GB2147415A

GB2147415A - Device and method for antibiotic sensitivity testing

Info

Publication number: GB2147415A
Application number: GB08321137A
Authority: GB
Inventors: Leslie S W Dodd
Original assignee: Powell and Scholefield Ltd
Current assignee: SHS International Ltd
Priority date: 1983-08-05
Filing date: 1983-08-05
Publication date: 1985-05-09
Also published as: GB2147415B; GB8321137D0

Abstract

A device for determining the sensitivity of organisms to antibiotics comprises a layer of a stabilized absorbent material, for example paper, impregnated with a tetrazolium compound and an electron accelerator compound. The antibiotics may be applied to the surface of a culture plate or an antibiotic may be included in the growth medium, then the device of the invention is applied to the surface of the plate and any colour change observed. The electron accelerator compound may be 5-methyl phenazonium methyl sulphate or NADPH.

Description

SPECIFICATION Device and method for antibiotic sensitivity testing The present invention relates to a device and method for antibiotic sensitivity testing. More particularly the present invention relates to a method for testing the sensitivity of organisms to antibiotics and to a device for use in such a method.

The rapid and accurate determination of the sensitivity of organisms to antibiotics is of importance both in clinical laboratories and in quality control of the antibiotic impregnated microbiological test discs.

At present the in vitro determination of the sensitivity of bacterial strains to antibiotics is normally carried out by using the agar diffusion test or the tube dilution test. In the agar diffusion test the effect of antibiotics on the growth of the bacteria on a solid agar growth medium is observed, normally by direct observation of bacterial growth with the naked eye.

However, in the tube dilution test the effect of antibiotics on the growth of the bacteria in a liquid growth medium is observed, again normally by direct observation of the bacterial growth with the naked eye.

Developments in the field of antibiotic sensitivity testing have produced a number of accelerated in vitro antibiotic sensitivity test methods based on the tube dilution test and observing the change in particular characteristics of the test organism in the growth medium in the presence of antibiotics. For example, microcalorimetric, intracellular ATP level and optical variation have been monitored and directly related to the reaction of the test organism with the antibiotic. As a consequence of these developments, a generation of automatic antibiotic sensitivity test systems are now available based on the tube dilution test.

2,3,5-triphenyl tetrazolium chloride (T.T.C.) can be used to indicate when organism growth has occurred and when no organism growth has occurred, the 2,3,5-triphenyl tetrazolium chloride being red in colour in the presence of organisms and being clear in colour in the absence of any organism. However, tube dilution tests carried out to observe the growth of standard strains of Staphylococcus Aureus, Escherichia Coli and Pseudomonas Aeruginosa bacteria in broth in the presence of sub-inhibitory concentrations of 2,3,5-triphenyl tetrazolium chloride resulted in no significant chlorimetric reaction (i.e., no significant difference in colour between samples having no bacterial growth and samples having bacterial growth), whereas higher concentrations of 2,3,5-triphenyl tetrazolium chloride, although resulting in an observable chlorimetric reaction, proved to be inhibitory to certain strains of test bacteria. That is, at concentrations of 2,3,5-triphenyl tetrazolium chloride sufficient such that an observable chlorimetric reaction occurs, the 2,3,5-triphenyl tetrazolium chloride itself can have an inhibitory effect on the growth of certain strains of organism. This possible inhibitory effect of 2,3,5triphenyl tetrazolium chloride itself is, of course, a disadvantage when carrying out tests to determine the sensitivity of organisms to antibiotics as it may not be clear whether any observed inhibition in the growth of the test organism is due wholly, or in part, to such an inhibitory effect of the 2,3,5-triphenyl tetrazolium chloride.

It has been found that tube dilution tests involving the addition of optimum concentrations of 2,3,5-triphenyl tetrazolium chloride to established broth cultures of standard strains of organisms produced minimum inhibitory concentration (M.l.C.) determinations within seven hours and that these determinations had a 90% correlation to within one double dilution. However, although quite good correlation in the M.l.C. determinations is found to occur, the test system still suffers from the above-stated disadvantages of possible inhibition of organism growth as a result of inhibition by the 2,3,5-triphenyl tetrazolium chloride itself.

In addition to utilizing 2,3,5-triphenyl tetrazolium chloride in antibiotic sensitivity tests based on the tube dilution test, it is also possible to utilize 2,3,5-triphenyl tetrazolium chloride as an organism growth indicator in antibiotic sensitivity tests based on the agar diffusion test. For example, in such antibiotic sensitivity tests based on the agar diffusion test, the 2,3,5-triphenyl tetrazolium chloride may be incorporated into the agar before contact of the test organism with the agar or it may be applied to established agar/organism cultures.

Agar diffusion tests involving the incorporation of 2,3,5-triphenyl tetrazolium chloride into Mueller-Hinton agar have resulted in uninhibited organism growth (as compared with control bacterial cultures containing no 2,3,5,-triphenyl tetrazolium chloride) and zone delineation at a concentration of 0.001% by weight of 2,3,5-triphenyl tetrazolium chloride in agar (10 ug T.T.C.

ml-' agar) for Staphylococcus Aureus and 0.01% by weight of 2,3,5-triphenyl tetrazolium chloride in agar (100 ug T.T.C. ml-' agar) for Escherichia Coli after 6 hours incubation at 37"C.

However, observations with other bacterial cultures suggested variable sensitivity of the bacteria to the 2,3,5-triphenyl tetrazolium chloride. It was, therefore, concluded that, whilst agar diffusion tests utilizing certain levels of 2,3,5-triphenyl tetrazolium chloride in agar resulted in good zone delineation with some bacterial isolates, the same levels of 2,3,5,-triphenyl tetrazolium chloride were inhibitory to the growth of other bacterial isolates and, consequently, that the use of 2,3,5-triphenyl tetrazolium chloride in the agar diffusion test is not satisfactory as a method for accelerated antibiotic sensitivity testing.

In the agar diffusion test an agar plate is seeded or inoculated with a test organism culture and an antibiotic is then placed on the surface of the agar plate, the antibiotic usually being applied to the surface of the agar plate in the form of one or more paper discs impregnated with the antibiotic. The effect, if any, of the antibiotic on the growth of the test organism is then observed. After the antibiotic has been applied to the surface of the agar plate it commences to diffuse through the agar, the rate of diffusion being a function of the diffusion coefficient of the specific antibiotic, the concentration of the antibiotic at source (i.e., at its point of application to the agar plate), and possibly the concentration of the test organism.If the test organism is sensitive to the antibiotic, the antibiotic will interact with the test organism as it diffuses through the agar and this will result in a zone being formed in which growth of the test organism is inhibited by the antibiotic. As the antibiotic diffuses through the agar and interacts with the test organism, the concentration of the antibiotic will decrease. Therefore, at a certain distance from the antibiotic source the antibiotic will reach a concentration which is sufficiently low that it can no longer inhibit the growth of the test organism and, at this point, the zone of inhibited organism growth will have attained its maximum size. The maximum size of the inhibition zone is a function of the sensitivity of the test organism to the antibiotic and also a function of the concentration of the test organism.At the outer periphery of the inhibition zone, and beyond its outer periphery, the test organism can survive as the concentration of the antibiotic is subinhibitory.

Although the maximum size of the inhibitiion zone is usually formed after only approximately 4-5 hours, it is not visible to the naked eye for approximately 1 8 to 24 hours after the commencement of the test. The reason for this is that after 4-5 hours the concentration of test organisms in the uninhibited regions of the agar plate is too low (approximately 106 organisms per square centimeter of plate surface) to be clearly observed by the naked eye and, hence, too low for the border between the inhibition zone and the organism growth region to be observed by the naked eye.It is only after approximately 1 8 to 24 hours that the concentration of test organisms in the uninhibited regions of the agar plate is sufficiently high (approximately 10'0 organisms per square centimetre of plate surface) to allow the border between the inhibition zone and the organism growth region to be clearly observed by the naked eye.

In view of the above, it is clear that a significant improvement in accelerated antibiotic sensitivity tests would be achieved if a method, based on the agar diffusion test, could be developed which avoids the disadvantage of the previously used accelerated antibiotic sensitivity tests, namely the possible inhibitory effect of the 2,3,5-triphenyl tetrazolium chloride indicator itself on the growth of the test organisms. Such an improved accelerated antibiotic sensitivity test has now been developed in accordance with the present invention.

According to the present invention there is provided a device, which comprises a layer of a stabilized absorbent material impregnated with a tetrazolium compound and an electron accelerator compound.

The stabilized absorbent material used in the device according to the present invention, may for example, be a stabilized fibrous absorbent material. Preferably the stabilized absorbent material is an absorbent paper, especially blotting or filter paper.

In one embodiment of the device according to the present invention, the layer of stabilized absorbent material is in the form of a circular disc, preferably approximately 8 cm in diameter.

The tetrazolium compound used in the device according to the present invention is preferably selected from 2, 3,5-triphenyl tetrazolium chloride, 2,2', 5, 5'-tetraphenyl-3, 3'-(3, 3'-dimethoxy- 4,4'-diphenylene)ditetrazolium chloride, and p,p'-diphenylene-bis-2-(3,5-diphenyl tetrazolium chloride).

The electron accelerator compound used in the device according to the present invention is a compound which is used as an electron carrier in biochemical metabolic pathways. Preferably the electron accelerator compound is selected from 5-methyl phenazonium methyl sulphate and dihydronicotinamide adenine dinucleotide phosphate (NADPH).

The device according to the present invention may, for example, be prepared by a method, which comprises sterilizing a layer of a stabilized absorbent material, impregnating the sterilized layer of the stabilized absorbent material with a solution comprising a tetrazolium compound, an electron accelerator compound and deionized water, and then drying the impregnated layer of the stabilized absorbent material.

The stabilized absorbent material, the tetrazolium compound and the electron accelerator compound used in the above method for preparing the device according to the present invention may, for example, be any of the materials hereinbefore mentioned in connection with the device according to the present invention. Similarly. the layer of the stabilized absorbent material used in the above method for preparing the device according to the present invention may, for example, have the configuration hereinbefore mentioned in connection with the device according to the present invention, i.e., the layer of the stabilized absorbent material, may for example, be in the form of a circular disc, preferably approximately 8 cm in diameter.

The solution used in the above method for preparing the device according to the present invention preferably comprises from 50 to 200 milligrams of the tetrazolium compound per millilitre of deionized water, more preferably from 75 to 1 50 milligrams of the tetrazolium compound per millilitre of deionized water.

The solution used in the above method for preparing the device according to the present invention preferably comprises from 2 to 20 milligrams of the electron accelerator compound per millilitre of deionized water, more preferably from 5 to 10 milligrams of the electron accelerator compound per millilitre of deionized water.

In the above method for preparing the device according to the present invention the layer of the stabilized absorbent material is preferably stabilized, prior to impregnation with the aqueous tetrazolium compound/electron accelerator compound solution, by heating it to a temperature of from 100"C to 140"C, more preferably from 110"C to 130"C, at a pressure of from 0.5 to 3 atmospheres, more preferably from 0.75 to 2.5 atmospheres.

Further, in the above method for preparing the device according to the present invention, the impregnated layer of the stabilized absorbent material is preferably dried at atmospheric pressure by heating it at a temperature of from 30"C to 80"C, more preferably from 50"C to 70"C, for a period of 1 to 7 hours, more preferably from 3 to 5 hours.

The device according to the present invention has been found to be particularly useful in the determination of the sensitivity of organisms to antibiotics.

Accordingly, the present invention further provides a method for determining the sensitivity of organisms to antibiotics, hereinafter referred to as the test method of the invention, which comprises contacting an antibiotic with a test organism on the surface of an organism growth medium, incubating the test organism at elevated temperature, applying a device according to the present invention to the surface of the organism growth medium such that the device contacts at least that portion of the surface of the organism growth medium on which the test organism/antibiotic contact takes place, and observing any subsequent colorimetric reaction occurring as a result of interaction between the test organism and the tetrazolium compound contained in the device according to the present invention.

After application of the device according to the present invention to the surface of the organism growth medium, the tetrazolium compound and the electron accelerator compound present in the device will diffuse from the device to the organism growth medium and, if in doing so the tetrazolium compound comes into contact with the test organism, a colour change will occur which can be observed and from which can be determined the sensitivity of the test organism to the antibiotic.

The organism growth medium suitable for use in the test method of the invention is preferably an agar growth medium, for example, Oxoid CM 337 marketed by Oxoid.

Examples of organism, the sensitivity of which to antibiotics can be determined by the test method of the invention, include Escherichia Coli, Staphylococcus Aureus, Pseudomonas Aeruginosa, Klebsiella Aerogenes and Proteus Vulgaris.

Examples of antibiotics suitable for use in the test method of the invention include cotrimoxazole, naladixic acid, sulphafurazole, nitrofuranton, penicillin, ampicillin, mecillinam, gentamicia, cephaloridine and amikacin.

According to a first preferred embodiment of the test method of the invention there is provided a method for determining the sensitivity of organisms to antibiotics, which comprises forming a test plate comprising a solid organism growth medium inoculated or seeded with a test organism, applying an antibiotic to a portion of the surface of the test plate, incubating the resulting test plate at elevated temperature, applying a device according to the present invention to the surface of the test plate such that the device contacts that portion of the test plate to which the antibiotic has been applied and also extends outwardly across the surface of the test plate, allowing at least a portion of the tetrazolium compound and the electron accelerator compound, which are present in the device according to the present invention, to diffuse from the device to the test plate, and observing any subsequent colorimetric reaction occuring as a result of interaction between the tetrazolium compound and the test organism.

In this first preferred embodiment of the test method of the invention the test plate may, for example, be formed by placing the organism growth medium, in fluid form, into a suitable container, for example a petri dish, then surface drying the organism growth medium, for example at approximately 37"C for approximately 30 minutes, and then inoculating, for example by means of a swab, the test organism onto the dried surface of the organism growth medium.

An alternative method for forming the test plate comprises seeding the organism growth medium, in liquid form, with the test organism, placing the seeded organism growth medium into a suitable container, for example a petri dish, and then surface drying the organism growth medium, for example at approximately 37"C for approximately 30 minutes.

In the above first preferred embodiment of the test method of the invention the antibiotic may, for example, be applied to the test plate in the form of an absorbent paper disc, e.g. a filter paper disc or a blotting paper disc, impregnated with the antibiotic.

After application of the antibiotic to the test plate in the above described first preferred embodiment of the test method of the invention, the test plate is preferably incubated at approximately 37"C. Preferably the period of incubation of the test plate is from 4 to 6 hours.

The use, in the above described first preferred embodiment of the test method of the invention, of a device according to the present invention comprising a tetrazolium compound allows the region of the test plate in which growth of the test organism is inhibited to be readily distinguished from the region of the test plate in which growth of the test organism is taking place. The reason for this is that the tetrazolium compound will interact with any test organism which it comes into contact with and, as a result of such an interaction, will change colour, e.g.

from colourless to red. Therefore, the region or regions of the surface of the test plate in which growth of the test organism is inhibited and which is/are contacted by the device according to the present invention will be one colour, e.g. colourless, and the region or regions of the surface of the test plate in which growth of the test organism is taking place and which is/are contacted by the device according to the present invention will be a different colour, e.g. red.

As the indicator (i.e. colour change) properties of the tetrazolium compound allow the region(s) of test organism growth and the region(s) of inhibited test organism growth to be readily distinguished from each other in a period of time much less than the 1 8 to 24 hours incubation time required if these regions were to be distinguished directly by the naked eye (i.e.

without the use of an indicator), for example after an incubation period of only 4 to 6 hours, and also as the use of the device according to the present invention allows the tetrazolium compound to be used in a sub-inhibiting amount (i.e. an amount low enough such that the tetrazolium compound itself does not inhibit the test organism growth) but still gives a clear distinction between the region(s) of the test organism growth and the region(s) of inhibited test organism growth after an incubation period of, for example, only 4 to 6 hours, it can be seen that the test method of the present invention provides a significant improvement over the known methods for determining the sensitivity of organisms to antibiotics.

In the above described first preferred embodiment of the test method of the invention the test plate may, for example, be inoculated or seeded with two or more test organisms, each of the test organisms occupying a different region of the test plate and being contacted by a single antibiotic. In this case, the antibiotic may, for example, be applied to the surface of the test plate in the form of one or more absorbent paper discs, e.g. filter paper discs or blotting paper discs, impregnated with the antibiotic, the or each antibiotic impregnated disc being placed on the surface of the test plate such that it contacts more than one of the test organism-containing regions of the test plate (i.e. the or each antibiotic impregnated disc bridges the border between two or more test organism-containing regions of the test plate).Alternatively, however, the antibiotic may, for example, be applied to the surface of the test plate in the form of a plurality of absorbent paper discs, e.g. blotting paper discs or filter paper discs, impregnated with the antibiotic, each of the test organism-containing regions of the test plate being contacted by at least one of the antibiotic impregnated discs and each of the antibiotic impregnated discs contacting only one test organism-containing region of the test plate (i.e. antibiotic impregnated disc is located wholly within a particular test organism-containing region of the test plate and each test organism-containing region of the test plate is contacted by at least one of the antibiotic impregnated discs.

In the above described first preferred embodiment of the test method of the invention the test plate may, for example, be inoculated or seeded with one or more test organisms, each test organism occupying a different region of the test plate when more than one test organism is utilized, and the or each test organism being contacted by two or more different antibiotics. For example, the antibiotics may be applied to the surface of the test plate in the form of absorbent paper discs, e.g. blotting paper discs or filter paper discs, impregnated with antibiotic and with each absorbent paper disc being impregnated with only one antibiotic.

According to a second preferred embodiment of the test method of the invention there is provided a method for determining the sensitivity of organisms to antibiotics, which comprises forming a test plate comprising a solid organism growth medium and an antibiotic, applying a test organism to the surface of the test plate, incubating the resulting test plate at elevated temperature, applying a device according to the present invention to the surface of the test plate such that the device contacts at least that portion of the surface of the test plate to which the test organism has been applied, allowing at least a portion of the tetrazolium compound and the electron accelerator compound, which are present in the device according to the present invention, to diffuse from the device to the test plate, and observing any subsequent colorimetric reaction occurring as a result of interaction between the tetrazolium compound and the test organism.

In this second preferred embodiment of the test method of the invention the test plate may, for example, be prepared by admixing the antibiotic and the organism growth medium, placing the resulting mixture into a suitable container, for example petri dish, and then surface drying the organism growth medium, for example at approximately 37"C for approximately 30 minutes.

After application of the test organism to the test plate in the above described second preferred embodiment of the test method of the invention, the test plate is preferably incubated at approximately 37"C. Preferably the period of incubation of the test plate is from 4 to 6 hours.

By following the procedure of the second preferred test method of the invention it is possible to determine qualitatively the sensitivity of the test organism to the antibiotic. This qualitative determination is achieved by observing whether there is any colorimetric reaction occurring as a result of interaction between the tetrazolium compound, which is contained in the device according to the present invention and which diffuses from the device to the test plate, and the test organism and directly relating the degree of any colour change of the tetrazolium compound to the sensitivity of the test organism to the antibiotic. For example, no observed colour change, only slight colour change or a strong colour change of the tetrazolium compound is interpreted as meaning that the test organism is sensitive, slightly resistant or resistant, respectively to the antibiotic.If this procedure is repeated utilizing a test organism of known sensitivity to the antibiotic, it is possible to obtain a direct comparison between the test organism of unknown sensitivity and the test organism of known sensitivity to the antibiotic.

If a quantitative determination of the sensitivity of a test organism to an antibiotic is required utilizing the procedure of the second preferred embodiment of the test method of the invention, then this may, for example, be achieved by preparing a series of test plates comprising the organism growth medium and the antibiotic, each test plate in the series being more dilute with respect to the antibiotic content than the next test plate along the series. Each of these test plates is then utilized in the procedure of the second preferred embodiment of the test method of the invention and any colorimetric reaction occurring as a result of interaction between the tetrazolium compound and the test organism is observed. In this way it is possible to determine the test plate which has the weakest concentration of the antibiotic but still prevents growth of the test organism.The concentration of the antibiotic in this test plate is then interpreted as being the minimum inhibitory concentration (M.l.C.) of antibiotic to the particular test organism.

Results of tests carried out to determine the M.l.C. of an antibiotic to a test organism utilizing the above procedure of the second preferred embodiment of the test method of the invention showed an 89.7% correlation with the results of the test carried out to determine the M.l.C. of the same antibiotic/test organism combination utilizing the known test procedure where the growth/inhibition of the test organism by the antibiotic is observed directly by the naked eye after an incubation period of from 18 to 24 hours.

In the first and second preferred embodiment of the test method of the invention the organism growth medium, the test organism and the antibiotic may, for example, be any of those mentioned above, i.e. any of those mentioned above in connection with the general test method of the invention.

If desired, the method in accordance with the present invention, for determining the sensitivity of organisms to antibiotics may, after the step of observing any colorimetric reaction occurring as a result of interaction between the test organism and the tetrazolium compound contained in the device according to the present invention, comprise the additional steps of further incubating the system comprising the test organism, organism growth medium, antibiotic and device according to the present invention, e.g. for a period of time of approximately 1 5 to 30 minutes, at elevated temperature, e.g. at approximately 37"C, and then observing either whether or not there has been any alteration in the colorimetric reaction observed after the initial incubation period, or if no colorimetric reaction was noted after the initial incubation period, whether or not any colorimetric reaction has taken place as a result of the further incubation period. As an alternative to retaining the device according to the present invention on the surface of the organism growth medium during the further incubation period, the device may be removed prior to the further incubation and a fresh device according to the present invention applied to the surface of the organism growth medium after the further incubation period but before the further observation.

By carrying out the further incubation and observation steps it is possible to investigate whether or not the initial incubation period has been sufficient to obtain the maximum inhibitory effect, if any, of the antibiotic on the test organism.

The present invention will now be further illustrated by way of the following Examples. In the Examples antibiotic impregnated absorbent paper discs were utilized, these discs being supplied by Scientific Hospital Supplies Limited and being impregnated with one of the antibiotics listed in Table 1 below.

TABLE 1 Levels discs-' Antibiotic (yg.disc- 1) Code Cotrimonazole 25 COT Naladixic Acid 30 NA Sulphafurazole 300 SF Nitrofurantoin 300 NF Penicillin 10* PG Ampicillin 10 AH Mecillinam 25 MC Gentamicin 10 GT Cephaloridine 30 CPM Amikacin 10 AK 'Penicillin disc levels in International units.

The antibiotic content of the impregnated discs was within + 5% of the stated content.

Also, in the following Examples the test organisms utilized are as indicated in Table 2 below.

TABLE 2 Organism Experimental Code Source Escherichia Coli 6 NCTC '1 Escherichia Coli "2 PS 451 URINE Escherichia Coli '3 BG 439 URINE Staphylococcus Aureus 42 NCTC Staphylococcus Aureus BG 643 WOUND Pseudomonas Aeruginosa 2 NCTC Pseudomonas Aeruginosa BG 662 SPUTUM Klebsiella Aerogenes BG 536 URINE Proteus Vulgaris PS 413 URINE *1-National Culture Type Collection *2-Powell and Scholefield stock cultures "3---Clinical isolates from Brooke General Hospital, Woolwich, London.

Further, in Examples 10 to 27 and 32 to 35, the indicator discs used were prepared by autoclawing, at a pressure of 2 atmospheres and a temperature of 121 C, 8 cm diameter Whatman grade 3 filter paper discs, impregnating the autoclawed discs by immersing them in a solution of 2,3,5-triphenyl tetrazolium chloride and 5-methyl phenazonium methyl sulphate in deionized water, blotting dry the impregnated discs and then drying the discs at a temperature of 55"C for 3 hours. The 2,3,5-triphenyl tetrazolium chloride was present in the aqueous solution in an amount of 100 mg ml-' of deionized water and the 5-methyl phenazonium methyl sulphate was present in the aqueous solution in an amount of 6 mg.ml-1 of deionized water.

EXAMPLES 1 to 9 (Comparitive) An overnight culture of the test organism was used. The overnight culture was inoculated with a swab onto the surface of a pre-dried (37"C for 30 minutes) Mueller-Hinton agar test plate formed in a petri-dish. 0.1 ml of standardized incolum having approximately 108 colony-forming units (cfu) ml-1 of inoculum was inoculated onto the test plate. An antibiotic impregnated disc was then applied to the surface of the test plate. The test plate was then incubated at 37"C for 1 8 hours and, after the incubation, the delineation between the zone of organism growth and any zone of inhibited- organism growth was observed directly by the naked eye. The sensitivity of the test organism to the antibiotic was then determined by the standard Kirby-Bauer method which relates inhibition zone size to the sensitivity of the organism to the antibiotic. This procedure was adopted for each of the test organism/antibiotic combinations and results obtained are given below in Table 3.

TABLE 3

Average Antibiotic inoculum Example Organism Code COT NA SF NF PG AH MEC GT CPH (cfu.ml-1) 1 E. Coli NCTC6 S S S S S S S S S 2.2 x 108 2 E. Coli PS451 S S S S S S S S S 4.7 x 108 3 E. Coli BG439 S S S S R R S S S 1.3 x 109 4 S. Aureus NCTC42 R R R R S S S S S 2.4 x 109 5 S. Aureus BG 643 R R R R R R R S R 7.5 x 108 6 P. Aeruginosa NCTC2 R R R R R R R S R 6.9 x 108 7 P. Aeruginosa BG 662 R R R R R R R S R 9.2 x 107 8 K. Aerogenes BF 536 R R R R R R S S S 6.1 x 108 9 P. Vulgaris PS 413 R S R R R R S R S 1.9 x 108 r = Resistant as determined by the standard Kirby-Bauer method.

S = Sensitive " " " " " " " " Each result recorded after triplicate experiments.

EXAMPLES 10 to 18 An overnight culture of the test organism was used. Mueller Hinton agar was seeded with 1 ml of test organism culture having approximately 108 colony-forming units (cfu) ml -1 of culture and then a test plate was formed from this seeded agar by placing the seeded agar in a petridish and surface-drying it at 37"C for 30 minutes. Four antibiotic impregnated test discs were then applied to the surface of the test plate and the test plate was subsequently incubated at 37"C for either 5 or 6 hours. At intervals during the incubation and after the incubation had been completed, the zone of the organism growth and any zone(s) of inhibited organisms growth were observed by placing, on the surface of the test plate an indicator disc as defined above.

After initially placing the device according to the present invention on the surface of the test plate, the device was retained on the test plate during the remainder of the incubation. The sensitivity of the test organism to the antibiotic was then determined by the standard Kirby Bauer method which relates inhibition zone size to the sensitivity of the organism to the antibiotic. This procedure was adopted for each of the test organism/antibiotic combinations and the results obtained are given below in Table 4.

TABLE 4

average Antibiotic inoculum Example Organism Code COT NA SF NF PG AH MEC GT CPH (cfu.ml-1) 10 E. Coli NCTC6 S S S S S S S S S 6.7 x 108 11 E. Coli PS 451 S S S S R R S S S 4.9 x 108 12 E. Coli BG 439 S S S S R R S S S 7.1 x 108 13 S. Aureus NCTC42 S* S* S* S* S S S S S 9.4 x 108 14 S. Aureus BG 643 S* R S* R R R R S R 2.3 x 109 15 P. Aeruginosa NCTC2 R R R R R R R S R 4.9 x 108 16 P. Aeruginosa BG 662 R R R R R R R S R 6.9 x 108 17 K. Aerogenes BG 536 R R R S R R S S R* 1.5 x 108 18 P. Vulgaris PS 413 R S R R R R S R R* 5.5 x 108 R = Resistant as determined by Kirby-Bauer method S = Sensitive " " " " " " Each result recorded after triplicate experiments * = variation from sensitivity reported in Table 3.

In each of Examples 10 to 1 2 and 1 5 to 1 8 the total incubation time was 5 hours but in each of Examples 1 3 and 14 the total incubation time was 6 hours.

The results obtained in Examples 10 to 18 suggest that the test method according to the present invention gives a 91.1 % correlation with the reported sensitivities given in Table 3 for the comparitive Examples 1 to 9.

EXAMPLES 19 to 27 An overnight culture of the test organisms was used. The overnight culture was inoculated with a swab onto the surface of a pre-dried (37"C for 30 minutes) Mueller-Hinton agar test plate formed in a petri-dish. 0.1 ml of standardized inoculum having approximately 108 colony forming units (cfu).ml-1 of inoculum was inoculated onto the test plate. An antibiotic impregnated disc was then applied to the surface of the test plate. The test plate was then incubated at 37"C for either 5 or 6 hours. At intervals during the incubation and after the incubation had completed the zone of organism growth and any zone of inhibited organism growth were observed by placing, on the surface of the test plate, an indicator disc as defined above. After initially placing the device according to the present invention on the surface of the test plate the device was retained on the test plate during the remainder of the incubation period. The sensitivity of the test organism to the antibiotic was then determined by the standard Kirby-Bauer method which relates inhibition zone size to the sensitivity of the organism to the antibiotic. This procedure was adopted for each of the test organism/antibiotic combinations and the results obtained are given below in Table 5.

TABLE 5

average Antibiotic inoculum Example Organism Code COT NA SF NF PG AH MEC GT CPH (cfu.ml-1) 19 E. Coli NCTC6 S S S S S S S S S 7.9 x 108 20 E. Coli PS 451 S S S S R R S S S 4.3 x 108 21 E. Coli BG 439 S S S S R R S S S 9.5 x 107 22 S. Aureus NCTC42 S* S* S* S* S S S S S 4.6 x 109 23 S. Aureus BG 643 S* R S* R R R R S R 4.4 x 109 24 P. Aeruginosa NCTC2 R R S* S* R R R S R 5.5 x 108 25 P. Aeruginosa BG 662 R R S* S* R R R S R 2.9 x 108 26 K. Aerogenes BG 536 R R R S R R S S R* 4.1 x 108 27 P. Vulgeis PS 413 R S R R R R S R R* 4.0 x 108 R = Resistant as determined by Kirby-Bauer method S = Sensitive " " " " " " Each result recorded after triplicate experiments * = variation from sensitivity reported in Table 3.

In each of Examples 19 to 21 and 24 to 27 the total incubation time was 5 hours but in each of Examples 22 and 23 the total incubation time was 6 hours.

The results obtained in Examples 1 9 to 27 suggest that the test method according to the present invention gives an 86.6% correlation with the reported sensitivities given in Table 3 for the comparative Examples 1 to 9.

EXAMPLES 28 to 35 These Examples utilize the so-called Stokes test wherein an organism of known sensitivity is directly compared with against an organism of unknown sensitivity using the same antibiotic impregnated disc. The unknown organism sensitivity is then determined by direct comparison of the inhibition zone formed by interaction between the organism of unknown sensitivity and the antibiotic with the inhibition zone formed by interaction between the organism of known sensitivity and the antibiotic. Depending on the result of this comparison the organism of unknown sensitivity is stated to be "sensitive", "moderately resistant" or "resistant".

In each of comparative Examples 28 to 31 two test cultures were inoculated onto the surface of a pre-dried agar test plate. An antibiotic impregnated disc was then applied at one place on the border where the test cultures meet. The test plate was then incubated at 37"C for 18 hours and then the sizers of the inhibition zones were observed by the naked eye. The sensitivity of the organism of unknown sensitivity was then determined.

In each of Examples 32 to 35 two test cultures were inoculated onto the surface of a predried agar test plate. An antibiotic impregnated disc was then applied at one place on the border where the test cultures meet. The test plate was then incubated for 4.5 hours (Examples 32, 33 and 35) or 5.5 hours (Example 34). After this time an indicator disc, as defined above, was applied to the surface of the test plate and any zones of inhibited organism growth was observed. The test plate was then incubated for a further 0.5 hour whilst retaining the device according to the present invention on the surface of the test plate and any zones of inhibited organism growth were again observed. The sensitivity of the organism of unknown sensitivity to the antibiotic was then determined.

The organisms and antibiotics used in Examples 28 to 35, and the results of these Examples are given in Table 6.

TABLE 6

Antibiotic average inoculum Example Organisms COT NA NF PG AH GT AK (cfu ml-1) 28 E. Coli NCTC / PS 251 = = = R R = = 6.2/5.5 x 108 29 E. Coli NCTC / BG 439 = = = R R = = 5.2/8.4 x 108 30 S. Aureus NCTC42/BG 643 S = S R R = = 6.0/1.2 x 108/109 31 P. Aeruginosa NCTC2/BG 662 = = = S S = = 7.4/2.4 x 108/109 32 E. Coli. NCTC / PS 451 = = = R R = = 6.2/5.5 x 108 33 E. Coli NCTC / BG 439 S = S R R = = 5.2/8.4 x 108 34 S. Aureus NCTC42/BG 643 S = S M M = M 6.0/1.2 x 108/109 35 P. Aeruginosa NCTC2/BG 662 = = = S S = M 7.4/2.4 x 108/106 R = Clinical isolate = more resistant than standard strain S = " " = " sensitive " " " M = " " equal sensitivity with " " The results of the comparative Examples 28 to 31 show good correlation with sensitivities determined by the Kirby-Bauer technique, except for BG 662/PG and BG 662/AH.

The results of Examples 32 to 35 show good correlation with the results of comparative Examples 28 to 31.

Further a comparison of the results of Examples 32 to 35 with the results of comparative Examples 28 to 31 show: i) 92.8% correlation of results when considering major discrepancies (i.e. R-S) between corresponding Examples; ii) 85.7% correlation of results when considering minor discrepancies (i.e. R-M) between corresponding Examples; and iii) 78.5% correlation of results when considering all discrepancies between corresponding Examples.

The results of the above Examples illustrate that, by use of the test method according to the present invention, organism growth/inhibited organism growth zone delineation may be satisfactorily observed in only approximately 5 hours for gram-negative organisms, e.g.

Escherichis Coli, Pseudomonas Aeruginosa, Kilebsiella Aerogenes and Proteus Vulgaris, and in only approximately 6 hours for gram-positive organisms e.g. Staphylococcus Aureus, as compared with approximately 1 8 to 24 hours with the previously used method involving observation, by the naked eye, of any zone inhibited organism growth.

Further, the test method according to the present invention allows the tetrazolium compound, i.e. the indicator, to be used in a sub-inhibitory amount, thereby avoiding any possible inhibitory effect of this compound itself on the growth of the test organism.

The test method according to the present invention also gives good correlation of results with the previously used method involving an approximately 1 8 to 24 hour incubation with subsequent direct observation, by the naked eye, of any zone of inhibited organism growth. For example, the results of Examples 10 to 1 8 show a 91.1 % correlation with the results obtained using the known method involving an incubation time of approximately 1 8 hours and subsequent direct observation, by the naked eye, of any zone of inhibited organism growth (compare Examples 1 to 9 and 10 to 18), the results of Examples 1 9 to 27 show an 86.6% correlation with the results obtained using the known method involving an incubation time of approximately 1 8 hours and subsequent direct observation, by the naked eye, of any zone of inhibited organism growth (compare Examples 1 to 9 and 1 9 to 27), and the results obtained in Examples 32 to 35, utilizing the so-called Stokes test, show a 92.8% correlation (i.e.

considering major discrepancies, see Examples 32 to 35) with the results obtained utilizing the known Stokes test procedure involving an incubation time of approximately 1 8 hours and subsequent direct comparison, by the naked eye, of the zones of inhibited organism growth (compare Examples 28 to 31 and 32 to 35).

A statistical analysis of inhibited organism growth zone measurements obtained in the above Examples 4, 1 3 and 22 and expressed as zone diameters in millimetres is given below in Table 7 and indicates the significance of the test method according to the present invention. The values given in brackets in Table 7 represent the standard deviations.

TABLE 7

Antibiotic Example COT NA SF NF PG AH MEC GT CPH 4 14 15 14 15 48 35 22 25 41 (0.24) (0.35) (0.45) (0.92) (0.01) (0.72) (0.32) (0.29) (1.21) 13 17 18 16 18 46 35 24 26 44 (0.29) (0.52) (0.79) (0.65) (1.05) (0.81) (0.41) (0.16) (1.01) 22 17 19 18 18 49 37 21 26 40 (0.41) (0.72) (0.95) (0.79) (0.95) (0.65) (0.50) (0.35) (1.35) A statistical analysis of inhibited organism growth zone measurements obtained in the above Examples 1, 10 and 1 9 and expressed as zone diameters in millimetres is given below in Table 8 and again indicates the significance of the test method according to the present invention.

The values given in brackets in Table 8 represent the standard deviations.

TABLE 8

Antibiotic Example COT NA SF NF PG AH MEC GT CPH 1 36 29 33 32 25 26 29 33 28 (0.75) (0.092) (3.52) (0.21) (1.21) (0.86) (0.42) (0.69) (0.41) 10 37 29 35 32 27 28 29 33 29 (1.01) (1.21) (3.07) (0.41) (1.25) (0.85) (0.55) (0.69) (0.92) 19 38 30 35 33 27 28 28 34 27 (1.25) (1.12) (4.25) (0.55) (1.17) (0.92) (0.93) (0.75) (1.52) In connection with the test method of the present invention, it is believed that the observation that gram-negative organisms may show an earlier dilineation of the organism growth, inhibited organism growth zones (e.g. after approximately 5 hours) than the gram-positive organisms (e.g.

delineation after approximately 6 hours) may be due to a combination of the different growth rates of the two organism types and the rates of the redox process occurring at the grampositive cell wall of the organism and at the gram-negative cell membrane of the organism by interaction between the organism and the tetrazolium compound used as indicator.

In conclusion, therefore, it can be seen that the device according to the present invention, and its use in the test method according to the present invention, allows the sensitivity of organisms to antibiotics to be determined more rapidiy than by using the known technique involving incubation for approximately 1 8 to 24 hours with subsequent direct observation, by the naked eye, of any zone of inhibited organism growth. The device according to the present invention, and its use in the test method according to the present invention, avoids the previously encountered disadvantage of rapid sensitivity test using 2,3,5-triphenyl tetrazolium chloride as the indicator, namely the inhibitory effect of the indicator itself under certain circumstances.

Claims

1. A device which comprises a layer of a stabilized absorbent material impregnated with a tetrazolium compound and an electron accelerator compound.

2. A device as claimed in claim 1, in which the stabilized absorbent material is a stabilized fibrous absorbent material.

3. A device as claimed in claim 2, in which the stabilized absorbent material is an absorbent paper.

4. A device as claimed in claim 3, in which the paper is blotting or filter paper.

5. A device as claimed in any of claims 1 to 4, in which the layer of stabilized absorbent material is in the form of a circular disc.

6. A device as claimed in claim 5, in which the circular disc is approximately 8 cm in diameter.

7. A device as claimed in any of claims 1 to 6, in which the tetrazolium compound is 2,3,5triphenyl tetrazolium chloride; 2,2', 5, 5'-tetraphenyl-3, 3'-(3, 3'-dimethoxy-4,4'-diphenylene)ditet- razolium chloride; or p,p'-diphenylene-bis-2-(3, 5-diphenyl tetrazolium chloride).

8. A device as claimed in any of claims 1 to 7, in which the electron accelerator compound is 5-methyl phenazonium methyl sulphate or dihydronicotinamide adenine dinucleotide phosphate (NADPH).

9. A device according to claim 1, substantially as herein before described with particular reference to any of Examples 10 to 27 and 32 to 35.

10 A method of preparing a device as claimed in any of claims 1 to 9, which comprises sterilizing a layer of the stabilized absorbent material, impregnating the sterilized layer of the stabilized absorbent material with a solution comprising the tetrazolium compound, the electron accelerator compound and deionized water, and then drying the impregnated layer of the stabilized absorbent material.

11. A method as claimed in claim 10, in which the solution comprises from 50 to 200 milligrams of the tetrazolium compound per millilitre of deionized water.

1 2. A method as claimed in claim 11, in which the solution comprises from 75 to 1 50 milligrams of the tetrazolium compound per millilitre of deionized water.

1 3. A method as claimed in any of claims 10 to 12, in which the solution comprises from 2 to 20 milligrams of the electron accelerator compound per millilitre of deionized water.

14. A method as claimed in claim 13, in which the solution comprises from 5 to 10 milligrams of the electron accelerator compound per millilitre of deionized water.

1 5. A method as claimed in any of claims 10 to 14, in which the layer of the stabilized absorbent material is stabilized, prior to impregnation with the aqueous tetrazolium compound/ electron accelerator compound solution, by heating it to a temperature of from 100"C to 140"C.

1 6. A method as claimed in claim 15, in which the heating is at from 110"C to 130"C.

1 7. A method as claimed in claim 1 5 or 16, in which the heating is conducted at a pressure of from 0.5 to 3 atmospheres.

1 8. A method as claimed in claim 17, in which the heating is conducted at a pressure of from 0.75 to 2.5 atmospheres.

1 9. A method as claimed in any of claims 10 to 18, in which the impregnated layer of the stabilized absorbent material is dried at atmospheric pressure by heating it at a temperature of from 30"C to 80"C.

20. A method as claimed in claim 19, in which the material is dried by heating at from 50"C to 70"C.

21. A method as claimed in claim 19 or 20 in which the drying is effected by heating for a period of 1 to 7 hours.

22. A method as claimed in claim 20, in which the drying is effected by heating for from 3 to 5 hours.

23. A method according to claim 10, substantially as hereinbefore described with particular reference to any of the Examples 10 to 27 and 32 to 35.

24. A device whenever produced by a method as claimed in any of claims 10 to 23.

25. A method for determining the sensitivity of organisms to antibiotics which comprises contacting an antibiotic with a test organism on the surface of an organism growth medium, incubating the test organism at elevated temperature, applying a device as claimed in any of claims 1 to 9 to the surface of the organism growth medium such that said device contacts at least that portion of the surface of the organism growth medium on which the test organism/antibiotic contact takes place, and observing any subsequent colorimetric reaction occurring as a result of interaction between the test organism and the tetrazolium compound contained in said device.

26. A method as claimed in claim 25, in which the organism growth medium is an agar growth medium.

27. A method as claimed in claim 25 or 26, in which the organism is Escherichia Coli, Staphylococcus Aureus, Pseudomonas Aeruginosa, Klebsiella Aerogenes or Proteus Vulgaris.

28. A method as claimed in any of claims 25 to 27 in which the antibiotic is cotrimoxazole, naladixic acid, sulphafurazone, nitrofuranton, penicillin, ampicillin, mecillinam, gentamicia, cephaloridine or amikacin.

29. A method as claimed in any of claims 25 to 28, which comprises forming a test plate comprising a solid organism growth medium inoculated or seeded with the test organism, applying the antibiotic to a portion of the surface of the test plate, incubating the resulting test plate at elevated temperature, applying the device as claimed in any of claims 1 to 9 to the surface of the test plate, such that said device contacts that portion of the test plate to which the antibiotic has been applied and also extends outwardly across the surface of the test plate, allowing at least a portion of the tetrazolium compound and the electron accelerator compound, which are present in said device, to diffuse from the device to the test plate, and observing any subsequent colorimetric reaction occurring as a result of interaction between the tetrazolium compound and the test organism.

30. A method as claimed in claim 29, in which the test plate is formed by placing the organism growth medium, in fluid form, into a container, then surface drying the organism growth medium, and then inoculating the test organism onto the dried surface of the organism growth medium.

31. A method as claimed in claim 29, in which the test plate is formed by seeding the organism growth medium, in liquid form, with the test organism, placing the seeded organism growth medium into a container, and then surface drying the organism growth medium.

32. A method as claimed in any of claims 29 to 31, in which the antibiotic is applied to the test plate in the form of an absorbent paper disc impregnated with the antibiotic.

33. A method as claimed in any of claims 25 to 32, in which, after application of the antibiotic to the test plate, said test plate is incubated at approximately 37"C.

34. A method as claimed in any of claims 29 to 33, in which the period of incubation of the test plate is from 4 to 6 hours.

35. A method as claimed in any of claims 29 to 34, in which the test plate is inoculated or seeded with two or more test organisms, each of the test organisms occupying a different region of the test plate and being contacted by a single antibiotic.

36. A method as claimed in claim 35, in which the antibiotic is applied to the surface of the test plate in the form of one or more absorbent paper discs, impregnated with the antibiotic, the or each antibiotic impregnated disc being placed on the surface of the test plate such that it contacts more than one of the test organism-containing regions of the test plate.

37. A method as claimed in claim 35, in which the antibiotic is applied to the surface of the test plate in the form of a plurality of absorbent paper discs impregnated with the antibiotic, each of the test organism-containing regions of the test plate being contacted by at least one of the antibiotic impregnated discs and each of the antibiotic impregnated discs contacting only one test organism-containing region of the test plate.

38. A method as claimed in any of claims 29 to 34, in which the test plate is inoculated or seeded with one or more test organisms, each test organism occupying a different region of the test plate when more than one test organism is utilized, and the or each test organism being contacted by two or more different antibiotics.

39. A method as claimed in claim 38, in which the antibiotics are applied to the surface of the test plate in the form of absorbent paper discs impregnated with antibiotic and with each absorbent paper disc being impregnated with only one antibiotic.

40. A method as claimed in any of claims 25 to 28, which comprises forming a test plate comprising a solid organism growth medium and the antibiotic, applying the test organism to the surface of the test plate, incubating the resulting test plate at elevated temperature, applying the device as claimed in any of claims 1 to 9 to the surface of the test plate such that said device contacts at least that portion of the surface of the test plate to which the test organism has been applied, allowing at least a portion of the tetrazolium compound and the electron accelerator compound, which are present in said device, to diffuse from the device to the test plate, and observing any subsequent colorimetric reaction occurring as a result of interaction between the tetrazolium compound and the test organism.

41. A method as claimed in claim 40, in which the test plate is prepared by admixing the antibiotic and the organism growth medium, placing the resulting mixture into a suitable container, and then surface drying the organism growth medium.

42. A method as claimed in claim 40 or 41, in which said test plate is incubated at approximately 37"C.

43. A method as claimed in claim 42, in which the period of incubation of the test plate is from 4 to 6 hours.

44. A method according to claim 25, substantially as hereinbefore described with particular reference to any of Examples 10 to 27 and 32 to 35.