DE3419327A1 - Method for the quantitative determination of the growth of bacteria - Google Patents

Abstract

The present invention relates to a method for the quantitative determination of the growth of aerobic Gram-negative bacteria in a suspension, wherein the bacteria are mixed with a substrate which can be enzymatically cleaved, and a cleavage product of the substrate is measured by photometry.

Description

Method for the quantitative determination of growth

of bacteria The invention relates to a method for quantitative Determination of the growth of aerobic gram-negative bacteria in a suspension.

The previously known method for determining the growth of bacteria are based on the determination of the number or mass of cells in general homogeneous suspensions of the bacteria are present in a liquid. Here receives either the bacterial concentration (number of cells / ml) or the bacterial density (mg / ml). From the increase in these sizes in a growing bacterial culture, the Division rate as the number of doubling the bacterial concentration / h and as you Reciprocal value determine the generation time as the time interval for doubling.

The most widely used method for determining total cell counts makes use of the microscopic counting in a thin layer in a counting chamber (e.g. according to Neubauer, Toma or Petroff-Hauser) spread cells. The relative count to known numbers of small particles, e.g. the erythrocytes of the blood (approx. 5 x 106 erythrocytes / ml) is one of the oldest procedures. Furthermore, the counting number can be determined with electronic counting devices. These make up the loss of conductivity an electrolyte solution, which occurs when a bacterium passes through a tight opening occurs. The membrane filter method can be used for cell counts below 106 cells / ml use.

The live cell count is usually determined using the Koch cast plate method certainly. Here is an aliquot in an appropriate manner diluted homogeneous cell suspension mixed with liquid nutrient agar and placed in Petri dishes poured out. The suspension can also be applied on the surface with a Drigalsky spatula be spat out. The cells can also be filtered through a filter which are then deposited on nutrient agar or nutrient pads is launched. In all cases the colonies are counted after incubation. the Application of the Koch plate casting process and derived methods is on the type of counting of the same cells from homogeneous suspensions is switched off and can be not transferred to the counting method of different individuals from mixed populations.

The choice of the method to be used to determine the bacterial mass depends on the context in which reference is made to the bacterial mass target. When determining cell yields (yields), the fresh weight is often used or dry weight determined. As a reference for metabolic and enzyme activities serve the protein or nitrogen content.

The cell mass can be determined directly or indirectly. With the former the fresh and wet weight is determined after the cells have been centrifuged off. Both Methods are subject to considerable systematic errors. Much more accurate the total nitrogen content (microkjedal method and microdiffusion of the Ammonia) and the total carbon content (according to van-Slike-Folch). The bacterial protein is often determined routinely. Modifications of the biuret method and other colorimetrically detectable color reactions serve well. Microprocesses are geared towards the measurement of representative protein building blocks (tyrosine, tryptophan) (after Lowry or Folin-Ciocal- teu). To the indirect methods The determination of the turbidity of a cell suspension counts for the biomass determination.

In routine operation, this is the optical density of a suspension measured as absorbance (absorbance measurement, turbidimetry). For some purposes it is also the scattered light measurement (nephelometry) more precisely. A linear dependency between the measured values and the bacterial mass exists in both methods only in Area of very low cell densities. Increasing cell densities have a rapidly decreasing one Result in accuracy. In particular, the turbidimetric measurement is very imprecise, since the linearity decreases very quickly above an absorbance of 0.3. Both procedures also have the disadvantage that unspecific cloudiness and air bubbles cause growth can pretend, especially since only one wavelength is used. Another one The problem with direct growth determination is that some types of bacteria in the presence of certain antibiotics (e.g. B-lactam antibiotics) a prolonged lag phase. As a result, the measured values are at low levels of germination so close together that it is difficult or even impossible to tell if it is growth or there is no growth. Another indirect method is based on metabolic parameters to measure that are directly related to growth (O2 uptake, production of CO2 or acid) and which therefore provide an adequate measure of the mass of microorganisms. Their measurement is indicated in cases where other methods fail, e.g. in very low cell densities. For measurement, titrim (trisclle, manometric and electrochemical processes are used.

Other known methods relate to the inhibition of growth by various inhibitors (e.g.

Antibiotics). For quantitative testing of the effect one The plate diffusion test is often used for antibiotics. To carry out of this test are dishes with a nutrient agar to which the test microbe suspension has been added filled to a certain height.

The test solutions are applied to these plates by either in punched holes (perforated plate test) or in attached glass or metal cylinders be pipetted (cylinder plate method) or sucked up in filter paper and be placed on the agar as filter paper disks. If the reaction is positive, will After incubation, an inhibition zone is visible, the diameter of which remains constant if maintained Test conditions (nutrient composition, layer thickness, sowing density, incubation time and temperature) proportional to the logarithm of the concentration of the antibiotic is. In the dilution series test, the antibiotic is inoculated with the test germ in one Nutrient solution diluted in a 1: 2 dilution and the minimum after incubation Concentration determined at which there is currently no growth (minimal bacteriostatic Concentration). The test can be performed on a macro or micro scale. Included are placed in different tubes or wells of a microtiter plate different Concentrations of an antibiotic. With a suitable selection of the dilution steps the minimum inhibitory concentration of the antibiotic can be determined. The duration of the test is usually 16 to 24 hours. The agar dilution test becomes similar to the serial dilution test carried out. The only difference is that instead of a liquid suspension, it is solid Culture media are used.

All of these methods for determining growth or

the growth inhibition of bacteria have disadvantages. You are namely sometimes very time-consuming and / or lead to inaccurate results.

The present invention has therefore set itself the task of a indirect method for the quantitative determination of the growth of aerobic gram-negative Develop bacteria that work quickly and accurately.

To solve the problem, the invention provides that the Bacteria mixed with an enzymatically cleavable substrate and a split piece of the substrate measured photometrically.

It has surprisingly been found that paranitroanilides are used as substrates can serve for aerobic gram-negative bacteria and the resulting split pieces form a direct measure of the growth of the bacteria.

L-leucine paranitroanilide and proline paranitronilide are preferred. L-alanine paranitroanilide is particularly preferred.

From DE-PS 26 53 047 test systems are known that are used for detection Use L-alanine-4-nitroanalide for gram-negative germs via their aminopeptidase activity. From J. Clin. Microbiol. 13 (1981) 483-490 is also known to use naphthylamine derivatives for-the identification of Enterobacteriaceae to be used. For quantitative determination However, these test systems are not used for bacterial growth. aside from that it is assumed that the test systems are not suitable for all gram-negative bacteria suitable is.

The bacteria split the paranitroanilide with the help of the aminopeptidase enzyme and release paranitroaniline. The enzyme aminopeptidase has been used in various gram-negative Bacteria detected for the first time by Teuber ud Cerny (Arch. Mikrobiol. Vol. 91, 235-240 (1973)).

The amino endopeptidase was discovered by Lazdunski and co-workers (Eur. J. Biochem. Vol. 60, 349-369 (1975)). While the paranitroanilide in Hardly any range from 380 to 420 nm in the concentrations used according to the invention absorb, the light absorption changes due to the cleavage of the paranitroaniline in a characteristic way. The increase in absorption per unit time is a direct measure of the increase or decrease in bacterial growth. In particular The process can also capture the synergistic and antagonistic Serving effect of various substances. The method is also suitable to determine the resistance of aerobic gram-negative bacteria to antimicrobial active compounds or growth inhibitors, in particular antibiotics and chemotherapeutics. In microbiological laboratory diagnostics, this is bacterial in addition to the identification Pathogens a very important test step to make statements about the To receive therapy options.

Our own studies have shown that the nitroanilides in concentrations from 0.5 to 5 mmol / l do not show any disruptive bacteriostatic effects of their own and should therefore preferably be used in this concentration range.

The measurement takes place at 2 different wavelengths, which are indicated by the Split pieces of the substrate are absorbed differently. Wavelengths are preferred in the range from 380 to 450 nm. It is particularly preferred that the one wavelength at 414 nm and the second at 450 nm. Instead of 414 nm, another wave length be used in which free paranitroaniline is absorbed. Absorbed at more than 450 nm Paranitroaniline only insignificantly. The two-wavelength measuring principle has the big one Advantage that all also unspecific turbidity values are subtracted, while with the turbidimetric methods usually used in microbiology (often incorrectly referred to as photometric method) air bubbles and solid components of the nutrient solution have a disturbing effect. The absorption values for the haze caused by the growth are roughly the same for the two wavelengths used. This becomes a pure enables photometric evaluation. This in turn has the advantage that the different Values for growth and growth inhibition clearly differ from one another. While in the case of pure turbidity measurement, absorption values of maximum within 5 hours 0.5 to 0.6 can be achieved, the values of the method according to the invention are between 1.4 and 2.3 and, if there is no or delayed growth, below 0.2.

For the assessment of whether there is growth or not, a special evaluation principle followed: (control) (antibiotic) = Q (control) If the quotient Q is above a threshold value, the germ cannot grow. The bacterium grows below this threshold. The thresholds are for the individual antibiotics differ, but are in the range from 0.85 to 0.92.

The invention is further illustrated by the following exemplary embodiments explained: Beisy? Iele I. Materials: 1. Microtitration plates with predefined antibiotic concentrations and one or more antibiotic-free microtitration plates. The substrates can be dried or frozen be.

2. Sterile Müller-Hinton broth with a concentration of 3 mmol / l L-alanine paranitroanilide; 3. Sterile saline solution; 4. McFarland standard 0.5 (turbidity standard); 5. pipette; 6. Plate shaker for microtitration plates with or without an incubator unit; 7. Microtitration plate photometer (Multiskan MC); 8. Computer unit connected to 7. with floppy disk drive, monitor and printer; 9. Bacterial cultures on agar plates.

II. Procedure A suspension is made with physiological saline solution (according to McFarland standard 0.5 108 microorganisms / ml). Manufacture of the 0.5 McFarland standard: 0.5 ml, 0.048 M BaCl2 (1.175% BaC12 2H2Og / v) and 99.5 ml, 0.18 M H2SO4 (1% v / v) are added put together and vigorously mixed. The standard can be locked well can be kept in the dark for several weeks.

100 μl of this suspension are added to 10 ml of Müller-Hinton broth 3.0 mmol / l analine paranitroanilide given (dilution factor 1: 100 corresponds approximately 106 germs / ml). Then add 50 μl of this with an automatic pipette Suspension (or another suitable volume) into the wells of the alicrotitration plate and then covers the plate with an adhesive film. The microtiter plates are placed on a plate shaker and with shaking for 5 hours at 35 bis 370C incubated. The plates are then evaluated with the photometer. The results are compiled in the following tables. The in table 1 indicated antibiotic concentrations worked. The results show that the absorption values measured in the presence and absence of L-alanine paranitroanilide clearly stand out from each other. In addition, the absorption values are the pure turbidity measurement according to Table 3, very low and close together even at 550 nm.

On the other hand, Table 2 shows clear gradations of the measured values with the addition of different amounts of antibiotics. So lets the increase in growth retardation even with only slightly increased antibiotic concentrations measure each other precisely.

Table 1 Antibiotic concentrations (nlg / .l) of agar plates A. - H agar plate H G F E D C B A 1. Ampicillin * 0.5 1 2 4 8 16 32 2. Piperacillin * 1 2 4 8 16 32 64 3. Mezlocillin * 1 2 4 8 16 32 64 4. Cefazolin * 1 2 4 8 16 32 64 5. Cefotaxime * 0.5 1 2 4 8 16 32 6. Lamoxactam * 1 2 4 8 16 32 64 7. Cefoxitin * 0.5 1 2 4 8 16 32 8. Gentamicin * 0.25 0.5 1 2 4 8 16 9. Amikacin * 1 2 4 8 16 32 64 10. Doxycycline * 0.25 0.5 1 2 4 8 16 11. Fosfonycin * 4 8 16 32 64 128 256 12. Chlorine * 0.5 1 2 4 8 16 32 amphenicol * blank T a b e l l e 2 absorption values with L-analin-paranitroanilide 1 2 3 4 5 6 7 8 9 10 11 12 A 0.151 0.805 0.704 0.077 0.059 0.060 0.061 0.058 0.049 0.066 0.059 0.060 B 0.292 0.858 1.009 0.078 0.068 0.064 0.060 0.058 0.059 0.064 0.067 0.055 C 0.406 0.829 0.771 0.089 0.086 0.063 0.065 0.048 0.049 0.061 0.072 0.049 D 0.661 0.788 0.728 0.100 +0.121 +0.078 0.090 0.055 0.054 +0.074 0.369 0.057 E 0.875 0.595 0.677 +0.089 0.285 0.313 +0.103 +0.076 0.060 1.223 1.736 0.055 F 0.966 0.643 0.634 0.175 0.611 0.798 1.350 1.187 +0.055 1.329 1.772 +0.068 G 1.340 0.678 0.602 0.619 0.902 0.927 1,918 1,979 0.279 1,927 1,838 0.162 H 1,868 1,788 1,800 1,794 1,837 1,767 1,872 1,882 1,866 1,991 2,024 1,817 Explanations: + = visually after 24 h ~ = after 5 h photometrish measurement at 414 nm and 450 nm; Letters A to H and numbers 1 to 12 according to table 1; Test organism: Escherichia coli T a b e l l e 3 absorption values without L-alanine paranitroanilide (pure turbidity measurement) 1 2 3 4 5 6 7 8 9 10 11 12 A 0.069 0.079 0.078 0.075 0.077 0.067 0.084 0.094 0.072 0.084 0.078 0.071 B 0.078 0.089 0.091 0.107 0.088 0.075 0.087 0.076 0.086 0.078 0.075 0.082 C 0.076 0.082 0.109 0.086 0.071 0.100 0.078 0.069 0.081 0.078 0.078 0.080 D 0.093 0.088 0.098 0.096 0.078 0.081 0.071 0.091 0.073 0.077 0.093 0.075 E 0.107 0.099 0.094 0.079 0.097 0.095 0.088 0.104 0.081 0.148 0.197 0.072 F 0.093 0.087 0.091 0.070 0.101 0.086 0.105 0.133 0.102 0.123 0.219 0.090 G 0.124 0.107 0.099 0.125 0.097 0.129 0.194 0.167 0.107 0.205 0.248 0.106 H 0.191 0.246 0.229 0.220 0.224 0.238 0.231 0.217 0.183 0.216 0.208 0.166 Explanations: Letters A to H and numbers 1 to 12 according to table 1; Measurement at 550 nm; Test organism: Escherichia coli

Claims (9)

Claims 1. A method for the quantitative determination of growth of aerobic gram-negative bacteria in a suspension, characterized in that that the bacteria are mixed with an enzymatically cleavable substrate and a Measures gap piece of the substrate photometrically. 2. The method according to claim 1, characterized in that the substrate L-alanine paranitroanilide, L-leucine paranitroanilide or proline paranitroanilide and the cleavage product paranitroaniline is measured. 3. The method according to claim 2, characterized in that the concentration of the substrate is 0.5 to 5 mmol / l. 4. The method according to any one of claims 1 to 3, characterized in that that measured at two different wavelengths through the gap of the substrate be absorbed differently. 5. The method according to claim 4, characterized in that the wavelengths range from 380 to 450 nm. 6. The method according to any one of claims 4 or 5, characterized in that that one wavelength is 414 nm and the second 450 nm. 7. The method according to any one of claims 4 to 6 for detecting the synergistic and antagonistic effects of various substances. 8. The method according to any one of claims 1 to 6 for determining the Resistance of aerobic gram-negative bacteria to antimicrobial compounds. 9. The method according to claim 8 for determining the aerobic resistance gram-negative bacteria versus antibiotics or chemotherapy drugs.