubated 18-24 hours at 37°C, the automated or displayed reaction readings are carried out.
- control well bacteria growth well
- Antibiotics are listed in Table 1.
- plates, test tubes and pipets may be decontaminated by a sodium hypochlorite incubation, incinerated or processed on autoclave.
- control strains are used from ATCC, Maryland, US.
- the kit may be stored for one year thereafter at a temperature ranging from 2°C up to 8°C.
- Example 2 TWO CONCENTRATION ANTIBIOTIC ASSAY ON URINE GERMS
- the system consists of 12 dual concentrated, dried antibiotics.
- the assay procedure is as in Example 1, but the antibiotics are listed in table 3:
- Example 3 QUICK ASSAY ON NEGATIVE BACTERIA
- the system consists of 12 dual concentrated, dried antibiotics, and allows to assay the sensitivity of the more common Gram negative bacteria (Negative Oxidase) to antibiotics.
- the procedures are the same described in the Example 1, but the concerned antibiotics are as listed in Table 5 herebelow.
- Example 4 STAPHYLOCOCCUS ASSAY The system consists of dual concentration dried antibiotics, and allows to carry out the antibiotic assay for the staphylococcus bacteria. The procedure is the same as described in the Example 1, but the antibiotics are used as listed in the Table 6 herebelow:
- the system contains 12 dual concentrated dried antibiotics, and allows to carry out the Streptococcus assay. The same procedure of the Exaple 1 is followed, but the system contains the antibiotic as listed in the Table 7 herebelow: Table 7 Antibiotic contained in the system
- the system consists of single concentrated dried antibiotics, and allows to assay the sensitivity of the gram positive bacteria to the antibiotics.
- Example 7 COMPARISON WITH OTHER ASSAY METHODS The following comparison tables are obtained using certified bacterial strains from American Tissue Culture Collection (ATCC), Maryland, US.
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Abstract
A method to assay a microorganism growth, or a microorganism growth inhibition, in the presence of an effective antibiotic amount, as a function of the pH changes in the culture medium and a color change of a color indicator, is described. The method and the kit therefom are to be used to assay samples from biological fluids, water, effluents, etc.
Description
ANTIBIOTIC ASSAY AND KITS FOR THE USE THEREOF
This invention relates to an antibiotic assay and to kits for the use thereof.
More particularly, this invention relates to a method to detect the growth of bacterial strains, which are resistant to one or more antibiotics, by means of pH variation sensitive colorimetric indicators, on samples from biological fluids, waters, emissions, etc.
In order to carry out proper therapies, it is crucial to identify the sensitivity of bacterial strains to various antibiotics in a sample. Purposely, reliable and standard assays are needed, allowing to identify the sensitivity to different antibiotics of the bacterial strain isolated from a sample. A largely used method of the previous art is the so-called Kirby and Bauer's technique, involving to cultivate one or more isolated bacterial species into a solid medium (Mϋller/Hinton's medium), in presence of a number of disks, each one impregnated with a different antibiotic at a given concentration. The detection of a typical growth inhibition area around the disk allows to classify the bacteria as either resistant, or sensitive, or partially sensitive to the antibiotic. Materials to carry out the method are commercialized by Biolife, Italy.
However, this method involves long times, art- skilled operators and high costs. Moreover the detection of bacterial growth inhibition areas does not lead to a clear and univocal evaluation. Finally, the Kirby and Bauer's method does not allow to assay the antibiotic minimum concentration which is able to inhibit microorganism proliferation.
Thus, systems were developed in the recent years, allowing to assay the microorganism sensitivity to different antibiotics at the same time and with a
higher precision. In all of these systems the bacterial antibiotic resistance is assayed by means of bacterium growth in a liquid medium in the presence of the antibiotic; being the bacterial growth detection evidetiated by means of a turbidity reading, either by sigth or automatic spectrophotometers. The kit derived therefrom is available on the market from Bio-Merieux, France.
This assay type shows some advantages, when compared with the conventional Kirby and Bauer's method; but it is also involving art-skilled operators and expensive equipment; furthermore the method does not allow a clear and univocal result evaluation, as it often shows intermediate values. The authors of the present invention developed a system to detect bacterial growth, or an inhibition of bacterial growth, in the presence of an effective amount of antibiotics, as function of pH changes in the culture medium. "Effective amount" here means a concen- tration, that is able to inhibit the growth of a microorganism strain, which is sensitive to such given antibiotic. pH changes, depending from the metabolism of sugars into the culture medium, are detected by means of color indicators. Results fully agree with the previously described methods and, in some cases, shows even a higher sensitivity. Further, the method is easy to apply, not expensive and no skilled operators are requested. Finally, the result readings are clear and univocal. It is an object of the present invention a method to detect the microorganism growth from a sample into a liquid culture medium in the presence of an effective amount of an antibiotics, wherein said microorganism growth is detected by identifying pH changes in said culture medium, so that the pH becomes more acidic in respect of the pH of the culture medium,
when no growth occurs. Preferably, pH changes are identified by a pH color indicator contained into said culture medium.
It is a further object of the invention a method to detect the microorganism growth from a sample, comprising the steps, as follows: to add a microorganism suspension to a culture medium suitable to grow said microorganism and containing a pH color indicator, preferably phenolsulfonphtalein (red phenol), to get a mixture;
- to load sterile containers with fractions of said mixture, each container containing an effective amount of an antibiotics; to incubate said mixture at a growth permissive temperature for said microorganism for at least 16 hours, preferably from 18 up to 24 hours; and
- to verify the color of said culture medium. Preferably, said culture medium for said microorganism comprises:
- Mϋller-Hinton' »s broth 21 g/i
- K2HP0 0.3 g/i
- Glucose 30 g/i
- MgCl2 0.25 g/i
- CaCl2 6.7 g/i
- Red phenol (0. .3 %) in H20 42 ml/1
- Horse serum 10 ml/1 being the pH in the range of 7.0 - 7.4.
Said containers may comprise test tubes, trays, microtitration plates, wherein the antibiotic was previously added steadily. For convenience, microtitration trays comprise different wells, each one containing a different amount of different antibiotics, in order to enable to assay various antibiotics at the same time. It is a further object of the invention a kit to detect growth a microorganism from a sample in the
presence of an effective amount of an antibiotics, comprising:
- a sterile microtitration plate, comprising different wells, each one containing an antibiotic effective amount in a stabilized state; said plate at least containing two different antibiotics in two different wells and at least one antibiotic-free well;
- a sterile container containing an adequate amount of a culture medium suitable to promote the growth of said microorganism as well as a pH indicator, preferably a red phenol.
According to a preferred embodiment of the invention, said antibiotic is contained at least into a pair of wells, at the minimum effective concentration in the former and at a higher concentration in the latter.
According to the invention, the plate is prepared by dissolving the antibiotic as instructed by the Supplier, diluting properly the obtained solution, filling wells at the same time with 50 μl of said solution, by using an automatic multichannel dosing device, and finally dehydrating said wells until the solvent is full evaporated, at a temperature not able to deactivate said antibiotics. Sterile plates could be maintained up to one year at a temperature ranging from 4°C up to 8°C.
The invention shall be described in the following by reference to some explicative, but not limiting, examples, which are related to different samples as well as to some comparison tables in respect of other assay tecniques.
Example 1 QUICK ANTIBIOTIC ASSAY ON URINE GERMS
The system consists of 17 dried antibiotics into various wells of a microtitration tray at single specific concentrations, allowing to assay the
"sensitivity to antibiotics of the most common bacteria,
that could be collected from an urine culture. Bacteria are resuspended in a culture medium, containing a growth indicator, and then loaded in each system wells. After having been incubated 18-24 hours at 37°C, the automated or displayed reaction readings are carried out.
Procedure:
1) to take a plate;
2) to obtain a bacterial suspension with an opacity corresponding to 0.5 of the McFarland's standard;
3) to transfer the following amounts of this suspension to the test tube, containing the culture broth with the red phenol: a) 10 μl of Gram negative bacteria; b) 200 μl of Gram positive bacteria;
4) to load each well with 0.2 ml of the bacterial suspension;
5) to put over the well a proper cover after having recorded the patient's name, the assay date and the bacterium type;
6) to incubate for 18-24 hours at 37°C;
7) to check the color change in the control well (bacterium growth well) and evaluate the results.
Antibiotics are listed in Table 1.
TABLE 1 Antibiotics contained in the system
WELL Nr. CODE ANTIBIOTIC mg/1
1 F Nitrofurantoin 100
2 NA Nalidixic acid 16
3 NOR Norfloxacillin 8
4 CIP Cyprofloxacillin 2
5 PEF Pefloxacillin 4
6 KF Chefalotin 32
7 CFD Cefonicid 32
8 CAZ ' Ceftadizime 32
9 CN Gentamicin 8
10 TOB Tobramycin 8
11 AK Amikacin 16
12 SXT Co-trimoxazole 8
13 ATM Aztreonam 32
14 AMP Ampicillin 16
15 AMX Amoxycillin 16
16 MEZ Mezlocillin 32
17 PRL Piperacillin 64
18 C Control
Readings and evaluation:
Red to yellow color changes are recorded for each well and results are evaluated according to the following table 2:
TABLE 2
COLOR BACTERIAL GROWTH THE BACTERIUM IS:
Red - S = sensitive Orange +/- I = partial sensitive
Yellow + R = resistant
Some no-glucose fermenting and oxidase negative bacteria cause no yellow shifts of red phenol, used as bacterial growth indicator; therefore the antibiotic- resistance is visualized by well turbidity as follows: CLEAR RED = S = SENSITIVE; and TURBID RED = R = RESISTANT
After the use, plates, test tubes and pipets may be decontaminated by a sodium hypochlorite incubation, incinerated or processed on autoclave.
In order to check the method standardization, control strains are used from ATCC, Maryland, US.
The kit may be stored for one year thereafter at a temperature ranging from 2°C up to 8°C.
Example 2 TWO CONCENTRATION ANTIBIOTIC ASSAY ON URINE GERMS
The system consists of 12 dual concentrated, dried antibiotics. The assay procedure is as in Example 1, but the antibiotics are listed in table 3:
TABLE 3 Antibiotics contained in the system
WELL NR. CODE ANTIBIOTIC μg/ml c C
1-2 F Nitrofurantoin 25 100
3-4 NA Nalidixic acid 8 16
5-6 NOR Norfloxacillin 1 8
7-8 AMX Amoxycillin 4 16
9-10 PRL Piperacillin 16 64
11-12 KF Chefalotin 8 32
13-14 CFD Cefonicid 4 32
15-16 CAZ Ceftadizime 4 32
17-18 CN Gentamicin 4 8
19-20 TOB Tobramicin 4 8
21-22 AK Amikacin 16
23 SXT Co-trimoxazole
24 C Control
Red to yellow shifts are recorded for each well contents and the results evaluated as in Table 4:
TABLE 4
COLORS OF THE BACTERIAL
TWO WELLS GROWTH T THH1E BACTERIUM IS
(each antibiotic) c C
Red/red - - S sensitive
Orange/red +/- - MS mid sensitive
Yellow/red + - LS lightsensitive
Yellow/orange + +/- MR mid resistant
Yellow/yellow + + R ' resistant
Red/yellow - + Test not in confor¬ mity (not valid) wherein: c = lower antibiotic concentration; C = higher antibiotic concentration; - = no bacterial growth (red) ;
+ = bacterial growth (yellow) ; and -/+ = small bacterial growth (orange) .
Example 3 QUICK ASSAY ON NEGATIVE BACTERIA The system consists of 12 dual concentrated, dried antibiotics, and allows to assay the sensitivity of the more common Gram negative bacteria (Negative Oxidase) to antibiotics. The procedures are the same described in the Example 1, but the concerned antibiotics are as listed in Table 5 herebelow.
TABLE 5 Antibiotics contained in the system
WELL NR. CODE ANTIBIOTIC μg/ l c C
1-2 AMP Ampicillin 4 1 6
3-4 MEZ Mezlocillin 8 32
5-6 KF Chefalotin 8 32
7-8 CAZ Ceftadizime 4 32 9 9--1100 C CFFDD C Ceeffoonniicciidd 4 32
11-12 CN Gentamicin 4 8
13-14 TOB Tobramicin 4
15-16 AK Amikacin 16
17-18 TET Tetracycline
1199--2200 CC CClloorraammpphheenniiccooll 8 16
21-22 CIP Cyprofloxacin 1 2
23 SXT Co-trimoxazole
24 C Control
The red to yellow shifts are recorded for each well contents and the results evaluated according to the criteria as described in the Example 2. Example 4: STAPHYLOCOCCUS ASSAY The system consists of dual concentration dried antibiotics, and allows to carry out the antibiotic assay for the staphylococcus bacteria. The procedure is the same as described in the Example 1, but the antibiotics are used as listed in the Table 6 herebelow:
TABLE 6 Antibiotics contained in the system
WELL NR. COD: ANTIBIOTIC μg/ml c C 1 1--22 E ERRYY Erythromycin 1 4
3-4 PEF Pefloxacin 1 4
5-6 CIP Cyprofloxacillin 1 2
7-8 SXT Co-trimoxazole 2 8
9-10 CFD Cefonicid 4 32 1 111--1122 T TEECC Teichoplanin 4 16
13-14 CN Gentamicin 4 8
15-16 AK Amikacin 8 16
17-18 FOS Phosphomycin 32 64
19-20 PRL Piperacillin 16 64 2 211--2222 A AMMSS Ampicillin/Sulbactam 8/4 16/8
23 OXA Oxacillin 2
24 Control
The red to yellow shifts are recorded for each well contents and the results evaluated according to the the criteria as described in Example 2. Example 5: STREPTOCOCCUS ASSAY
The system contains 12 dual concentrated dried antibiotics, and allows to carry out the Streptococcus assay. The same procedure of the Exaple 1 is followed, but the system contains the antibiotic as listed in the Table 7 herebelow:
Table 7 Antibiotic contained in the system
WELL NR. CODE ANTIBIOTIC μg/m] c C
1-2 ERY Erythromycin 1 4
3-4 PEF Pefloxacillin 1 4
5-6 CIP Cyprofloxacillin 1 2
7-8 SXT Co-trimoxazole 2 8
9-10 CFD Cefonicid 4 32
11-12 CAZ Ceftadizime 4 32
13-14 TEC Theicoplanin 4 16
15-16 RD Rifamycin 4 16
17-18 AMS Ampicillin/Sulbactam 8/4 16/8
19-20 TCC Ticarcillin/Clavulanic acid 16/1 64/4
21-22 PRL Piperacillin 16 64
23 OXA Oxacillin 2
24 Control
The red to yellow shifts are recorded for each well contents and the results evaluated according to the criteria as described in the Example 2. Example 6: GRAM POSITIVE ASSAY
The system consists of single concentrated dried antibiotics, and allows to assay the sensitivity of the gram positive bacteria to the antibiotics. The procedure followed as in Example 1, however the concerned antibiotics are enlisted as in the Table 8 herebelow. Table 8
Antibiotics contained in the system
WELL NR. CODE ANTIBIOTIC μg/ml
1 CN Gentamicin
2 AK Amikacin 16
3 TOB Tobramycin 8
4 FOS Phosphomycin 64
5 TEC Theicoplanin 16
6 TET Tetracycline 8
7 RD Rifamycin 16
8 ERY Erythromycin 4
9 CIP Cyprofloxacillin 2
10 PEF Pefloxacillin 4
11 SXT Co-trimoxazole 8
12 CFD Cefonicid 32
13 CAZ Ceftadizime 32
1144 AAMMSS Ampicillin/Sulbactam 16/8
15 TCC Ticarcillin/Clavulanic acid 64/4
16 PRL Piperacillin 64
17 OXA Oxacillin 2
18 C Control
The red to yellow shifts are recorded for each well contents and the results evaluated according to the criteria as described in the Example 1. Example 7: COMPARISON WITH OTHER ASSAY METHODS The following comparison tables are obtained using certified bacterial strains from American Tissue Culture Collection (ATCC), Maryland, US.
BACT.: E.Coli (ATCC 25922)ATR-1 API-BIOMER. HINTON'S DISK
F Nitrofuroantoin 100 S s s NA Nalidixic acid 16 S s s NOR Norfloxacillin 8 S s s CIP Cyprofloxacillin 2 S s s PEF Pefloxacillin 4 S s s KF Chefalotin 32 s s s CFD Cefonicid 32 s s s CAZ Ceftadizime 32 s s s CN Gentamicin 8 s s s TOB Tobramicin 8 s s s
AK Amikacin 16 S S s
SXT Co-trimoxazole 8 S S s
ATM Aztreonam 32 S S s
AMP Ampicillin 16 R R R
MX Amoxycillin 16 R I R
MEZ Mezlocillin 32 S s S
PRL Piperacillin 64 S s S
C Control GROWTH +
S = sensitive (red) R - resistant (yellow) I = intermediate (orange
BACT. :Streptofecalis(ATCC19433)ATR-l API-BIOM. HINTON'S
DISK
F Nitrofurantoin 100 S s S
NA Nalidixic acid 16 R R R
NOR Norfloxacillin 8 S s S
CIP Cyprofloxacilin 2 S s S
PEF Pefloxacillin 4 S S S
KF Chefalotin 32 S S s
CFD Cefonicid 32 R R R
CAZ Ceftazidime 32 I R R
CN Gentamicin 8 R R R
TOB Tobramicin 8 S S S
AK Amikacin 16 R R R
SXT Co-trimoxazole 8 S S S
ATM Aztreonam 32 R R R
AMP Ampicillin 16 S S S
AMX Amoxycillin 16 R R R
MEZ Mezlocillin 32 S S S
PRL Piperacillin 64 S S S
C Control GROWTH +
BACT. Providencia Stuartii ATR-1 API-BIOM. HINTON'S
DISK
F Nitrofurantoin 100 S S S
NA Nalidixic acid 16 S S S
NOR Norfloxacillin 8 s S S
CIP Cyprofloxacillin 2 s S s
PEF Pefloxacillin 4 s S s
KF Chefalotin 32 R R R
CFD Cefonicid 32 S S s
CAZ Ceftazidime 32 S S s
CN Gentamicin 8 S S s
TOB Tobramicin 8 S s s
AK Amikacin 16 S s s
SXT Co-trimoxazole 8 S s s
ATM Aztreonam 32 S s s
AMP Ampicillin 16 R R R
AMX Amoxycillin 16 R R R
MEZ Mezlocillin 32 S S S
PRL Piperacillin 64 S S S
C Control GROWTH +
BACT. E. Coli ATR-2 API-BIOM . HINTON'S
DISK
F Nitrofurantoin 25 100 S S S
NA Nalidixic acid 8 16 S S S
NOR Norfloxacillin 1 8 S s S
AMX Amoxycillin 4 16 I I I
PRL Piperacillin 16 64 S s s
KF Chefalotin 8 32 I I I
CFD Cefonicid 4 32 s s s
CAZ Ceftazidime 4 32 s s s
CN Gentamicin 4 8 s s s
TOB Tobramicin 4 8 s s s
AK Amikacin 8 16 s s s
SXT Co-trimoxazole 8 s s s
C Control GROWTH +
BACT. : E. Cloacae ATR-2 API-BIOM. HINTON'S
DISK
F Nitrofurantoin 25 100 I I I
NA Nalidixic acid 8 16 S s S
NOR Norfloxacillin 1 8 S s S
AMX Amoxycillin 4 16 R R R
PRL Piperacillin 16 64 S S S
KF Chefalotin 8 32 R R R
CFD Cefonicid 4 32 R R R
CAZ Ceftadizime 4 32 S S S
CN Gentami—cin 4 8 S S S
TOB Tobramicin 4 8 S S S
AK Amikacin 8 16 s S S
SXT Co-trimoxazole 8 s S S
C Control GROWTH +
BACT. : Ent. Cloacae ATR-3 API- -BIOM. HINTON'S
DISK
AMP Ampicillin 4 16 R R R
MEZ Mezlocillin 8 32 S S S
KF Cefalotin 8 32 R R R
CAZ Ceftazidime 4 32 S S S
CN Gentamicin 4 8 S S S
TOB Tobramicin 4 8 S S S
AK Amikacin 8 16 S S S
TET Tetracycline 4 8 S S S
C Chloramphenicol 8 16 S S S
CIP Cyprofloxacillin 1 2 S s S
SXT Co-trimoxazole 8 S s S
C Control GROWTH -
BACT. : Staphylococcus ATR-4 API- -BIOM. HINTON'S aureus DISK
ERY Erythromycin 1 4 S S S
PEF Pefloxacillin 1 4 S S S
"CIP Cyprofloxacillin 1 2 s S S
SXT Co-trimoxazole 2 8 S S S
CFD Cefonicid 4 32 S S S
TEC Theicoplanin 4 16 S S S
CN Gentamicin 4 8 S S S
AK Amikacin 8 16 S S S
FOS Phosphomycin 32 64 I I R
PRL Piperacillin 16 64 S S S
AMS Ampicillin/
Sulbactam 8/4 16/8 S S S
OXA Oxacillin 2 S S S
C Control GROWTH +
Claims
1. A method to assay the microorganism growth from a sample in a liquid culture medium in the presence of an effective amount of an antibiotics, wherein said growth is detected by identifying pH changes in said culture medium, so that the pH becomes more acidic in respect of the pH of the culture medium, when no growth occurs.
2. A method to assay the microorganism growth from a sample according to claim 1, wherein said pH changes are detected by means of a pH color indicator contained whitin said culture medium.
3. A method to assay the microorganism growth from a sample according to claim 2, wherein said pH color indicator is red phenol.
4. A method to assay the microorganism growth from a sample according to any of previous claim, comprising the steps as follows: to add a microorganism suspension in a culture medium permissive to the growth of said microorganism and containing a pH color indicator, preferably red phenol, to get a mixture;
- to load sterile containers with fractions of said mixture, each container containing an effective amount of an antibiotics; to incubate said mixture at a growth permissive temperature for said microorganism for at least 16 hours, preferably from 18 up to 24 hours; and
- to verify the color of said culture medium.
5. Method to assay the microrganism growth from a sample according to the claim 4, wherein said suitable culture medium to promote the growth of said microorganism comprises:
- Muller-Hinton broth 21 g/1 - K2HP04 0.3 g/1
- Glucose 30 g/1 - MgCl2 0.25 g/1
- CaCl2 6.7 g/1
- Red phenol (0.3%) in H20 42 ml/1
- Horse serum 10 ml/1 ranging the pH of the culture medium from 7.0 to 7.4.
6. A method to assay the microorganism growth from a sample according to any claim 4 or 5, wherein said containers include test tubes, trays, microtitration trays to which the antibiotic was previously added steadily.
7. A method to assay the microorganism growth from a sample according to the claim 6, wherein said microtitration trays comprise a number of wells, each one containing at least a preset antibiotic concentration to allow to assay various antibiotics at the same time.
8. A kit to assay a microorganism growth from a sample in the presence of an effective antibiotic amount comprising: - a sterile microtitration plate, comprising different wells, each one containing an antibiotic effective amount in a stabilized state; said plate at least containing two different antibiotics in two different wells and at least one antibiotic-free well; - a sterile container containing an adequate amount of a culture medium suitable to promote the growth of said microorganism as well as a pH indicator, preferably a red phenol.
9. A kit according to the claim 8, wherein said antibiotic is contained at least into a pair of wells, at the minimum effective concentration in the former and at a higher concentration in the latter.
10. A kit according to the claims 8 or 9, wherein said plates are prepared by dissolving the antibiotic as instructed by the Supplier, by diluting properly the obtained solution, by filling wells at the same time with 50 μl of said solution, by using an automatic multichannel dosing device, and finally by dehydrating said wells until the solvent is full evaporated, at a temperature not able to deactivate said antibiotics.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU60438/94A AU6043894A (en) | 1993-01-04 | 1994-01-03 | Antibiotic assay and kits for the use thereof |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ITRM930002A IT1261158B (en) | 1993-01-04 | 1993-01-04 | ANTIBIOGRAM AND DIAGNOSTIC KIT FOR ITS REALIZATION. |
| ITRM93A000002 | 1993-01-04 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1994016097A1 true WO1994016097A1 (en) | 1994-07-21 |
Family
ID=11401372
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/IT1994/000001 WO1994016097A1 (en) | 1993-01-04 | 1994-01-03 | Antibiotic assay and kits for the use thereof |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU6043894A (en) |
| IT (1) | IT1261158B (en) |
| WO (1) | WO1994016097A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1589113A1 (en) * | 2004-04-19 | 2005-10-26 | Lohmann Animal Health GmbH & Co. KG | Device for determining the sensitivity of bacterial pathogens and vaccine strains of poultry |
| US6984499B2 (en) | 1997-10-02 | 2006-01-10 | Idexx Laboratories, Inc. | Method and apparatus for concurrently detecting pathogenic organisms and antimicrobial susceptibility |
| WO2009037369A1 (en) | 2007-09-21 | 2009-03-26 | Consejo Superior De Investigaciones Cientificas | MANIPULATION OF THE FUNCTION OF AtDBP1 IN ORDER TO GENERATE POTYVIRUS RESISTANCE |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1132455A (en) * | 1965-10-30 | 1968-11-06 | Albert John Oliver | Means for testing the action of chemical compounds on micro-organisms |
| DE2822433A1 (en) * | 1978-05-23 | 1979-12-20 | Peters Hans Volker Dipl Biolog | Increasing antibacterial activity of chemotherapeutic agents - against resistant bacteria, by combination with plant growth factors, e.g. gibberellin A3 |
| SU1171525A1 (en) * | 1983-12-27 | 1985-08-07 | Кишиневский государственный медицинский институт | Method of determining minimum inhibiting concentration of antibiotics |
| FR2638170A1 (en) * | 1988-10-24 | 1990-04-27 | Var Diffusion Bacteriologie | System and process for the detection of bacteria in a medium |
| EP0373543A2 (en) * | 1988-12-16 | 1990-06-20 | BASF Corporation | Diagnostic media and assay for detecting fungicide resistance |
| SU1693059A1 (en) * | 1989-07-17 | 1991-11-23 | Белгородский сельскохозяйственный институт | Method for determination of cow mastitis pathogens sensitivity to antibiotics |
| WO1992006213A1 (en) * | 1988-07-27 | 1992-04-16 | George Saperstein | Antibiotic sensitivity test, apparatus for use with the same and antibiotic sensitivity test kit containing the same |
| US5206151A (en) * | 1990-06-11 | 1993-04-27 | Nalco Chemical Company | Rapid selection of biocide using a reduction oxidation indicator system |
-
1993
- 1993-01-04 IT ITRM930002A patent/IT1261158B/en active IP Right Grant
-
1994
- 1994-01-03 WO PCT/IT1994/000001 patent/WO1994016097A1/en active Application Filing
- 1994-01-03 AU AU60438/94A patent/AU6043894A/en not_active Abandoned
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1132455A (en) * | 1965-10-30 | 1968-11-06 | Albert John Oliver | Means for testing the action of chemical compounds on micro-organisms |
| DE2822433A1 (en) * | 1978-05-23 | 1979-12-20 | Peters Hans Volker Dipl Biolog | Increasing antibacterial activity of chemotherapeutic agents - against resistant bacteria, by combination with plant growth factors, e.g. gibberellin A3 |
| SU1171525A1 (en) * | 1983-12-27 | 1985-08-07 | Кишиневский государственный медицинский институт | Method of determining minimum inhibiting concentration of antibiotics |
| WO1992006213A1 (en) * | 1988-07-27 | 1992-04-16 | George Saperstein | Antibiotic sensitivity test, apparatus for use with the same and antibiotic sensitivity test kit containing the same |
| FR2638170A1 (en) * | 1988-10-24 | 1990-04-27 | Var Diffusion Bacteriologie | System and process for the detection of bacteria in a medium |
| EP0373543A2 (en) * | 1988-12-16 | 1990-06-20 | BASF Corporation | Diagnostic media and assay for detecting fungicide resistance |
| SU1693059A1 (en) * | 1989-07-17 | 1991-11-23 | Белгородский сельскохозяйственный институт | Method for determination of cow mastitis pathogens sensitivity to antibiotics |
| US5206151A (en) * | 1990-06-11 | 1993-04-27 | Nalco Chemical Company | Rapid selection of biocide using a reduction oxidation indicator system |
Non-Patent Citations (3)
| Title |
|---|
| DATABASE WPI Week 8609, Derwent World Patents Index; AN 86-060413 * |
| DATABASE WPI Week 9242, Derwent World Patents Index; AN 92-347830 * |
| K.G.WHITHEAR ET AL.: "Evaluation and Use of a Micro-broth Dilution Procedure for Testing Sensitivty of Fermemtative Avian Mycoplasmas to Antibiotics", AVIAN DISEASES, vol. 27, no. 4, 1983, pages 937 - 949 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6984499B2 (en) | 1997-10-02 | 2006-01-10 | Idexx Laboratories, Inc. | Method and apparatus for concurrently detecting pathogenic organisms and antimicrobial susceptibility |
| EP1589113A1 (en) * | 2004-04-19 | 2005-10-26 | Lohmann Animal Health GmbH & Co. KG | Device for determining the sensitivity of bacterial pathogens and vaccine strains of poultry |
| WO2009037369A1 (en) | 2007-09-21 | 2009-03-26 | Consejo Superior De Investigaciones Cientificas | MANIPULATION OF THE FUNCTION OF AtDBP1 IN ORDER TO GENERATE POTYVIRUS RESISTANCE |
Also Published As
| Publication number | Publication date |
|---|---|
| ITRM930002A1 (en) | 1994-07-04 |
| ITRM930002A0 (en) | 1993-01-04 |
| AU6043894A (en) | 1994-08-15 |
| IT1261158B (en) | 1996-05-09 |
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