HK1084698A - Method for universal detection of micro-organisms and reaction medium therefor - Google Patents

Description

Method for the universal detection of microorganisms and reaction environment for carrying out said method

The present invention relates to the field of microorganisms, and in particular to methods for detecting and identifying microorganisms in different environments where they may be found.

Many methods for detecting microorganisms have been developed to address different needs. In this connection, mention may be made of medical sample analysis, quality control in the agrofoods industry and water treatment work afterwards.

An ideal method for detecting microorganisms should be rapid, specific (should not have false positives), sensitive, and easy to implement. The method should allow detection of living and dead microorganisms in different environments. Finally, an additional advantage would be the preliminary identification of the type of bacteria involved.

The method of culturing in a petri dish or in liquid phase allows to detect with good sensitivity all bacteria in growth phase in most environments. Theoretically, a single bacterium is sufficient to ensure that a positive result is obtained after cultivation, and the cultivation in liquid phase can be automated (g.aubert et al, 1993). However, the time required to obtain the results is sometimes very long. Therefore, detection of Propionibacterium strains (Propionibacterium) in blood products requires a four day multiple culture time (me. brecheret al, 2001). For mycobacteria (Mycobacterium), detection takes more than twenty days (h.saitoh et al, 2000). The growth of the bacteria is also largely regulated by the selected culture environment, which may be simple or charge-enhanced (enriched), with or without antimicrobial inhibitors. The culture conditions are also specific for the bacteria to be detected. Thus, different culture temperatures and aerobic or anaerobic conditions are applied. Identification of the microorganism should be carried out by these methods at a second time after cultivation. Finally, this technique does not allow the detection of dead or non-regenerable bacteria.

The method of molecular biology is rapid and sensitive because several hours of incubation is sufficient to produce a positive sample and at least ten microorganisms can be detected in a single reaction.

Performing a Polymerization Chain Reaction (PCR) using fluorescent probes specific for the target DNA allows for the immediate detection of bacterial contamination in the sample (q.he et al, 2002). Purification of the sample is necessary to protect the polymerase necessary in the reaction to amplify the potential inhibitor. For example, a number of PCR inhibitors are found in plasma (wa. al-Soud et al, 2002). This preliminary purification step makes the method for detecting microorganisms using PCR difficult to implement. Thus, if the sample contains bacteria phagocytosed by leukocytes, any residual traces of DNA will give a positive result in the sample, which will greatly impair the specificity of the method.

Hybridization techniques allow the universal and/or specific detection of bacteria (EB Braun-Howland et al, 1992; S.Poppert et al, 2002). Similar to PCR, the sample preparation step again becomes a limiting step in the method. The presence of residual nucleic acids is again a source of false positives.

The main limitation of molecular biology techniques lies in the choice of primers, the specificity of which must be sufficiently high to allow genetic testing, and which are specific to the microorganism to be tested in order to avoid false positive reactions. Usually a mixture of different primers is required, which creates technical limitations.

Immunohistochemical or immunocytochemical labeling methods (enzyme-linked immunosorbent assay, ELISA) using antibodies against the bacterial cell wall are limited by the specificity of the antibodies. Indeed, no antibodies are currently available for the universal detection of microorganisms. This technique can only be used to accurately identify bacterial strains (K.Kakinoki et al, 2001; J.Guarner et al, 2002). It also requires special preparation of the cells or tissues to be analyzed, including for example fixation of the sample and cell infiltration, which would lead to the intervention of solvents of the acetone, formaldehyde and methanol type.

Visible morphological identification of the type of contaminating bacteria can be performed using colorimetric microscopic methods, using, for example, GRAM stains or essential (visual) stains or fluorescent dyes (p.fazii et al, 2002). However, they lack sensitivity and require extended operating times and several days of microbial growth to ensure visibility (s.mirrett et al, 1982).

Microorganisms can be detected in a rapid and simple manner using cytometry (DT. Reynolds et al, 1999; H.Okada et al, 2000).

However, this method is limited to the marking process. In fact, neither antibodies specific for the cell wall of the target strain nor intercalator-type DNA markers (molecules that can insert themselves between platforms formed of nucleic acid base pairs (plasteaus)) enable universal detection of microorganisms. However, the latter option requires pre-manipulation of the bacteria in order for the marker to penetrate the cell wall of the bacteria (d. marie et al, 1996).

To overcome the above drawbacks, the applicant has devised a universal method for detecting microorganisms, using a marker that is universal for all bacteria, yeasts, molds, parasites, such as an intercalator compound of DNA that is non-specific for a particular nucleic acid sequence.

The present detection method can be applied to any biological fluid. In the present invention, the term "biological fluid" means any fluid containing one or several microorganisms, such as ionic, medium, physiological, such as blood or derivatives thereof, such as platelet concentrates or erythrocytes or plasma, and therefore relates to different fields of application, such as the analysis of medical samples, the quality control in the agri-food industry and the subsequent operations of water treatment.

The microorganism detection method of the present invention is preferably applied to blood or its derivatives such as platelet concentrate or red blood cells or plasma.

The method for labelling microorganisms which forms the subject of the present invention uses a reaction environment comprising a labelling agent, a cell-penetrating agent which facilitates the entry of the labelling agent into the molecular channels of the genome of the microorganism independently of the nature of the microorganism. In a particularly preferred method, the labeling method of the invention allows the structure of the microorganism, in particular the bacteria, to be preserved intact.

This reaction environment allows the passage of the labeling reagents:

the cytoplasmic membrane, i.e. the bilayer structure of the lipid molecules and the membrane proteins of whatever microorganism;

the cell wall of gram-positive bacteria, mostly consisting of peptidoglycans or mureins, is in contact with the cytoplasmic membrane and possibly covered by a polysaccharide surface layer;

the outer membrane of gram-negative bacteria, which contains a number of phospholipids, lipoproteins and lipopolysaccharides, is separated from the cytoplasmic membrane by a periplasmic space, in which proteins are present and which is penetrated by a number of pores. Such walls are impermeable to most substances except for the substances passing through the pores.

This novel method of microbial labeling allows for universal labeling of both live and dead or non-regenerable microorganisms.

Microorganisms labeled in this way can be analyzed, for example, by microscopic fluorescence techniques using epifluorescence microscopy and/or liquid cytometry and/or solid phase cytometry.

The method of the invention comprises an initial preparation of the microorganisms starting from a sample containing the microorganisms. Different reagents are used in the same step for penetrating the microorganisms without changing their morphology and causing them to fluoresce.

The method of the invention allows the structure of the bacteria to be preserved in an intact manner for analysis according to cell biology techniques that allow visual identification of a large class of microorganisms: bacillus, coccus, spore, yeast.

The present method simultaneously allows for the detection and morphological identification of microorganisms based on their shape and size. The method can be used for detecting microorganisms in different physiological, culture and ionic environments.

The present method preferably and simultaneously allows the detection and morphological identification of microorganisms based on their shape and size present in blood or its derivatives such as platelet concentrates or erythrocytes or plasma.

The general detection method for microorganisms comprises 4 or 5 steps.

The microorganisms suspended in water in buffer, physiological serum, culture environment, blood, plasma or blood derivatives are placed in a single reaction environment comprising an intercalating agent and at least one cell penetrating reactant.

In the context of the present invention, a "cell-penetrating reagent" is intended to mean a solution comprising at least a mixture of at least one permeabilizing agent (agent), a detergent, an ion-chelating agent and a preservative.

More precisely, the invention relates to a method for detecting microorganisms that may be present in a biological fluid, comprising the following steps:

a) the method comprises the steps of extracting a biological liquid sample,

b) placing the sample in contact with a reaction environment comprising a labeling reagent and a cellular reactant that penetrates the membranes of the microorganisms,

c) filtering the sample on a filter capable of retaining the labelled microorganisms which may be present in the sample, and

d) detecting the microorganisms labeled and retained on the filter in step c).

The labeling agent is preferably an intercalator compound of DNA selected from the group consisting of: cyanine derivatives, propidium iodide, acridine orange and ethidium bromide. The cyanine derivative is selected from the group consisting of PicoGreen, SYBR Green and YOPRO 1. As to their preferred concentration, the concentration of the cyanine derivatives is from 0.001% to 0.5% (v/v), preferably from 0.003% to 0.05%. The concentration of propidium iodide, acridine orange or ethidium bromide is from 0.1. mu.g/ml to 100. mu.g/ml, preferably from 1. mu.g/ml to 40. mu.g/ml.

The labeling agent is preferably PicoGreen.

In the following and in the claims and in the examples without indication to the contrary, "preferred concentration" means the concentration of the product in the final reaction environment "biological sample and reaction environment (labeling reagent + cell penetration reactant)". The skilled person knows how to easily adjust the concentration of the different components in the breakthrough reactants, e.g. in the concentrated mother liquor.

The cell-penetrating reactant of the microorganism is preferably a solution comprising at least a mixture of at least one permeabilizing agent, a detergent, an ion chelating agent and a preservative.

According to the invention, the percentage amounts (by weight) of permeabilizing agent, detergent, ion-chelating agent and preservative in the final reaction mass are comprised from 1X 10^-4％/0.03％/0.02％/6×10^-4% to 2.5X 10^-3％/0.8％/0.6％/0.015％。

The permeabilizing agent is selected from the group consisting of polyethylene glycol (PEG), digitonin (digitonine), monensin, Polyethylenimine (PEI), sodium hexamethophate (sodium hexamethophosrate), and benzalkonium chloride (benzalkonium chloride).

Preferred concentrations of these permeabilizing agents are as follows:

-the concentration of PEG is from 0.01% to 1%, preferably from 0.05% to 0.5%;

-the concentration of digitonin is from 0.01 to 10 μ g/ml, preferably from 0.05 to 5 μ g/ml;

-concentration of monensin from 0.1 to 5 μ g/ml, preferably from 0.5 to 1 μ g/ml;

-the concentration of PEI ranges from 1. mu.g/ml to 400. mu.g/ml, preferably from 5. mu.g/ml to 120. mu.g/ml;

-the concentration of sodium hexametaphosphate is from 0.005% to 1%, preferably from 0.01% to 0.1%;

-benzalkonium chloride at a concentration from 0.001% to 0.1%, preferably from 0.005% to 0.05%;

the permeabilizing agent is preferably Polyethylenimine (PEI).

Among detergents, preferred are those as follows: n-octyl- β -D-glucopyranoside (NOG), saponin (saponin), Tween, Triton, Igepal and CHAPS. Their preferred concentrations are as follows:

-the concentration of saponin or Tween is from 0.005% to 10%, preferably from 0.05% to 0.5%;

-concentration of NOG from 0.01% to 10%, preferably from 0.1% to 0.5%;

-concentration of Triton from 0.0001% to 0.05%, preferably from 0.0008% to 0.002%;

-the concentration of Igepal is from 0.01% to 20%, preferably from 1% to 5%;

the detergent is preferably N-octyl- β -D-glucopyranoside (NOG).

For the ion chelating agent, those of the group comprising EDTA and EGTA are preferred.

The concentration of the ion-chelating agent preferably comprises from 0.05% to 0.8%.

The ion chelating agent is preferably EDTA.

-the concentration of EDTA is from 0.1mM to 50mM, preferably from 0.2mM to 7.5 mM.

The preservative is selected from the group comprising: pyrrolidone iodine (betadine), cetrimide (cetrimide), tea tree oil, terpinen-4-ol, chlorohexidine (chlorohexidine), polymyxin B and rifampicin.

The preservative is preferably chlorohexidine.

-chlorhexidine at a concentration of from 0.0005% to 0.5%, preferably from 0.001% to 0.05%;

-the concentration of cetrimide is from 0.01% to 5%, preferably from 0.05% to 1%;

-the concentration of pyrrolidone iodine is from 0.0001% to 0.001%, preferably from 0.0005% to 0.005%;

-the concentration of tea tree oil is from 0.0001% to 0.1%, preferably from 0.0005% to 0.05%;

-the concentration of terpinen-4-ol is from 0.05% to 10%, preferably from 0.5% to 5%;

the concentrations of polymyxin B and rifampicin are from 0.1. mu.g/ml to 100. mu.g/ml, preferably from 1. mu.g/ml to 50. mu.g/ml.

The penetration reactant may also include an enzyme or a bacteriocin.

The enzyme is preferably lysozyme and the bacteriocin is preferably nisin.

The concentration of lysozyme is advantageously from 0.5. mu.g/ml to 200. mu.g/ml, preferably from 0.05. mu.g/ml to 20. mu.g/ml, and the concentration of nisin is advantageously from 0.005. mu.g/ml to 10. mu.g/ml, preferably from 0.005. mu.g/ml to 0.05. mu.g/ml.

In order to effectively penetrate the bacterial cell wall according to the invention, cryoprotective agents, such as DMSO or ions (NaCl, KC) can also be usedl，MgCl₂Sodium hypochlorite) or sucrose.

The concentration of DMSO is from 0.05% to 20%, preferably from 0.5% to 5%;

the concentration of sucrose is from 0.5% to 70%, preferably from 5% to 20%;

the concentration of sodium hypochlorite is from 0.001% to 5%, preferably from 0.005% to 0.5%;

the concentration of potassium citrate is from 0.5mM to 200mM, preferably from 5mM to 50 mM.

According to one embodiment of the invention, step b) of the method for the detection of microorganisms can be carried out in two substeps b ') and b').

In step b'), the sample is contacted with a reaction environment comprising a labeling reagent and a osmopolymer selected from polyethylene glycol (PEG) or Polyethylenimine (PEI). Preferably Polyethylenimine (PEI) is used.

In step b "), a mixture comprising at least one detergent, an ion-chelating agent, a preservative and another permeabilizing agent selected from nisin, digitonin, sodium hexametaphosphate and benzalkonium chloride is added to the reaction environment.

When step b) of the process of the invention is carried out in two substeps b') and b "), the enzyme is added to step b").

The invention also relates to a reaction environment for labelling microorganisms, comprising a labelling agent and a reactant for penetrating the microbial cells.

According to the present invention, it is preferable that the labeling reagent for labeling microorganisms and the permeation reagent and their concentrations or their components contained in the reaction environment are the same as those used in the aforementioned detection method of the present invention.

The cell-penetrating reactant according to the present invention preferably comprises:

PicoGreen (molecular Probe) from-1/22000;

-PEI at a final concentration of 5.5 μ g/ml;

final concentration of 4.5X 10^-4% of chlorhexidine diacetate;

-N-octyl glucopyranoside at a final concentration of 0.16%;

nisin at a final concentration of 0.018 μ g/ml;

-EDTA at a final concentration of 0.45 mM;

phosphate buffer (PPS) in an amount sufficient to achieve the desired final volume.

The invention will be illustrated by means of the following examples and figures, in which the concentrations referred to refer to the concentrations of the reactants that penetrate:

FIG. 1 shows the effect of addition of nisin on the detection of Staphylococcus epidermidis (Staphylococcus epidermidis) and Escherichia coli (Escherichia coli). The results are expressed as the number of bacteria detected by cytometry in solid phase (FIG. 1A) and as a percentage of bacteria detected by enzymatic assay (FIG. 1B).

FIG. 2 shows the effect of EDTA alone on the detection of Staphylococcus epidermidis and Escherichia coli prepared in different experimental environments.

FIG. 3 is a graph showing the results of an experiment of improved detection of gram-negative bacteria (E.coli) with different concentrations of nisine in combination with different concentrations of EDTA. The results are expressed as a percentage of detection relative to the enzymatic assay.

FIG. 4 is a graph showing the results of tests for the detection of gram-negative bacteria (E.coli and Serratia marcescens) and gram-positive bacteria (Staphylococcus epidermidis) with different concentrations of nisine in combination with a fixed concentration of 7.5mM EDTA. The results are expressed as the number of bacteria detected by cytometry in solid phase.

FIG. 5 is a graph showing the effect of pH on the detection of E.coli using a DNA fluorescent marker in the presence of nisine 0.2. mu.g/ml EDTA7.5 mM.

FIG. 6 shows the detection of the gram-negative bacteria Escherichia coli, Serratia marcescens, Enterobacter aerogenes, Pseudomonas aeruginosa, Proteus mirabilis using DNA fluorescent markers in the presence of nisine 0.2. mu.g/ml EDTA7.5mM, pH 4.8.

FIG. 7 shows the results of an experiment with N-octyl glucopyranoside as cell penetration reactant in combination with nisine 0.2. mu.g/ml and EDTA7.5mM for improving the labelling of Staphylococcus epidermidis (gram positive) and Pseudomonas aeruginosa (gram negative) (N/E. nisine 0.2. mu.g/ml/EDTA 7.5mM solution).

FIG. 8 shows the results of using chlorohexidine as a cell penetration reactant for improving the labeling of Escherichia coli, Pseudomonas aeruginosa and Serratia marcescens (gram-negative bacterial strains) and the effect on Staphylococcus epidermidis.

FIG. 9 shows DNA labeling and detection of bacteria (P.aeruginosa) in different environments.

Figure 10 shows DNA labeling and detection of bacteria in chlorohexidine demonstrating the importance of the combination of NOG to improve the penetration ability and penetration of the marker. Fig. 10A shows the bacterial suspension in labeled PBS and fig. 10B shows the bacterial suspension in platelet concentrate.

FIG. 11 shows the effect of different concentrations of PEI on the detection of Serratia marcescens using fluorescent DNA markers.

FIG. 12 is a graph showing the effect of PEI on DNA labeling and fluorescence detection of E.coli.

FIG. 13 shows the results of detection of Staphylococcus epidermidis and Escherichia coli in the presence of a labeling composition comprising nisine/EDTA/CLX/NOG/PEI in different environments.

The method for detecting microorganisms in a sample can be carried out by subjecting the sample to a two-step process, the first step of labeling/cell penetration, by adding to the sample a composition comprising a labeling reagent and a first cell penetration reactant, and after a period of incubation, a second step in which a composition comprising a further cell penetration reactant is added.

The method can be implemented, for example, according to the following scheme:

3ml of the sample to be treated were incubated in 1ml of the first cell penetrating/labelling solution (PicoGreen0.5ml/l, PEI 60mg/l, PBS solution) for 40 minutes. This step is carried out at room temperature with stirring.

In the second step, 7ml of a composition in solution are added, which makes it possible to carry out subsequent labeling (nisine 0.2mg/l, NOG 2.5g/l, EDTA 1.86g/l, chlorhexidine diacetate 50 mg/l). Incubate at room temperature for 20 minutes. The sample is then filtered on a charcoal filter, such as polycarbonate or polyester, and analyzed by solid phase cytometry.

The method of detecting a microorganism in a sample may also be carried out by subjecting the sample to a treatment in a single step, i.e. by adding to such a sample a composition comprising a labelling agent and one or more cell penetrating agents.

The method may be implemented, for example, according to the following scheme:

8ml of the sample to be treated were incubated for 60 minutes at room temperature with 3ml of a cell penetrating/labeling solution (PicoGreen0.17ml/l, PEI 20mg/l, EDTA 4.34g/l, nisine 0.47mg/l, NOG 5.83g/l, chlorhexidine diacetate 116.7 mg/l). The sample was then filtered on a polycarbonate charcoal filter and analyzed by solid phase cytometry.

All these treatments can be carried out indifferently in open devices such as in test tubes or closed devices such as syringes or a device for preparing platelets for bacteriological analysis (hermosystem, ref. spk 01).

Microbial detection

Determining optimal compositions for labeling/cell penetrating reaction environments

1-labelling in the Presence of nisine

Nisin is used alone as permeabilizing agent facilitating the penetration of the labeling agent.

I reactants

Marking solution

PicoGreen solution (molecular probes) was prepared at 1/2000 in PBS buffer (phosphate buffer, pH7.4).

Nisin solutions

A series of nisin dilutions (starting material 2.5% w/w) were prepared with distilled water:

0.1g nisin was dissolved in 50ml distilled water to 50. mu.g/ml solution,

0.02g nisin was dissolved in 50ml distilled water to 10. mu.g/ml solution,

0.004g of nisin is dissolved in 50ml of distilled water which is 2 mug/ml solution,

preparation of bacterial suspensions in PBS

Escherichia coli (CIP105901)

Staphylococcus epidermidis (68.21)

Adjusting the article to obtain 10⁴Bacteria/ml suspension

II Process

1.2ml of marking solution

+3ml bacterial suspension

Incubation at 22 ℃ for 15 min

+7ml nisine solution

Filtration was carried out with a carbon filter having a pore size of 0.4 μm

III analysis and results

After filtration the filter was analysed by cytometry in solid phase and the results expressed as the number of bacteria detected by cytometry in solid phase and as a percentage of bacteria detected relative to the enzyme detection method.

These results show the effect of nisin addition on the detection of Staphylococcus epidermidis and Escherichia coli and are graphically depicted in FIGS. 1A, 1B.

It was determined that the addition of nisin allows for a good labeling of gram positive bacteria and preferably at low concentrations.

2-use of EDTA itself as permeabilizing agent facilitating the penetration of the labeling agent.

I reactants

Marking solution

PicoGreen solution (molecular probes) was prepared in PBS buffer at 1/2000.

EDTA solution

EDTA 5 mM: 0.093g disodium EDTA, QSP (Latin ═ sufficiency) 50ml distilled water

Bacterial suspensions were prepared in PBS, distilled water, TSB (tryptone soy broth) environment, and plasma.

Escherichia coli (CIP105901)

Staphylococcus epidermidis (CIP68.21)

Adjusting the article to obtain 10³Bacteria/ml suspension

II Process

1.2ml of marking solution

+3ml bacterial suspensions in different environments

Incubation at 22 ℃ for 15 min

+7ml EDTA solution

Filtration was carried out with a carbon filter having a pore size of 0.4 μm

III analysis and results

After filtration, the filters were analyzed by cytometry in solid phase and the results expressed as the number of fluorescent bacteria.

These results show the effect of EDTA alone on the detection of Staphylococcus epidermidis and Escherichia coli prepared in different detection environments and are graphically represented in FIG. 2.

It was determined that EDTA by itself did not allow for the correct labeling of gram positive and negative bacteria.

3-use of nisine/EDTA in combination as permeabilizing agent facilitating the penetration of the labeling agent.

I reactants

Marking solution

PicoGreen solution (molecular probes) was prepared at 1/2000 in PBS buffer (phosphate buffer, pH7.4).

nisin/EDTA solution

Nisin 10 μ g/ml: 0.02g nisine (starting material 2.5% w/w) QSP 50ml distilled water.

EDTA 20 mM: 0.372g disodium EDTA, QSP 50ml distilled water,

a series of EDTA (concentration range 0.1; 1; 5 and 7.5mM) was prepared with 0.25, 2.5, 12.5 and 18.75ml of 20mM EDTA,

adding 50, 250, 500. mu.l or 1ml of nisine at 10. mu.g/ml (concentration range 0.10; 0.05; 0.1 and 0.2. mu.g/ml),

QSP 50ml of distilled water.

Bacterial suspension prepared in PBS

Escherichia coli (CIP105901)

Adjusting the article to obtain 10³Bacteria/ml suspension

II Process

1.2ml of marking solution

+3ml of bacterial suspension

Incubation at 22 ℃ for 15 min

+7ml of nisine solution or nisine/EDTA solution

Filtration was carried out with a carbon filter having a pore size of 0.4 μm

III analysis and results

After filtration the filters were analyzed by cytometry in solid phase and the results expressed as the number of bacteria detected by cytometry in solid phase and as the percentage of bacteria detected relative to the enzyme detection method.

These results show the effect of the addition of nisine in combination with EDTA on the detection of E.coli and are graphically depicted in FIG. 3.

It was determined that labeling in the presence of the nisine/EDTA mixture had a synergistic effect on bacterial detection. It was also determined that the percentage of marked E.coli was maximal at a nisine concentration of 0.1. mu.g/ml and EDTA of 7.5 mM.

Optimum concentration of 4-nisine/EDTA combination as cell-penetrating reactant for detecting bacteria

I reactants

Marking solution

PicoGreen solution (molecular probes) was prepared at 1/2000 in PBS buffer (phosphate buffer, pH7.4).

nisin/EDTA solution

0.02g nisine (starting material 2.5% w/w) was dissolved in 50ml distilled water i.e. 10. mu.g/ml,

nisin 0.05 μ g/ml/EDTA 7.5 mM: 250 ul nisine 10 ug/ml +0.140g disodium EDTA, QSP 50ml distilled water,

nisin 0.1. mu.g/ml EDTA7.5 mM: 500. mu.l nisine 10. mu.g/ml +0.140g disodium EDTA, QSP 50ml distilled water,

nisin 0.5 μ g/ml/EDTA 7.5 mM: 2.5ml nisine 10. mu.g/ml +0.140g disodium EDTA, QSP 50ml distilled water,

bacterial suspensions prepared in PBS

Escherichia coli (CIP105901)

Staphylococcus epidermidis (CIP68.21)

Serratia marcescens (CIP 103716)

Adjusting the article to obtain 10³Bacteria/ml suspension

II Process

1.2ml of marking solution

+3ml of bacterial suspension

Incubation at 22 ℃ for 15 min

+7ml of nisine/EDTA solutions of different concentrations

Filtration was carried out with a carbon filter having a pore size of 0.4 μm

III analysis and results

After filtration, the filter was analyzed by cytometry in solid phase, and the results were expressed as the number of bacteria detected by cytometry in solid phase. The results of this experiment show the results of the detection of gram-negative bacteria (E.coli, Serratia marcescens) and gram-positive bacteria (Staphylococcus epidermidis) in the presence of different concentrations of nisine in combination with EDTA7.5mM are shown in FIG. 4.

It was determined that the percentage of labelling in E.coli was greatest at a nisine concentration of 0.1. mu.g/ml, and that better detection results were obtained for all tested bacteria when nisine was used at 0.2. mu.g/ml in combination with EDTA7.5 mM.

Effect of 5-pH on bacterial labelling in the Presence of nisine/EDTA

I reactants

Marking solution

1/2000 PicoGreen solution (molecular probes) was prepared in PBS buffer (phosphate buffer, pH7.4).

nisin/EDTA solution

Nisin 10 μ g/ml: 0.02g nisine (starting material 2.5% w/w) QSP 50ml distilled water.

EDTA 20 mM: 0.372g disodium EDTA, QSP 50ml distilled water,

18.75ml EDTA 20mM

+1ml nisine 10. mu.g/ml

QSP 50ml distilled water

The pH of this solution was 4.8

Buffered with NaOH (1M) until pH6, pH7, pH 8.

Bacterial suspensions prepared in PBS

Escherichia coli (CIP105901)

Staphylococcus epidermidis (CIP68.21)

Serratia marcescens (CIP 103716)

Enterobacter aerogenes (CIP 60.86T)

Pseudomonas aeruginosa (CIP 76110)

Proteus mirabilis (CIP 104588)

Adjusting the article to obtain 10³Bacteria/ml suspension

II Process

1.2ml of marking solution

+3ml of bacterial suspension

Incubation at 22 ℃ for 15 min

At various pH +7ml nisine/EDTA solution

Filtration was carried out with a carbon filter having a pore size of 0.4 μm

III analysis and results

After filtration, the filters were analyzed by cytometry in solid phase and the results expressed as the number of fluorescent bacteria detected.

The results of this experiment showing the effect of pH on the detection of E.coli with a DNA fluorescent marker in the presence of nisine 0.2. mu.g/ml EDTA7.5mM are shown in FIG. 5.

It was confirmed that increasing the pH under the predetermined conditions did not improve the labeling of E.coli.

The detection of the gram-positive bacteria Staphylococcus epidermidis and the gram-negative bacteria Escherichia coli, Serratia marcescens, Enterobacter aerogenes, Pseudomonas aeruginosa, Proteus mirabilis with the DNA fluorescent marker in the presence of nisine 0.2. mu.g/ml/EDTA 7.5mM at pH4.8 is shown in FIG. 6.

It can be determined that the marking is homogeneous (homogone) between different strains of gram-negative bacteria at pH 4.8. Gram-positive bacteria staphylococcus epidermidis are better detected than negative bacteria.

6-nisine/EDTA/N octyl glucopyranoside combination

I reactants

Marking solution

1/2000 PicoGreen solution (molecular probes) was prepared in PBS buffer (phosphate buffer, pH7.4).

nisin/EDTA/NOG solutions

Nisin 100 μ g/ml: 0.2g nisine (starting material 2.5% w/w) QSP 50ml distilled water.

EDTA 100 mM: 1.86g disodium EDTA, QSP 50ml distilled water,

5% of N-octyl glucopyranoside: 2.5g of this solution was dissolved in 50ml of distilled water

20, 10, 5 or 2.5ml of 5% NOG

+3.75ml EDTA 100mM

+0.1ml nisine 100. mu.g/ml

QSP 50ml distilled water

The pH of this solution was 4.8.

Bacterial suspensions prepared in PBS

Staphylococcus epidermidis (CIP68.21)

Pseudomonas aeruginosa (CIP 76110)

Adjusting the article to obtain 10³Bacteria/ml suspension

II Process

1.2ml of marking solution

+3ml of bacterial suspension

Incubation at 22 ℃ for 15 min

+7ml of nisine/EDTA solution or nisine/EDTA/NOG solution

Filtration was carried out with a carbon filter having a pore size of 0.4 μm

III analysis and results

After filtration, the filters were analyzed by cytometry in solid phase and the results expressed as the number of fluorescent bacteria.

The results of this test, in which the composition of the reaction environment with N-octyl glucopyranoside as the reactant for the penetration of the microbial cells, combined with nisine 0.2. mu.g/ml and EDTA7.5mM, improved the marking of Staphylococcus epidermidis (gram-positive) and of Pseudomonas aeruginosa (gram-negative), are shown in FIG. 7.

It was determined that the addition of 0.25% and 0.5% of N-octyl glucopyranoside had a positive effect on the marking of staphylococcus epidermidis (gram-positive) and pseudomonas aeruginosa (gram-negative).

7-labeling in the Presence of Chlorhexidine

To facilitate penetration of the bacterial labeling agent, tests were conducted using only chlorhexidine as the permeabilizing agent.

I reactants

Marking solution

1/2000 PicoGreen solution (molecular probes) was prepared in PBS buffer (phosphate buffer, pH7.4).

Chlorhexidine solution

Chlorhexidine diacetate 5%: 1g of distilled water dissolved in 20ml of distilled water

50. 25 or 10 μ l of 5% chlorhexidine diacetate was dissolved in 50ml distilled water to obtain 0.01%; 0.005% or 0.001% concentration range.

Bacterial suspensions prepared in PBS, platelet concentrate and autologous plasma

Escherichia coli (CIP105901)

Staphylococcus epidermidis (CIP68.21)

Serratia marcescens (CIP 103716)

Pseudomonas aeruginosa (CIP 76110)

Adjusting the article to obtain 10³Bacteria/ml suspension

II Process

1.2ml of marking solution

+3ml of bacterial suspension

Incubation at 22 ℃ for 15 min

+7ml chlorohexidine solutions of different concentrations

Filtration was carried out with a carbon filter having a pore size of 0.4 μm

III analysis and results

After filtration, the filters were analyzed by cytometry in solid phase and the results expressed as the number of fluorescent bacteria. Counting was performed on a Petri dish (Petri dish) at 48 hours using the reference method.

The results of this experiment with a composition comprising chlorohexidine as cell penetration reactant to improve the marked reaction environment of Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens (gram-negative bacterial strains) and Staphylococcus epidermidis are shown in FIG. 8.

It was determined that the optimum concentration of chlorohexidine for the detection of gram-negative bacteria was 0.005%. However, this concentration is toxic to gram-positive bacteria and can destroy them.

From fig. 9, it can be determined that the presence of plasma antagonizes the effect of chlorohexidine on the cell penetration of the pseudomonas aeruginosa marker. Chlorohexidine alone cannot be used for universal labeling in different environments including biological fluids.

Combination of 8-chlorohexidine and N-octyl glucopyranoside

I reactants

Marking solution

1/2000 PicoGreen solution (molecular probes) was prepared in PBS buffer (phosphate buffer, pH7.4).

Chlorhexidine/N octyl glucopyranoside solutions

Chlorhexidine diacetate 5%: 1g of distilled water dissolved in 20ml of distilled water

1% of N-octyl glucopyranoside: 0.5g of the extract was dissolved in 50ml of distilled water

50 or 25. mu.l of chlorhexidine diacetate 1% (final concentration 0.001% or 0.0005%)

+ N octyl glucopyranoside 5% (final concentration 0.25%)

QSP 50ml distilled water

Bacterial suspensions prepared in PBS and mechanical platelet (Apheresis Platelets) concentrates

Escherichia coli (CIP105901)

Staphylococcus epidermidis (CIP68.21)

Serratia marcescens (CIP 103716)

Pseudomonas aeruginosa (CIP 76110)

Adjusting the article to obtain 10³Bacteria/ml suspension

II Process

1.2ml of marking solution

+3ml of bacterial suspension

Incubation at 22 ℃ for 15 min

+7ml chlorohexidine/NOG solution

Filtration on a charcoal filter with 0.4 μm pores

III analysis and results

After filtration, the filters were analyzed by cytometry in solid phase and the results expressed as the number of fluorescent bacteria.

The test results showing the labeling of DA and the detection of labeled bacteria in the presence of a composition containing a reactive environment of chlorohexidine and NOG used to enhance the penetration ability and penetration of the label are shown in fig. 10A and 10B.

It can be seen that the best marking effect of the bacteria is obtained at the highest concentration of chlorohexidine.

9-labeling in the Presence of PEI Only

I reactants

Marking solution

A PicoGreen solution of 1/2000 (molecular probes) was prepared in PBS buffer (phosphate buffer, pH7.4) and PEI was added to obtain final concentrations of 40, 80, 100, 120, 140 and 160. mu.g/ml.

Bacterial suspensions prepared in PBS

Serratia marcescens (CIP 103716)

Sample for analysis

Bacterial suspension was diluted > 1/20 in platelet concentrate samples to obtain 10⁴Final concentration of Serratia marcescens/ml.

II Process

1.2ml of marking solution

+3ml samples incubated for 45 minutes at 23 ℃

Filtration at 5 μm (PALL filter 32mm)

Incubate for 20 min in 7ml PBS

Filtration was carried out with a porosity of 0.4. mu.m.

III analysis and results

After filtration, the filters were analyzed by cytometry in solid phase and the results expressed as the number of fluorescent bacteria detected.

The results of this experiment showing the effect of different concentrations of PEI on the detection of Serratia marcescens using fluorescent DNA markers are shown in FIG. 11.

It was determined that the concentration of PEI ranges from 40 to 100. mu.g/ml for obtaining the best bacterial detection of the bacteria.

Combination of 10-nisine/EDTA/N octyl glucopyranoside/chlorohexidine/PEI

The goal of this assay was to determine the optimal concentration range of PEI for the labeling of E.coli

I reactants

Marking solution

1/2000 PicoGreen solution (molecular probes) was prepared in PBS buffer (phosphate buffer, pH7.4) and PEI was added to obtain final concentrations of 100, 80 and 60. mu.g/ml.

Chlorhexidine/N octyl glucopyranoside/EDTA solution

0.5% Chlorhexidine diacetate 500. mu.l (final concentration of Chlorhexidine diacetate 5X 10)^-3％)

+1ml or 500. mu.l 25% N-octyl glucopyranoside (final concentration of N-octyl glucopyranoside 0.5 or 0.25%)

+ 500. mu.l nisine 20. mu.g/ml (final nisine concentration 0.2. mu.g/ml)

+ 500. mu.l of 0.5M EDTA (final EDTA concentration 5mM)

QSP 50ml PBS

Preparation of bacterial suspensions in PBS

Escherichia coli (CIP105901) with a concentration of 10⁴Bacteria/ml.

Sample for analysis

3ml of bacterial suspension +27ml of platelet concentrate or bacterial suspension diluted 1/10 in a sample of platelet concentrate to obtain a final concentration of bacteria of 10⁵/ml。

II Process

1.2ml PEI labeling solution with a concentration of 60, 80 or 100. mu.g/ml, respectively

+3ml sample

Incubation at 23 ℃ for 45 min

Filtration at 5 μm (PALL filter 32mm)

Incubation in 7ml of cell penetrating solution of 0.5% or 0.25% NOG for 20 min

Filtration through a 0.4 μm pore (Whatman monocolor s carbon filter).

III analysis and results

After filtration, the filters were analyzed by cytometry in solid phase and the results expressed as the number of fluorescent bacteria detected.

The results of this experiment showing the effect of PEI on DNA labeling and fluorescence detection of E.coli are shown in FIG. 12.

It was determined that, whatever the concentration of NOG, a PEI concentration of 60. mu.g/ml was the optimal concentration for the penetration of the DNA marker.

11-general labeling of bacteria under different environments

I reactants

Marking solution

1/2000 PicoGreen solution (molecular probes) was prepared in PBS buffer (phosphate buffer, pH7.4) and PEI was added to obtain a final concentration of 60. mu.g/ml.

Chlorhexidine N octyl glucopyranoside/EDTA/nisine solutions

0.5% Chlorhexidine diacetate 500. mu.l (final concentration of Chlorhexidine diacetate 5X 10)^-3％)

+ 500. mu.l 25% N-octyl glucopyranoside (final concentration of N-octyl glucopyranoside 0.25%)

+ 500. mu.l nisine 20. mu.g/ml (final nisine concentration 0.2. mu.g/ml)

+ 500. mu.l of 0.5M EDTA (final EDTA concentration 5mM)

QSP 50ml PBS

Bacterial suspension prepared in PBS

Escherichia coli (CIP105901) with a concentration of 10⁴Bacteria/ml.

Staphylococcus epidermidis (68.21)

Sample for analysis

In biological fluidsBacterial suspension was diluted 1/10 in the sample to obtain a final bacterial concentration of 10⁵The ratio of/ml or:

3ml of bacterial suspension +27ml of distilled water

3ml bacterial suspension +27ml PBS

3ml bacterial suspension +27ml culture environment (tryptone soya broth)

3ml bacterial suspension +27ml human plasma

3ml bacterial suspension +27ml platelet concentrate

II Process

1.2ml of the marking solution +3ml of the sample

Incubation at 23 ℃ for 45 min

Filtration at 5 μm, incubation for 20 min in 7ml of cell penetrating solution

Filtration was carried out with a porosity of 0.4. mu.m.

III analysis and results

After filtration, the filters were analyzed by cytometry in solid phase and the results expressed as the number of fluorescent bacteria detected.

The results of this experiment showing the detection of Staphylococcus epidermidis and Escherichia coli in different environments are shown in FIG. 13.

The protocol defined in this way allows the detection of gram-positive and negative bacteria in different ionic, culture and physiological environments. This detection is equivalent for both types of bacteria.

Reference to the literature

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Fazii P，Ciancaglini E，Riario Sforza G Differential fluorescent stainingmethod for detection of bacteria in blood cultures，cerebrospinal fluid andother clinical specimens.Eur J Clin Microbiol Infect Dis 2002 May；21(5)：373-8.

Mirrett S，Lauer BA，Miller G A，Reller LB.Comparison of acridineorange，methylene blue，and Gram stains for blood cultures.J ClinMicrobiol 1982 Apr；15(4)：562-6

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Claims (25)

1. A method for detecting microorganisms which may be present in a biological fluid, characterized in that

a) A sample of the biological fluid is taken,

b) contacting the sample with a reaction environment for labelling microorganisms, the reaction environment comprising a labelling agent and a cell-penetrating reactant which penetrates the membranes of the microorganisms,

c) filtering the sample on a filter capable of retaining the labelled microorganisms that may be present in the sample, and

d) detecting the microorganisms which are labeled and which remain on the filter in step c).

2. The method of claim 1, wherein said labeling agent is an intercalator compound of DNA.

3. The method of claim 2, wherein the DNA intercalator is selected from the group consisting of cyanine derivatives, propidium iodide, acridine orange and ethidium bromide.

4. Method according to claim 3, characterized in that the cyanine derivative is selected from the group consisting of PicoGreen, SYBR Green and YOPRO 1.

5. Method according to claim 3, characterized in that the concentration of cyanine derivatives comprises from 0.001% to 0.5%, preferably from 0.003% to 0.05%, and the concentration of propidium iodide, acridine orange or ethidium bromide comprises from 0.1 μ g/ml to 100 μ g/ml, preferably from 1 μ g/ml to 40 μ g/ml.

6. The method according to any one of claims 1 to 5, characterized in that the reactant of cell penetration of the microorganism is a solution comprising at least a mixture of at least a permeabilizing agent, a detergent, an ion chelating agent and a preservative.

7. Method according to claim 6, characterized in that the ratio between permeabilizing agent, detergent, ion chelating agent and preservative in the final reactant in said cell penetration reactant is comprised from 1 x 10^-4％/0.03％/0.02％/6×10^-4% to 2.5X 10^-5％/0.8％/0.6％/0.015％。

8. The method according to claim 6 or 7, characterized in that said permeabilizing agent is selected from the group consisting of polyethylene glycol (PEG), digitonin, monensin, Polyethylenimine (PEI), sodium hexametaphosphate and benzalkonium chloride.

9. Method according to claim 8, characterized in that the concentration of PEG comprises from 0.01% to 1%, preferably from 0.05% to 0.5%, the concentration of digitonin comprises from 0.01 μ g/ml to 10 μ g/ml, preferably from 0.05 μ g/ml to 5 μ g/ml, the concentration of monensin comprises from 0.1 μ g/ml to 5 μ g/ml, preferably from 0.5 μ g/ml to 1 μ g/ml, the concentration of PEI comprises from 1 μ g/ml to 400 μ g/ml, preferably from 5 μ g/ml to 120 μ g/ml, the concentration of sodium hexamethylphosphate comprises from 0.005% to 1%, preferably from 0.01% to 0.1%, the concentration of benzalkonium chloride comprises from 0.001% to 0.1%, preferably from 0.005% to 0.05%.

10. The method according to any one of claims 6 to 9, wherein the detergent is selected from the group comprising N-octyl- β -D-glucopyranoside (NOG), saponin, Tween, Triton, Igepal and CHAPS.

11. Method according to claim 10, characterized in that the concentration of saponin or Tween comprises from 0.005% to 10%, preferably from 0.05% to 0.5%, the concentration of NOG comprises from 0.01% to 10%, preferably from 0.1% to 0.5%, the concentration of Triton comprises from 0.0001% to 0.05%, preferably from 0.0008% to 0.002%, and the concentration of Igepal comprises from 0.01% to 20%, preferably from 1% to 5%.

12. Method according to any one of claims 5 to 11, characterized in that the ion chelating agent is selected from the group comprising EDTA and EGTA.

13. Method according to claim 12, characterized in that the concentration of EDTA comprises from 0.1mM to 50mM, preferably from 0.2mM to 7.5 mM.

14. Method according to any one of claims 5 to 13, characterized in that the preservative is selected from the group comprising: pyrrolidone iodine, cetrimide, tea tree oil, terpinen-4-ol, chlorohexidine, polymyxin B and rifampicin.

15. Method according to claim 14, characterized in that the concentration of chlorohexidine comprises from 0.0005% to 0.05%, preferably from 0.001% to 0.05%, the concentration of cetrimide comprises from 0.01% to 5%, preferably from 0.05% to 1%, the concentration of pyrrolidone iodine comprises from 0.0001% to 0.001%, preferably from 0.0005% to 0.005%, the concentration of tea tree oil comprises from 0.0001% to 0.1%, preferably from 0.0005% to 0.05%, the concentration of terpinen-4-ol comprises from 0.05% to 10%, preferably from 0.5% to 5%, the concentration of polymyxin B and rifampicin comprises from 0.1 μ g/ml to 100 μ g/ml, preferably from 1 μ g/ml to 50 μ g/ml.

16. The method of any one of claims 1 to 15, wherein the permeation reagent further comprises an enzyme or a bacteriocin.

17. The method according to claim 16, characterized in that the enzyme is lysozyme, the lysozyme concentration preferably being comprised from 0.5 μ g/ml to 200 μ g/ml, particularly preferably from 0.05 μ g/ml to 20 μ g/ml.

18. Method according to claim 16, characterized in that the bacteriocin is nisin, the concentration of nisin preferably comprising from 0.005 μ g/ml to 10 μ g/ml, particularly preferably from 0.005 μ g/ml to 0.05 μ g/ml.

19. The method according to any one of claims 1 to 18, characterized in that the penetration reactants further comprise a cryoprotecting agent, such as DMSO or ions (NaCl, KCl,MgCl₂sodium hypochlorite) or sucrose.

20. Method according to claim 19, characterized in that the concentration of DMSO comprises from 0.05% to 20%, preferably from 0.5% to 5%, the concentration of sucrose comprises from 0.5% to 70%, preferably from 5% to 20%, and the concentration of sodium hypochlorite comprises from 0.001% to 5%, preferably from 0.005% to 0.5%.

21. The method according to any one of claims 1 to 20, characterized in that step b) of the method for detecting microorganisms can be achieved by two sub-steps b ') and b "), wherein step b') consists in contacting the sample with a reaction environment comprising a labeling reagent and a osmopolymer selected from polyethylene glycol (PEG) or Polyethylenimine (PEI), preferably Polyethylenimine (PEI), and step b") consists in adding to the reaction environment a mixture comprising at least one detergent, one ion chelating agent, one preservative and another permeabilizing agent selected from nisin, digitonin, sodium hexametaphosphate and benzalkonium chloride.

22. Reaction environment for labelling microorganisms, characterized in that it comprises a labelling agent and at least one cell-penetrating reactant of these microorganisms.

23. The reaction environment according to claim 22, characterized in that said labeling reagent is according to any one of claims 2 to 5.

24. The reaction environment according to claim 22, characterized in that the cell-penetrating reactants of said microorganisms are as defined in any one of claims 6 to 21.

25. A cell-penetrating reactant comprising

PicoGreen (molecular Probe) from-1/22000;

-PEI at a final concentration of 5.5 μ g/ml;

final concentration of 4.5X 10^-4% of chlorhexidine diacetate;

-N-octyl glucopyranoside at a final concentration of 0.16%;

nisin at a final concentration of 0.018 μ g/ml;

-EDTA at a final concentration of 0.45 mM;

phosphate buffer (PPS) in an amount sufficient to achieve the desired final volume.