NO885400L - PROCEDURE FOR THE DISPOSAL OF BACTERIA AND SUSPENSION. - Google Patents

Description

The present invention relates to the detection of bacteria and fungi in a sample, especially in aqueous, physiological fluids. The invention relates in particular to a method for lysing the bacterial cell walls and the fungal cell walls using a combination of a lytic enzyme and a surface-active agent (detergent) and determining the bacterial enzyme activity and the fungal enzyme activity with an enzyme detection system.

The most frequently occurring bacterial and fungal infections are in the urinary tract. Many different microorganisms can infect the urinary tract. These organisms include gram-negative bacteria such as Escherichia coli, Proteus sp., Klebsiella sp. and Pseudomonas sp., gram-positive bacteria such as Staphylococcus sp. and Enterococcus sp. and fungi such as the yeast Candida.

These infections are usually confirmed in clinical laboratories using time-consuming procedures that include culturing the samples.

The importance of quantitative microbiological examination of the urine is often complicated by sample contamination. Colony counts of 10,000 (l.O<4>) to 100,000 (IO<5>) or organisms per milliliters indicates infection when the urine samples are collected by the "clean-voided" technique or by catheterisation. Colony counts of less than 10,000 (IO<4>) per ml usually indicates contamination. Bacteriogen-free testing procedures are often necessary to associate an acceptable statistical probability of bacteriuria (bacteria in urine) when the colony counts are below 10<5>per. ml. In addition, quantitative results require interpretation with respect to the patient's symptoms, timing, urine sampling methodology, quantitation methodology, and administration of therapeutic drugs, if any exist.

Various culture-based methods are used for the quantification of bacteriuria. The pour plate dilution technique is the most accurate, but requires 24 to 48 hours for a determination. Other quantitative methods use a standardized calibrated inoculation loop and streak plate. Semi-quantitative methods include dip-slide, filter paper, pipette, cup and pad culture. All of these methods require the growth of bacteria on media. Chemical procedures are based on the Griess test for nitrate, triphenyltetrazolium reduction, glucose oxidase reduction with glucose and catalyst-hydrogen-peroxide interaction. However, these methods are not as reliable, sensitive or specific as the culture test. A number of methods using different instruments are also available, but these require relatively expensive items and experienced technicians.

It is therefore necessary to have a simple, reliable and cheap method for rapid detection of bacteria which avoids the disadvantages of known systems.

The present invention provides a method for detecting bacteria and fungi in a sample by the enzymatic activity of the bacteria or fungi. The suspected microorganism in a sample is first lysed with a combination of a lytic enzyme and surfactant to destroy the cell walls. The bacteria or fungi are then detected via the enzymatic activity of the microorganism in an enzyme detection assay.

The inventors have discovered that known bacterial or fungal enzyme detection assays are enhanced by using the combination of surfactants and lytic enzymes to lyse the cell walls of the microorganisms. The detection signal is thus considerably increased by treating the microorganism according to the method according to this invention.

The present invention relates to a method for detecting bacteria or fungi in a sample. The sample is lysed with a combination of lytic enzyme(s) and surfactant(s) that destroy the microbial cell walls. The enzymatic activity of the lysed bacteria or fungi can then be detected in an enzyme analysis.

The sample believed to contain bacteria or fungi may include food, tissue, groundwater, cooling water, pharmaceutical products, sewage, etc. The invention is particularly useful for the detection of bacteria in aqueous fluids, such as human and animal biological fluids, such as urine , spinal fluid, blood etc. and also faecal secretions and suspensions of human or animal tissue. The preferred biological fluid used for carrying out this invention is urine.

To practice this invention, it is preferred that the sample is first processed to remove large or excess contaminating debris by centrifugation or filtration. This debris can mask, compete or otherwise interfere with the microbial lysis or enzyme detection system. The sample can e.g. be passed through a filter to separate the bacterial cells from other cellular debris present in the sample....The filtrate will then mainly contain the bacteria to be detected and other unicellular organisms. It may then be preferred to concentrate the bacteria by passing the filtrate through another, finer filter, which can capture the bacteria on a small surface area. Bacteria are detected by applying the lytic enzyme, the surfactant and the enzyme detection assay components to the second filter.

Lytic enzymes are enzymes that can destroy the microbial cell walls by hydrolysis of the cell wall components. Enzymes that have lytic activity include autolysins, glycosidases, amidases and endopeptidases. Among the specific lytic enzymes that can be used in the present invention are lysozyme, mutanolysin, lysostatin and chitinase.

Surface-active agents according to the definition here are any of the many synthetic water-soluble or liquid organic preparations such as e.g. can emulsify oils, keep particles in suspension and act as wetting agents. Surfactants that can be used in the invention can be ionic, non-ionic or zwitterionic and can include, but are not limited to, the following: Tween-20 (polyoxyethylene sorbitan monolaurate), Tergitol NP-40 (a polyglycol ether surfactant) and Triton X -114 (octylphenoxypolyethoxyethanol). "Tergitol" is a registered trademark of Union Carbide Corporation. "Triton" is a registered trademark of the Rohm & Haas Company.

The concentration of the lytic enzyme can vary from 10 ng/ml to 5 mg/ml, and the concentration of the surfactant can vary from 0.001 to 2%. Furthermore, one or more lytic enzymes can be used with one or more surfactants according to the present invention.

In the first step of the method for detecting bacteria and fungi according to this invention, the sample suspected of containing the bacteria or fungus is contacted with a combination of lytic enzyme(s) and surfactant(s) to destroy or lyse the microbial the cell wall. In another embodiment of this invention, lysis of the microbial cell wall can be performed simultaneously with the application of the enzyme detection assay. It may be that enzyme detection assays known in the art, which are adversely affected by the simultaneous lysis and detection, and therefore, where the lysis step and the enzyme detection step are performed one after the other. The enzyme detection assays used to carry out the present invention are known in the art and many of them are commercially available. In a preferred embodiment of this invention, the assay used detects the dehydrogenase activity of lysed bacteria. In this system, the dehydrogenase activity of the bacteria is coupled to a cationic dye, nitro blue tetrazolium (NBT), a color indicator. The coupling is carried out via an intermediate carrier, Meldola blue (8-dimethylamino-2, 3-benzophenoxazine), an electron carrier. Other electron carriers that may be used include resorufin, phenazine methosulfate and phenazine methosulfate. NBT is particularly useful in this invention because it is converted by reduction from a light yellow, almost colorless, water-soluble compound to a dark blue precipitate. This blue reduced form remains at the reduction site as insoluble formazan. Reagent in excess does not affect the sensitivity of the analysis because the reagent cannot dilute the insoluble reaction product.

Another enzyme detection system that can be used in this invention is the 2,6-dichloroindophenol assay system. This analysis system also detects dehydrogenase activity, and requires the use of electron carriers such as phenazine ethosulfate. On reduction, 2,6-dichloroindophenol is converted from blue to colourless.

The bacterial or fungal detection limit will generally depend on the microbial concentration in the sample, method of concentration of the microorganism (if any), species and metabolic state of the microorganism(s), and the sensitivity of the enzyme detection system. For example, bacteria can be detected in the urine at a level of 10,000 (104 ) per mL with the nitro blue tetrazolium assay when bacteria from a 1 mL sample are collected on a 3 mm diameter filter disk.

The substances used in the method according to this invention are suitable for the production of a kit. Such a kit may include a carrying device which is compartmentalized to receive in close association one or more container devices, such as containers, tubes and the like. Each of the container devices includes one or the separate elements that are used in the method according to this invention. One of the container devices can e.g. contain the substance to concentrate the microorganisms, such as filters ... or semi-permeable membranes to retain bacteria or fungi. The second container may contain the combination of the lytic enzyme and the surfactant. A third or fourth container device may contain the various components of the enzyme detection assay.

In addition, the carrier device can also contain a number of container devices, each of which contains a different, predetermined amount of known bacterial or fungal enzyme that can be used as a standard. These containers can then be used to prepare a standard curve that can be compared against the results obtained using the method of this invention to detect the presence of bacteria in a sample.

When using the kit, the user adds a premeasured amount of a sample suspected of containing the bacteria or fungus to a container, and concentrates the sample and/or removes interfering substances, if necessary. The user then adds the contents of the first container containing the combination of the lytic enzyme and the surfactant, and the contents of the second and third containers containing the enzyme detection assay. After an appropriate time for color development, according to the particular enzyme detection system used, the lysed bacteria or fungi are detected by measuring the enzymatic activity.

It should be noted that not every surfactant or lytic enzyme combination will enhance every enzyme detection assay for every microbial species. The determination of whether a given lytic enzyme and surfactant combination and enzyme detection assay may work within the scope of the present invention can be performed using a single test. The test involves adding the lytic enzyme and the surfactant to a microbial sample and running a control sample at the same time* and observing whether the enzyme detection assay is enhanced by the combination of the lytic enzyme and the surfactant. In general, an increase in the intensity of the assay's detection signal is indicative of amplification of the enzyme detection system.

The following examples are intended to illustrate, but not limit, the method according to the present invention.

EXAMPLE 1

Test system

In Examples 2-6, bacteria were detected using the general procedure.

The bacteria were clinical strains obtained from Newport (Rhode Island) Nåvel Hospital and cultured on nutrient agar plates (Scott Laboratories, Fiskeville, Rhode Island). Samples for testing were bacterial suspensions, usually at a concentration of 10<7> to 10<8> CFU (colony-forming unit) per mL, in phosphate-buffered saline. Bacterial concentrations were calculated by assuming an absorbance of 0.400 at 650 nm for suspension of 1 x 10<8>CFU per mL and was confirmed by seeding on culture dishes. These suspensions were kept at 2-8°C until use.

The analysis solution was prepared fresh in water, and contained the following: 0.17 mg/ml nitro blue tetrazolim (NBT)

(Calbiochem, San Diego, Calif.), 0.311 pg/ml Meldola blue

(Boehringer Mannheim Biochemicals, Indianapolis, Indiana), 0.096 mg/ml L-lactic acid (Sigma Chemical Co., St. Louis, Missouri), 1.35 mg/ml L-maleic acid (Sigma), 0.130 mg/ml glucose (Fisher Scientific, Pittsburg, PA), 0.56 mg/ml sodium phosphate monobasic monohydrate (Fisher), 4.42 mg/ml 3-nicotinamide adenine dinucleotide (Boehringer Mannheim), and 0.3 M 2-amino-2(hydroxymethyl)-1,3 -propanediol (Sigma). Lytic enzymes and/or surfactants (all from Sigma) were added at levels as indicated. The pH of the solution was adjusted to 9.0, and the solution was kept at 2-8°C until use.

The assays were performed in 96-well microtiter plates (Dynatech Laboratories, Alexandria, Virginia) by mixing 80 µl of assay solution and 20 µl of sample, and incubating for 30 minutes at 37" C. The absorbances of the wells were determined in a Dynatech MR-600 microplate reader at 570 nm, and ranged from 0.05 to 1.00 for positive samples.These absorbances were corrected for background by subtracting the absorbances of the negative controls in which 20 µl of phosphate buffered saline replaced the bacterial sample, pg by subtracting the contribution of Baerebacter len.

EXAMPLE 2

Suspensions of six bacterial species were tested as described in Example 1. The effect of addition of the lytic enzymes lysozyme (0.1 mg/ml) and the surfactant Tween-20 (0.05$) on color development was then determined. The results appear in Table I.

EXAMPLE 3

In this example, two bacterial suspensions were analyzed according to Example 1. The effect of color development upon addition of the lytic enzyme lysozyme (0.1 mg/ml) and to the two surfactants, Tergitol-NP40 ($0.05) or Triton X -114 (0.05$) was determined with and without added lysozyme. The results appear in table II. "Tergitol" is a registered trademark of Union Carbide Corporation. "Triton" is a registered trademark of the Rohm & Haas Company.

EXAMPLE 4

In this example, one suspension of Pseudomonas aeruginosa was analyzed using a modification of the method in example 1. Bacteria in phosphate-buffered saline were pretreated with the lytic enzyme lysostaphin (0.5 mg/ml) and/or the surfactant Tween -20 (0.05$) for 30 minutes at 37°C before the assay solution was added as described in Example 1. The effect of the color development of NBT by lysostaphin and Tween-20 was determined and is summarized below in Table III.

EXAMPLE 5

In this example, an Esherichia col1 suspension was assayed according to Example 1 and treated with a color developing reagent containing tetranitro blue tetrazolim (Sigma) at 0.34 mg/ml instead of NBT. The effects on color development by adding lysozyme 0.1 mg/ml and Tween-20 0.05$ were then determined. The results appear in table IV.

EXAMPLE 6

In this example, two bacterial suspensions were analyzed using a modification of Example 1.

The analysis solution used in this example was prepared fresh in water, and contained 0.022 mg/ml 2,6-dichloroindophenol (Sigma), 0.096 mg/ml L-lactic acid (Sigma), 1.35 mg/ml L-malic acid (Sigma ), 0.30 mg/ml glucose (Fisher), 4.42 mg/ml P-nicotinamide adenine dinucleotide (Boehringer Mannheim), 0.076 mg/ml phenazine ethosulfate (Sigma), 10 mM sodium phosphate (Fisher), and 0.15 M sodium chloride. The pH of the solution was adjusted to 7.5 and the solution was kept at 2-8°C until use. The samples were prepared as 1 example 1.

The assays were performed in 96-well microtiter plates (Dynatech) by mixing 80 µl assay solution and 20 µl sample and incubating for 10 to 30 minutes at 37°C. The absorbance changes in the wells were determined in a Dynatech MR-600 microplate reader at 630 nm. Assays containing only buffer showed no changes in absorbance. The analyzes containing bacteria showed an increase in absorbance. The effect on absorbance changes when adding the lytic enzyme lysozyme (0.1 mg/ml) and the surfactant Tween-20 (0.05$) is presented in Table V.

Although the invention has been described in detail using illustrations and examples to improve the understanding, it is obvious that certain changes and modifications can be made within the scope of the invention, and limited only by the scope of the appended claims.

Claims (14)

1. Procedure for detecting bacteria or fungi in a sample, characterized by: lyses a sample suspected of containing bacteria or fungi with a combination of lytic enzyme and surfactant; contacting said lysed sample with an enzyme detection system which detects the bacterial enzyme activity or the fungal enzyme activity; and demonstrates the presence of bacteria or fungi in said sample. 2. Method according to claim 1, characterized in that said lysis is carried out with said lytic enzyme and surfactant and said contacting with said enzyme detection system one after the other. Method according to claim 1, characterized in that said lysis and said contacting with said enzyme detection system are carried out one after the other. 4. Method according to claim 1, characterized in that said sample is selected from the group consisting of fluids and tissues from animals and humans. 5. Method according to claim 1, characterized in that said lytic enzyme is selected from the group consisting of autolysins, glycosidases, amidases and endopeptidases. 6. Method according to claim 1, characterized in that said surfactant is selected from the group consisting of ionic, non-ionic and zwitterionic surfactants. 7. Method according to claim 1, characterized in that said enzyme detection system contains nitro-blue-tetrazolium and an electron carrier. 8. Method according to claim 7, characterized in that said electron carrier is selected from the group consisting of 8-dimethylamino-2,3-benzophenoxazine, resorufin, phenazine methosulfate and phenazine methosulfate. 9. Method according to claim 1, characterized in that said enzyme detection system consists of 2,6-dichloroindophenol and an electron carrier. 10. - Method according to claim 1, characterized in that said sample is filtered before lysis. 11. Method according to claim 10, characterized in that said filtered sample is filtered again to concentrate the bacteria or fungi contained in said filtered sample. 12 . Procedure for affecting bacteria or fungi in a sample, characterized by: filtering a sample believed to contain bacteria or fungus to remove interfering debris in said sample and to concentrate said bacteria or fungus in said sample; lighter said filtered sample with a combination of lytic enzyme and surfactant; contacting said lysed sample with an enzyme detection system which detects the bacterial enzyme activity or the fungal enzyme activity; and demonstrates the presence of said bacteria or fungus in said sample. 13. Kit for the detection of bacteria or fungi in a sample, characterized in that it includes a carrier device which is divided to receive in close connection therein one or more containers in which (a) a first container contains a lytic enzyme and surfactant and (b) another container contains an enzyme detection system. 14 . Kit according to claim 13, characterized in that it further contains a device for filtering said sample to remove interfering debris from said sample and to concentrate said bacteria or fungus that is inside said sample.