JP2009148303A - Antibiotic sensitivity testing - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a test in particular for the sensitivity of bacteria to an antibiotic or a biostatic agent, a test to assess the growth stage of the bacteria and the health state, and to provide methods of carrying out these tests and kits for achieving them. <P>SOLUTION: The invention relates to the use of an assay for adenylate kinase in an in vitro test for the effect of external conditions on the growth characteristics of bacterial cells. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to a method for testing bacterial growth characteristics, and in particular, to a susceptibility test for a specific antibiotic or biostatic agent, and a kit for use in the method.

Bacteria with antibiotic resistance are becoming an increasingly serious problem. Current methods for determining antibiotic resistance of a strain of bacteria are very time consuming. It first requires the isolation of the organism in pure culture. A bacterial “lawn” is then prepared and grown in the presence of a set of antibiotics. A zone of inhibition of growth around a particular antibiotic indicates that the bacterium is sensitive (the size of the zone indicates the degree of sensitivity). The absence of growth inhibition in the presence of antibiotics indicates resistance. This process takes at least two days to complete and is far from ideal, especially in clinical situations where the optimal treatment of infected individuals is determined by the results of these trials.
There is a need for a test that can determine antibiotic resistance or sensitivity fairly quickly, for example within a few hours.

この検定法では、ＡＤＰは試薬として、好ましくはマグネシウムイオンの存在下、試験する試料に加えられる。上記アデニル酸キナーゼ反応の結果として生成されるＡＴＰは、例えばホタルの生物発光を用いて検出することができる。このため、ルシフェリン／ルシフェラーゼの組合せのような試薬を、通常、例えば約５分のような短いインキュベーション時間後に混合物に加え、生じる発光シグナルを観察する。
この検定法の感度はアデニル酸キナーゼの自然放射能レベル及び使用する試薬の純度によってのみ制限される。例えば、モデル系として大腸菌を使用すると、１００以下の細胞由来のアデニル酸キナーゼ活性が、後述する図１に示されるように、５分のインキュベーション検定で測定された。この図を作成するために使用した試験では、試料体積は２００μlであった。 Assays for the detection of microorganisms by measuring adenylate kinase are known, for example, from international patent application numbers PCT / GB94 / 00118 and PCT / GB94 / 01513. Adenylate kinase is an essential enzyme in all living cells and catalyzes the production of ATP in the presence of ADP, as shown below.

In this assay, ADP is added as a reagent to the sample to be tested, preferably in the presence of magnesium ions. ATP produced as a result of the adenylate kinase reaction can be detected using, for example, firefly bioluminescence. For this reason, reagents such as the luciferin / luciferase combination are usually added to the mixture after a short incubation period, for example about 5 minutes, and the resulting luminescent signal is observed.
The sensitivity of this assay is limited only by the natural radioactivity level of adenylate kinase and the purity of the reagents used. For example, when Escherichia coli was used as a model system, adenylate kinase activity derived from 100 cells or less was measured by a 5-minute incubation assay as shown in FIG. In the test used to create this figure, the sample volume was 200 μl.

Applicants have found that adenylate kinase can be used as a susceptibility marker for biomass and that the assay described above can be used in studies that give much more detailed information on the growth characteristics of bacterial cells.
Accordingly, the present invention provides the use of an adenylate kinase assay in an in vitro test for the effect of external conditions on the growth characteristics of bacterial cells.

Adenylate kinase assays provide a fast and sensitive means of examining many aspects of bacterial growth and inhibition. There are various types of external conditions that can be examined using the present invention. For example, an adenylate kinase assay may be used in a method to determine the susceptibility of a particular bacterial strain or mixed culture to a particular antibiotic or biostatic agent, or because of antibiotic or biostatic properties. Alternatively, it may be used in a method that can be adapted to the application for screening reagents. In addition, since the comparison between the extracellular adenylate kinase content of the cell culture and the total intracellular and extracellular content is an indicator of the growth state and health status of the cell culture, using the adenylate kinase assay, It was found that these characteristics can be determined.

The test is structured taking into account the nature of the investigation being performed, the type of bacterial sample available, the nature of the reagents to be tested, if any, and whether they have a lytic or non-lytic effect on the cells. Various forms of these tests will be described in more detail later.
In particular, however, the present invention is a method for determining the susceptibility of a bacterium to a test substance, comprising assaying adenylate kinase released by lysis of the bacterium from said reagent-containing culture, and determining the result. Similar adenylate for any of the lysed bacteria from the same culture at different time points, and / or lysed bacteria from the same culture without the reagent A method comprising comparing to results obtained from a kinase assay is provided.

The reagent to be tested may be a known antibiotic or biostatic drug, or may be a new compound or reagent not previously known as an antibiotic, so that the test may form part of the screening program .
Some reagents, for example, antibiotics such as penicillin, for example β-lactam antibiotics such as ampicillin and amoxicillin, will lyse bacteria in culture. However, if this does not occur, it may be necessary to lyse the bacteria prior to performing the assay. This can be done in a variety of ways known in the art, including treatment with lysing agents and physical methods such as subjecting the bacteria to a magnetic or electric field, or ultrasound.

Agents that lyse bacteria include surfactants and enzymes such as lysin. However, they are not specific and will liberate AK from all living material present in the sample. This may be appropriate when the sample contains a pure culture. However, other strategies may be employed when the bacterium under investigation is a component of a mixed culture. Specific measurements from target cells in a mixed sample, for example: 1) After measurement of non-specific adenylate kinase, the cells in question are specifically captured and separated from contaminating organisms 2) measuring only adenylate kinase from them using a method that only lyses the target cells; or 3) using them in combination.

Adenylate kinase from target bacterial cells only (above 2) can be obtained using a lytic agent that is specific for the particular bacterium under investigation, eg, a bacteriophage that is specific for the target cell and lyses the bacterium. Can be liberated. These bacteriophages are viruses that infect bacteria, lyse the cells and release intracellular components including adenylate kinase to the external medium. This release occurs about 30-60 minutes after infection. Using this method for an assay that took 40 minutes, it was found that fewer than 500 cells were detected.

Phages can infect target cells equally well in pure or mixed cultures. By comparing the amount of adenylate kinase that can be chemically extracted from the sample with the amount released after a set time due to phage infection, the presence or absence of the target cell and the test substance contribute to the growth of the target cell. The effect can be determined.
In order to regenerate the bacteriophage and thereby cause lysis, the host cell must be in the logarithmic phase of growth. If growth is inhibited, for example as a result of the presence of a bacteriostatic agent or antibiotic, the bacteriophage will not be able to grow or lyse the cells. This can be used as a basis for further embodiments of the invention, as will be shown below.

Alternatively or additionally, the mixed culture may be subjected to a pretreatment step in which the cells of the target bacteria are enriched and / or separated from the culture. Such a process is well known in the art. For example, separation may involve an immunocapture method that uses antibiotics specific for a particular bacterium or binding fragments thereof to immobilize the cells on a solid surface such as a bead, microtiter plate, filter membrane or column. Can be achieved using. Magnetic beads may provide a particularly preferred solid surface. For separation of the beads, it is appropriate to use a magnetic separation method, which leads to substantial isolation of the target bacteria, as described below. Typically, using magnetic beads as a solid support has been found to be able to achieve detection of 1000 cells or less in a total assay time of about 30 minutes. Other substances can also be used as the solid support.

Further specificity may be obtained through the use of selective growth media. This can be used in an enrichment process to establish a healthy growth culture prior to infection with an immunocapture assay or bacteriophage. In addition, selective media can be used throughout the process of bacteriophage infection.
Such a medium will minimize the overgrowth by non-target organisms that may be present, sometimes much in excess of the target bacteria.
As mentioned above, the present invention can be adapted for use in testing bacterial susceptibility to certain antibiotics or bacteriostatic agents.

Antibiotics such as β-lactams, such as penicillin, function by disrupting cell wall synthesis, thereby losing cell integrity and lysing during replication. This occurs fairly quickly, approximately 10-15 minutes after exposure to antibiotics, provided that the bacteria are actively growing.
Other antibiotics such as chloramphenicol do not cause cell lysis, but inhibit cell proliferation in other ways that biostatic drugs do. The mode of action of any particular drug used is generally understood. The present invention can be adapted for use in any susceptibility test against bacteria for any of these agents.

A particular embodiment of the present invention is a method for determining the susceptibility of a bacterium to a lytic antibiotic, comprising the following steps: (i) separating the bacterium from other microbial species using, for example, an immunocapture step (Ii) determining the extracellular adenylate kinase content of the bacterial culture; (iii) adding a lytic antibiotic to the culture, which is sufficient to exert its lytic effect on the antibiotic. And (iv) determining the extracellular adenylate kinase content of the culture to determine whether lysis has occurred.

In this test, susceptible bacteria will be lysed by antibiotics, generally within about 15 minutes, after the addition of antibiotics. Thus, the content of free adenylate kinase in the culture will increase significantly as bacterial cells break open of free intracellular adenylate kinase after addition of antibiotics. Optimal measurement of adenylate kinase levels will take just before antibiotic addition and then again at least 15 minutes after antibiotic addition. The free adenylate kinase content of resistant bacterial cultures will remain largely constant. Thus, the content of only the low level extracellular adenylate kinase that is normally present is measured as described above, which is nearly stable for healthy proliferating cells. Using this method, antibiotic sensitivity can be determined in about 40 minutes.
The bacterial culture used in this method may include a selective growth medium that favors the aforementioned bacteria, which will minimize false positive results that are the result of contamination.

Another embodiment of the present invention is a method for determining the susceptibility of a bacterium to non-lytic antibiotics or biostatic agents, wherein (i) the bacterium is (Ii) incubating the bacterial culture in the presence of the non-lytic antibiotic or biostatic agent and optionally a selective growth medium in favor of the bacteria; (iii) Whether the total adenylate kinase content of the culture increased over time of incubation by taking samples intermittently, lysing bacteria in these samples and assaying for adenylate kinase in the samples Determining whether it has decreased.

In this case, the amount of adenylate kinase obtained after chemical lysis over a period of time, for example about 1 hour, will remain approximately constant if the bacterium is sensitive to the antibiotic. This is because bacteria cannot grow in the presence of the antibiotic. However, in the case of resistant bacteria, the bacteria will continue to grow and adenylate kinase levels will increase steadily over time.
Thus, the susceptibility of a pure culture to a specific antibiotic, either lytic or non-lytic, can be rapidly determined using the methodology of Step I above.

Other embodiments of the invention obviate the use of bacterial isolation or pure culture. Specifically, the present invention further provides a method for determining the susceptibility of a bacterium to an antibiotic or a biostatic agent, comprising: (a) a first sample of the bacterial culture, a first sample in the presence of the antibiotic. Incubating two samples, a third sample in the presence of a bacteriophage that specifically lyses the target bacteria, and a fourth sample in the presence of both the bacteriophage and the antibiotic, (b) after incubation Determining the adenylate kinase content for each of the first to fourth samples, and (c) based on the adenylate kinase assay results and based on the mode of action of the antibiotic or biostatic agent, Providing a method comprising determining the susceptibility or resistance of.

In order for bacteria to be sensitive to the effects of antibiotics, they must be actively growing, so selective media could be used for the initial enrichment step. The enrichment step may include about 1 hour incubation. After this time, the target cells may be enriched in fresh selective medium by immunocapture if desired. The effect of adding antibiotics to the target bacteria can be determined by measurement of adenylate kinase combined with bacteriophage-mediated lysis.

In order for the bacteriophage to regenerate, the host cell must be in log phase growth. For example, if growth is inhibited by the presence of antibiotics, the phage will not be able to replicate and therefore will not lyse the host. Bacteriophage and adenylate kinase assays can be used to determine bacterial antibiotic susceptibility within 3 hours. Additional time is required to establish healthy proliferating cells before exposure to antibiotics or infection with phage. Two types of test results may depend on the mode of action of the antibiotic concerned.

The results obtained are summarized in FIG. In the figure, “−” indicates a result consistent with detection of extracellular adenylate kinase alone, and “+” indicates a moderate positive result consistent with detection of intracellular and extracellular adenylate kinase in a non-proliferating living sample. And “++” indicates the detection of elevated levels of adenylate kinase consistent with lysis of the growth culture.

As is clear from FIG. 4, if the mode of action of the drug (lytic or non-lytic) is known from the pattern of results obtained using this series of tests, it is easy to distinguish sensitive bacteria from resistant bacteria. it can. As explained in more detail in the examples below, various effects are caused as a result of the interaction of various reagents with bacteria.
Comparison of the extracellular adenylate kinase content of the cell culture with the total intracellular and extracellular content was found to indicate the growth and health status of the cell culture.

Accordingly, yet another embodiment of the present invention is a method for determining the growth phase of a bacterial culture, comprising: (a) exposing a first sample of said bacterial culture to a lysing agent to lyse bacterial cells therein. (B) assaying adenylate kinase in the first sample and in a second sample of the culture that has not been exposed to a lysing agent; and (c) the results obtained from the first and second cultures. A method comprising comparing and determining the growth phase of the culture is provided.

Healthy log phase cultures have relatively low extracellular adenylate kinase levels (about 1% of total adenylate kinase), and the ratio of extracellular adenylate kinase remains relatively constant throughout the log phase. And increases as the culture approaches stationary phase. A stationary phase culture can have as much as one third of the total adenylate kinase in the culture medium. Thus, adenylate kinase can be used to quickly determine the health status and number of cells.

This method can be used to confirm that a particular cell culture grows well, for example, in other processes where, for example, optimal growth is required, such as fermentation or cell production. Apart from this, when screening for antibiotics or bacteriostatic compounds it is necessary to make sure that the cells grow very well in order to avoid false positive results due to the use of weak or stationary cultures in the test. might exist. In addition, it may be used to determine what effective environmental factors, such as temperature or culture medium, for any particular culture growth.
In each case, the adenylate kinase content is determined by taking a sample of the culture and performing an adenylate kinase assay as described, for example, in International Patent Application Nos. PCT / GB94 / 00118 and PCT / GB94 / 01513. You may judge.

The present invention also provides a test kit for achieving the method of the present invention. The test kit includes appropriate components that can perform a particular assay. An antibiotic susceptibility test kit may include a series of antibiotics, eg, lyophilized or other storage conditions. It may also include reagents for extracting adenylate kinase from a sample such as a surfactant or other chemical lysing agent, and reagents for assaying adenylate kinase such as luciferin / luciferase and the like. Also, for use in cases where mixed bacterial cultures are to be tested, the kit can include the appropriate bacteriophage in a storage state such as lyophilized bacteriophage. In addition, the kit may also include a suitable selective growth medium. Reagents may be supplied in a suitable reaction vessel such as a multi-well plate. The present invention will now be described in detail by way of example with reference to the accompanying drawings.

It is a graph which shows the experimental result for measuring the adenylate kinase activity from E. coli cells. Shows the results of a magnetic bead immunocapture assay for Salmonella typhimurium in pure culture in the presence of 3.5 × 10 ⁵ Bacillus subtilis var niger proliferating cells; where ■ indicates adenylate by cells captured by the beads Indicates the kinase activity; □ indicates the adenylate kinase activity by the remaining cells in the sample (ie, the left graph is derived from uncaptured Salmonella cells, the right graph is derived from these plus non-specific cells). FIG. 6 is a graph showing experimental results for investigating the time course of phage-mediated release of adenylate kinase from cultures of E. coli cells. It is a schematic diagram of the antibiotic test result obtained by one Embodiment of this invention. FIG. 5 shows a measurement plate for testing bacteria for antibiotic resistance. Shows the effect of phage 10359 and 50 mg / liter ampicillin on ampicillin sensitive and resistant cultures of E. coli 10243, where ○ represents the results of sensitive E. coli 10243 with ampicillin and phage 10359, and ▽ represents the results without lysing agent. And □ represents the results of resistant E. coli 10243 with ampicillin and phage. The effect of chloramphenicol (34 mg / liter) and phage 10359 on the culture of E. coli 10243 is shown, where ◯ represents only phage, ▽ represents phage 10359 and chloramphenicol, and □ represents chloramphenicol. Represents only call, and ◇ represents no phage or chloramphenicol.

Example 1
Comparison of extracellular and total contents of adenylate kinase in culture.
For example, in one embodiment, the bacterial cell sample is divided into first and second samples. A first sample of bacterial cells is mixed with a solution containing ADP and a surfactant in the presence of magnesium ions. This extracts the adenylate kinase from all cells present in the sample, thus causing an ATP generating reaction. The reaction is allowed to run for a required time, for example 5 minutes, after which a bioluminescent reagent is added and the resulting light is measured with a luminometer. The assay performed in this way determines the total amount of extracellular or intracellular adenylate kinase in the sample.
The second sample is subjected to the same assay except that no surfactant is present, and only extracellular adenylate kinase is measured.

Example 2
Use of immunocapture to separate target cells from mixed suspensions
Immunocapture assays for Salmonella typhimurium were performed with pure samples and samples that also contained 3.5 × 10 ⁵ Bacillus Subtilis var niger (BG) proliferating cells. The immunocapture assay was performed with a total volume of 300 μl. The immunocapture step on magnetic microbeads coated with S. typhimurium specific antibody took 10 minutes. The immobilized beads were washed to remove unbound particles and a non-specific lysis step was performed to liberate adenylate kinase from the bound material. This was detected by a 5 minute adenylate kinase assay.
The results are shown in FIG. The graph shows that about 70% of the target cells are selectively removed from the suspension and this is less affected by the presence of contaminants (BG cells).

Example 3
Use of phage-mediated lysis
The time course of adenylate kinase release from cultures of E. coli cells, some of which were infected with E. coli-specific bacteriophages, was examined. 100 μl samples (each containing exactly 350 cells) were intermittently removed from cultures infected with E. coli specific bacteriophage and assayed for extracellular adenylate kinase activity after 40 minutes. The results are shown in FIG. 3, where ○ = infected culture, ● = uninfected control.
From these results, it was found that 500 or less cells were detected by this method.

Example 4
Antibiotic susceptibility assay:-Lytic antibiotics Sample cells (whether mixed or pure) were split into two fractions and one was infected with bacteriophage. Each of these fractions was further divided into two fractions, one exposed to antibiotics and the other untreated. The relative level of extracellular adenylate kinase produced at a given time indicates the effect of antibiotics and bacteriophages on target cells. The actual test results obtained are shown in Table 1.
The results show that the antibiotic resistance status of both cells and controls guarantees that this test worked correctly.

Example 5
Antibiotic susceptibility tests:-Non-lytic antibiotics or biostatic drugs
Similar methods accelerate the susceptibility of bacteria to antibiotics that do not lyse cells but otherwise inhibit cell growth (and other chemicals such as biostatic drugs that inhibit bacterial cell growth). Can be determined.
Testing susceptibility of cells in mixed cultures to non-lytic antibiotics could be performed in a manner similar to testing for susceptibility to lysing antibiotics. However, susceptibility to antibiotics will be indicated by the lack of phage-mediated lysis in the treated sample, since the bacteria must be actively growing to allow bacteriophage infection.
The results that would be observed are summarized in Table 2.

Example 6
Antibiotic resistance test kit
A suitable test kit includes a sample container in which a number of wells are formed as shown in FIG. 5, typically a plastic plate. The total volume of liquid that can be held in each well may be about 0.5 ml. The plate is preferably pre-prepared so that certain wells also contain appropriate lyophilized (or other storage) preparations of antibiotics and / or bacteriophages, and optionally selective growth media. .
In a plate such as that shown in FIG. 5, four wells are arranged in each row and are used to test resistance to a particular antibiotic, and this plate is used to test three antibiotics simultaneously. be able to.

A test sample with a volume of about 0.2 ml is used, which may be a pure culture, a preparation enriched by immunocapture, a sample from selective media, or a suitable neat sample, added to each well. For example, a reagent for measuring adenylate kinase activity may be added after 1 hour incubation at 37 ° C. These may be reagents that produce a colorimetric signal in the presence of adenylate kinase, more preferably a reagent that produces a bioluminescent signal.
Specifically, after 5 minutes with luciferin and luciferase, ADP and magnesium ion source are added. The light emission is determined by moving one well into the tube and measuring in a tube luminometer, or preferably the plate is automatically calibrated in a plate luminometer, or using a CCD camera system. Image as a whole.

Example 7
Comparison of the effects of lytic and non-lytic antibiotics on culture growth.
The effects of lytic antibiotics (ampicillin) and non-lytic antibiotics (chloramphenicol) on E. coli culture growth were examined.
A plasmid (PUC18) encoding ampicillin resistance was introduced into a pure culture of E. coli 10243 to make it resistant without changing the host specificity of the phage. Resistant strains were also tested to ensure that plasmid transport did not alter its growth rate or infection with phage 10359, and the resistant strains were found to be similar to susceptible strains in terms of growth and infection.
Adenylate kinase released from sensitive (non-transported) and resistant bacteria in the presence of E. coli bacteriophage 10359 and compared to either lytic antibiotics (ampicillin) or non-lytic antibiotics (chloramphenicol) .

Log-phase cultures of both resistant and susceptible strains of E. coli 10243 were infected with 10 ⁵ phage 10359 at T ₀ and 50 μl / ml ampicillin at T ₅ . Cell lysis by ampicillin is evident at T ₁₀ in susceptible strains and is prominent at T ₂₀ and masks any tytic effects due to bacteriophages. Resistant strains, but were not infected by the antibiotic showed lysis by phage infection after 20 minutes, which was the unit T ₄₀ is not significant. The results are shown in FIG. FIG. 6 shows that the sensitivity of the culture of E. coli 10243 to ampicillin can be determined after only 40 minutes of incubation in the presence of ampicillin and phage.

A log phase culture of E. coli 10243 was incubated for 80 minutes in the presence of phage 10359 and / or chloramphenicol (34 μg / ml) and adenylate kinase was assayed as before. The results are shown in FIG.
When the culture was incubated with both bacteriophage and chloramphenicol, more cell lysis was shown compared to chloramphenicol alone. Chloramphenicol is not a lytic antibiotic but showed cell lysis, possibly due to cell death, throughout the incubation. The degree of phage-mediated lysis was much less in antibiotic-containing cultures. This is because the bacteria did not grow well and prevented the phage from achieving the replication cycle.

According to the results obtained with ampicillin resistant mutants, chloramphenicol resistant mutants will behave similarly in the presence or absence of antibiotics. Thus, after 60 minutes of incubation, the increase in spontaneous radioactivity emission will indicate whether the culture is sensitive to chloramphenicol.

Claims (37)

  Use of an adenylate kinase assay in an in vitro test for the effect of external conditions on the growth characteristics of bacterial cells.   The use according to claim 1, wherein the test is for the susceptibility of bacteria to antibiotics or biostatic drugs.   Use according to claim 1, wherein the test is to determine the stage of bacterial growth.   A method for determining the susceptibility of a bacterium to a reagent, comprising: assaying adenylate kinase released by lysis of the bacterium from a culture containing said reagent; and culturing the result prior to addition of the reagent, and Results from similar adenylate kinase assays for either lysed bacteria from the same culture at different time points and / or lysed bacteria from similar cultures that do not contain the reagents A method comprising comparing.   The method of claim 4, wherein the bacteria are lysed using a chemical lysing agent.   The method of claim 4, wherein the lysing agent is specific for a particular bacterium.   7. The method of claim 6, wherein the lysing agent is a bacteriophage that infects and lyses a specific bacterial genus, species or strain.   The method of claim 4, wherein the bacteria are lysed by an enzyme.   The method according to claim 8, wherein the enzyme is lysin.   The method according to any one of claims 3 to 9, wherein the bacterium is first subjected to a separation step in order to substantially remove any non-target bacterium in the culture.   The method of claim 10, wherein the separation is performed using an immunocapture method.   12. The method of claim 11, wherein the bacterium is concentrated on a solid surface on which an antibody or binding fragment thereof that is specific for the target bacterium is immobilized.   13. The method according to any one of claims 4 to 12, wherein the culture further comprises a growth medium advantageous for the selective cells.   A method for determining the susceptibility of a bacterium to a lytic antibiotic, comprising: (i) separating the bacterium from other microbial species; (ii) determining the extracellular adenylate kinase content of the bacterium culture; (iii) adding the lytic antibiotic to the culture and incubating it for a time sufficient for the antibiotic to exert its lytic effect; and (iv) determining the adenylate kinase content of the culture. The method of claim 4, further comprising the step of determining whether dissolution has occurred.   The method according to claim 14, wherein in step (i), the bacteria are separated by immunocapture.   16. A method according to claim 14 or 15, wherein the culture of bacteria comprises a selective growth medium advantageous to the bacteria.   A method for determining the susceptibility of a bacterium to a non-lytic antibiotic or biostatic agent, comprising: (i) separating the bacterium from other microbial species; (ii) culturing the bacterium, (Iii) intermittently removing samples during the incubation, lysing bacteria in these samples, and adenylate kinase in said samples 5. The method of claim 4, comprising determining whether the content of all adenylate kinases in the culture is increased or decreased by assaying.   18. The method of claim 17, wherein the bacteria are lysed using a chemical lysing agent.   The method according to claim 17 or 18, wherein, in step (i), the bacteria are separated by an immunocapture method.   20. A method according to any one of claims 17 to 19, wherein the culture of bacteria comprises a selective growth medium advantageous to the bacteria. A method for determining the susceptibility of a bacterium to an antibiotic or a biostatic agent, comprising: (a) specifically lysing the first sample of the bacterium, a second sample in the presence of the antibiotic, and the target bacterium Incubating a third sample in the presence of bacteriophage and a fourth sample in the presence of both said bacteriophage and said antibiotic;
(b) determining the adenylate kinase content of each of the first to fourth samples after culturing; and (c) based on the results of the adenylate kinase assay, and the antibiotic or biostatic Determining the susceptibility or resistance of the bacteria based on the mode of action of the drug;
Comprising a method.   The result obtained in step (c) is compared with the result shown in FIG. 4 to determine whether the bacterium is sensitive or resistant to the antibiotic or biostatic agent. The method of claim 21.   23. A method according to claim 21 or 22, wherein the concentration of the bacteria in the culture is increased by an immunocapture method prior to step (a).   24. The method according to any one of claims 21 to 23, wherein the sample further comprises a selective growth medium that favors the growth of the bacteria in preference to other bacterial species. A method for determining the growth phase of a bacterial culture, comprising: (a) subjecting a first sample of said bacterial culture to a lysing agent and lysing bacterial cells therein;
(b) assaying adenylate kinase in the first sample;
(c) assaying adenylate kinase in a second sample of the culture that has not been exposed to the lysing agent;
(c) A method comprising the steps of comparing the results obtained from the first and second cultures and determining the growth phase of the cultures.   Results showing in step (c) that adenylate kinase level in the second sample, which is on the order of 1% of the level found in the first sample, refers to healthy log phase culture and 1% excess level 26. The method of claim 25, wherein is indicative of progression to stationary phase.   27. The method of claim 25 or 26, wherein the lysing agent comprises a bacteriophage that specifically infects and lyses cells of a surfactant and a particular target bacterium.   A test kit for testing the susceptibility of bacteria to antibiotics, comprising one or more antibiotics and one or more reagents necessary for the assay of adenylate kinase.   The test kit according to claim 28, wherein the reagents necessary for the assay of adenylate kinase include ADP, a magnesium ion source, luciferin and luciferase.   30. The test kit according to claim 28 or 29, wherein the antibiotic is lyophilized.   The test kit according to any one of claims 28 to 30, further comprising a lysing agent.   32. The test kit according to claim 31, wherein the lysing agent comprises a chemical.   32. The test kit of claim 31, wherein the lysing agent comprises a bacteriophage specific for a particular target bacterial cell.   34. The test kit according to any one of claims 28 to 33, further comprising a multi-well plate.   Use according to claim 1, substantially as described above with reference to an embodiment.   5. A method according to claim 4, substantially as described above with reference to an embodiment.   A test kit substantially as described above with reference to Example 6.

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