WO2014118544A1

WO2014118544A1 - Quantification of bacteria

Info

Publication number: WO2014118544A1
Application number: PCT/GB2014/050250
Authority: WO
Inventors: Anthony Robert STEWART; Peter Salmon
Original assignee: Blood Analysis Ltd
Priority date: 2013-01-31
Filing date: 2014-01-30
Publication date: 2014-08-07
Also published as: GB2510365A; GB201301722D0

Abstract

A method for treating bacteria in order to improve the uptake and retention of fluorescent dyes and so to allow accurate quantitative fluorescent analysis of said bacteria involves applying heat to bacteria in the presence of a fluorescent dye.

Description

QUANTIFICATION OF BACTERIA

Description

FIELD OF THE INVENTION

[0001] The present invention relates to a method for quantifying bacteria in a sample. In particular, the present invention relates to a method for quantifying bacteria using fluorescent imaging techniques.

BACKGROUND TO THE INVENTION

[0002] Fluorescent imaging and quantification of bacteria is a rapid analytical technique that can replace traditional time-consuming culture, and can be used for the identification of non-culturable or slow-growing organisms. In particular, nucleotide staining with fluorescent dyes or probes permits universal detection of all species of microbes in a single test.

[0003] Fluorescent dyes may be cell-permeable or cell-impermeable; in either case, one issue associated with their use in staining microbes is a lack of retention of the stain by the cell during the analytical process. Bacteria possess a group of efficient efflux pumps responsible for the extrusion of antibiotics and other potentially toxic chemicals from the cell. These bacterial efflux pumps are however equally effective at removing nucleotide stains from within the bacteria and can therefore reduce the accuracy of detection and enumeration of bacteria in a sample population by fluorescence-based detection systems.

[0004] There are five major classes of bacterial efflux pumps, some of which are energy dependent while others are coupled to an electrochemical potential across a cell wall. For the purpose of quantifying bacteria using fluorescent imaging techniques, it would be useful to inhibit the activity of the bacterial efflux pumps and so improve the accuracy of detection and enumeration of bacteria. However, the different mechanisms of the bacterial efflux pumps makes it problematic to design a solution which inhibits all major classes of bacterial efflux pump mechanism, and has a universal application to all species. [0005] Various chemical reagents are known to be effective at inhibiting or reducing bacterial efflux pump activity. For example, it is known that phenothiazines inhibit efflux pumps mediated by P-glycoprotein in Escherichia coli (Molnar et al (1997) "Inhibition of the transport function of membrane proteins by some substituted phenothiazines in E. coli and multidrug resistant tumour cells" (Anticancer Research, 17: 481)). Calcium antagonists including Verapamil are known as inhibitors of ATP-dependent efflux pumps (Choudhuri et al (1999) "Isoniazid accumulation in Mycobacterium smegmatis is modulated by proton motive force driven and ATP dependent extrusion systems" (Biochemical & Biophysics Research Communications, 256: 682)). Proton pump inhibitors including Omeprazole are known to inhibit the NorA efflux pump (Aeschlimann et al (1999) "The effects of NorA inhibition of the activities of levofloxacin, ciprofloxacin and norfloxacin against two genetically related strains of

Staphylococcus aureus" (Journal of Antimicrobial Chemotherapy, 44: 343)). Piperine analoges are known to inhibit the NorA efflux pump (Sangwan et al (2008) "Piperine analogues as potent Staphylococcus aureus NorA efflux pump inhibitors" (Bioorganic & Medicinal chemistry, 16: 9847)). Reserpine is known to inhibit the NorC efflux pump (Patel et al (2010) "Ethidium bromide MIC screening for enhanced efflux pump gene expression or efflux activity in

Staphylococcus aureus" (Antimicrobial Agents & Chemotherapy, 54: 5070)). Phenylalanyl-arginyl- -naphthylamide competes as a pump substrate for the MexAB efflux pump (Lemovskaya et al. (2001) - "Identification and

characterisation of inhibitors of multidrug resistance efflux pump in Pseudomonas aeruginosa: novel agents for combination therapy" (Antimicrobial Agents & Chemotherapy, 45: 105)). Molecules bearing a trichloromethylamine group are known to inhibit the NorA efflux pump (Markam et al (1999) "Multiple novel inhibitors of the NorA multidrug transporter of Staphyloccoccus aureus"

(Antimicrobial Agents & Chemotherapy, 43 : 2404)).

SUMMARY OF THE INVENTION

[0006] In the quantitative fluorescent analysis of bacteria there will only be a small signal for detection, given the small size of most microbes. Efflux of stain from the bacteria significantly reduces the intensity of the light signals emitted, and can result in a signal below the threshold of detection by even the most sensitive photodetectors. There is therefore a need for means for inhibiting the active efflux systems and maintaining cell structure in order to enhance the accuracy of quantitative fluorescent analysis methods.

[0007] In accordance with the present invention, a method for treating bacteria in order to improve the uptake and retention of fluorescent dyes and so to allow accurate quantitative fluorescent analysis of said bacteria involves applying heat to bacteria, in the presence of a fluorescent dye.

DISCLOSURE OF THE INVENTION

[0008] In a first aspect, the present invention provides a method for treating bacteria for quantitative fluorescent analysis, which method comprises subjecting a bacterial sample for analysis to a heating and cooling cycle in the presence of a fluorescent dye, which heating and cooling cycle comprises heating the bacterial sample to a first predetermined temperature; holding said first predetermined temperature for a specified period of time; and cooling the bacterial sample to a second predetermined temperature for analysis.

[0009] The bacterial sample so treated retains the fluorescent dye and facilitates accurate quantitative analysis of said bacteria.

[0010] In a second aspect, the present invention provides a method for the quantitative analysis of bacteria in a sample, which method comprises:

1. obtaining a bacterial sample for analysis;

2. mixing said bacterial sample with a fluorescent dye;

3. heating said bacterial sample to a first predetermined temperature;

4. holding the predetermined temperature for a specified period of time;

5. cooling the bacterial sample to a second predetermined temperature;

6. analysing the cooled bacterial sample at said second predetermined temperature, to obtain a bacterial count.

[0011] Preferably, the bacterial sample is heated to a first predetermined temperature of at least 35°C and up to 95°C; for example at least 35°C and up to 90°C; or at least 35°C and up to 70°C. For example, the bacterial sample may be heated to a first predetermined temperature of up to 90°; up to 85°C; up to 80°C, up to 75°C, up to 70°C, up to 65°C; up to 60°C; up to 55°C, or up to 50°C; for example, the bacterial sample may be heated to a first predetermined temperature of at least 35°C, at least 40°C, at least 45°C, or at least 50°C. Preferably, the bacterial sample is heated to a first predetermined temperature of 45 °C to 70 °C; more preferably 50 °C to 70 °C. Alternatively, the bacterial sample is heated to a first predetermined temperature of 50°C to 90°C. Optimally, the bacterial sample is heated to a first predetermined temperature of around 50°C. Alternatively, the bacterial sample is heated to a first predetermined temperature of around 70°C.

[0012] Preferably, a method according to the present invention is initiated at room temperature. Once the desired first predetermined temperature is reached, said temperature is maintained for a specified period of preferably at least 0.5 minutes and up to 5.0 minutes; for example, the temperature may be maintained for a specified period of at least 1.0 minute; at least 1.5 minutes; at least 2.0 minutes; at least 2.5 minutes; or at least 3.0 minutes; for example the temperature may be maintained for a specified period of up to 4.5 minutes; up to 4.0 minutes; up to 3.5 minutes; or up to 3.0 minutes. Preferably, the first predetermined temperature is maintained for a period of 2.5-5.0 minutes; more preferably 2.5-4.0 minutes. Optimally, the temperature is maintained for a specified period of around 3.0 minutes.

[0013] At the end of the desired heating period, the bacterial sample is cooled to a second predetermined temperature of room temperature or lower. For example, the cooling may be to a second predetermined temperature of around 10°C, or around 4°C; or to a temperature of between 4°C and 10°C.

[0014] Heating may be achieved by any suitable means, for example, by using a thermal cycler of standard laboratory specification, programmed to increase the temperature to a first pre-determined point for a specified period of time, followed by a decrease to a second pre-determined hold temperature.

[0015] The fluorescent dye may be any fluorescent dye suitable for staining nucleotides, including, for example, any of the following: a cyanine dye (for example, "Picogreen"); an amine reactive dye (for example, 6-((7-amino-4- methylcoumarin-3-acetyl)amino)hexanoic acid; succinimydl ester); an intercalating dye (for example, ethidium bromide; a DNA major groove dye (for example, methylene green); a DNA minor groove dye (for example, Hoechst 33258); and other dyes reactive with nucleotides within the bacterial cell (for example, 7-actinomycin D).

[0016] In one embodiment, a method according to the present invention may be carried out in the presence of a bacterial cell wall permeability agent. By "bacterial cell wall permeability agent" is meant any chemical reagent that can influence the uptake of cell impermeable dyes, for example as discussed in Helander EVI & Mattila-Sandholm T (2000) "Fluorometric assessment of Gram negative bacterial permiabilization". (Journal of Applied Microbiology, 88: 213).

[0017] Suitable bacterial cell wall permeability agents include, for example, any of the following: ethylene diamine tetra-acetic acid (EDTA); an ionic detergent (for example, sodium dodecyl sulphate); a non-ionic detergent (for example, Triton X-100); a Zwitterionic detergent (for example, 3-((3- cholamidopropyl)dimethylammonio)-l-propansulphonate); various alcohols (for example, propan-2-ol); certain alkanes (for example, hexane); and enzymes such as lysozyme and achromopeptidase .

[0018] The source of the bacterial sample for use in the present invention may include, but is not limited to, a clinical sample, a food sample, a water sample, and an air sample. As one example, the bacterial sample may be a mammalian fluid such as blood or a blood product.

[0019] In the method of the second aspect of the present invention, the bacterial count may be carried out using methods known in the art. Suitable methods are those employing means to excite the fluorescent dye with one wavelength of light and detecting the emitted light for the bacteria bound by dye at a second, different, wavelength. Such methods include, for example, flow cytometry, laser scanning cytometry, and microscopy involving the imaging of fluorescent light, but are not limited thereto.

EXAMPLES

Example 1

Overnight cultures of bacteria were diluted in ringer's solution (quarter strength), to obtain a working concentration, and then re-suspended in stain solution (lOmM HEPES, 30mM EDTA, lmM dithiothreitol 1 : 1000 dilution Picogreen). Two hundred microlitres of bacteria in stain solution were placed in a thermal cycler (MJ Research, DNA engine tetrad) with a programme to increase the temperature to a first pre-determined point for a specified period of three minutes, followed by a decrease to a second predetermined hold temperature of 10°C, with maximum ramping. The samples were analysed in either a laser scanning cytometer

(Mirrorball, TTP LabTech) or flow cytometer (Cube6, Partec), both with standard 488nm lasers for excitation. The scanning laser cytometer used a detection channel of wavelength 488 - 540 to count particles on size and fluorescence intensity (FL1). In flow cytometry, bacteria were detected and gated on forward scatter (FSC), side scatter (SSC) and FL1 channels. Bacterial counts were confirmed by colony forming unit (CFU) pour plate cultures.

To determine the optimum temperature which would maximise the sensitivity of the staining procedure, a series of bacterial samples were subject to a range of first predetermined temperatures from 25°C to 90°C in the presence of stain solution. Samples were analysed by flow cytometry. The results for ten different species of bacteria are given below in table la.

TABLE la

Species 3 min @ 3 min @ 3 min @ 3 min @ 3 min @

25°C 35°C 50°C 70°C 90°C

% % % % % increase increase increase increase increase over no over no over no over no over no heat heat heat heat heat treatment treatment treatment treatment treatment

Klebsiella < > 12 44? 434 437

Klebsiella 6 87 527 497 495

2054 20- >7 258 270 274 marcescens

Escheriachia 0 0 0 0 0 coli Staphylococcus 111 III 111 aureus

Pseudomonas 0

aeruginosa

Bacterial samples were subject to a range of first predetermined temperatures from 35°C to 70°C in the presence of stain solution. Samples were analysed by flow cytometry. The results for Serratia marcescens are given below in Table lb.

TABLE lb

Temperature (°C) Bacteria / ml

50 1.98 x 10

II II I D!

On the basis of the above, a temperature of 50°C was identified as optimal.

To determine the optimum time period for maintaining said temperature, bacterial samples were treated as above for varying lengths of time. The results for Morganella morganii and Escherichia coli are given below in Table 2.

TABLE 2

TIME PERIOD ISmlriiii ml: Bacteria / ml:

at 50 °C Morganella mt/rganii Escherichia coli

5 min 10126 13899

4 min 14433 14208

3.5 min 14551 11539

3 min 14643 14649

2.5 min I2<"44

2 min 11054 11488

1.5 min IT62

1 min 7871 8291

0.5 min 2481 ⁷⁽¹⁴

0 min 156 4038

Further species of bacteria were treated and analysed in the same way. The results of bacterial counts are presented in Table 3 below.

TABLE 3

Species No heat 3 min at 50 °C % increase treatment

Klebsiella oxytoca 3 1 \ 1-·' 2.1 \ Hi' 6674%

Enterobacter cloacae 2.11 \ ll 2.54x 10' 1103%

Escherichia c li 2 ι>χ Ιι·" 2.2 \ 1ιι' 10% Heat pre-treatment experiments were repeated using a laser scanning cytometer on a range of bacteria to confirm the above results obtained by flow cytometry. These results are presented in Table 4.

TABLE 4

Species No heat 3 min at 50°C % increase treatment

Staphylococcus aureus 1 I h l u ¹ 14" ,

Morganella morganii 29 1 .55 \ I D¹ 534^>%

pneumoniae

The results show that heat treatment of bacteria in the presence of a cell permeant DNA stain increases the sensitivity of cytometric analysis to quantify the bacteria. This effect is demonstrable even when using cell permeant dyes to stain the bacteria. The heat treatment does not destroy or alter the other physical characteristics of bacteria in cytometric analysis. They have an identical FSC and SSC to non-heat treated bacteria. Pre-heat treatment for a short period offers an effective means to increase the sensitivity of a bacterial analysis assay.

Claims

1. A method for treating bacteria for quantitative fluorescent analysis, comprising subjecting a bacterial sample for analysis to a heating and cooling cycle in the presence of a fluorescent dye, which heating and cooling cycle comprises heating the bacterial sample to a first predetermined temperature; holding said first predetermined temperature for a specified period of time; and cooling the bacterial sample to a second predetermined temperature for analysis.

2. A method for the quantitative analysis of bacteria in a sample, which method comprises:

1. obtaining a bacterial sample for analysis;

2. mixing said bacterial sample with a fluorescent dye;

3. heating said bacterial sample to a first predetermined temperature;

4. holding the predetermined temperature for a specified period of time;

5. cooling the bacterial sample to a second predetermined temperature;

3. A method as claimed in claim 1 or claim 2, wherein the bacterial sample is heated to a first predetermined temperature of up to 90°C.

4. A method as claimed in any one of claims 1 to 3, wherein the bacterial sample is heated to a first predetermined temperature of at least 35°C.

5. A method as claimed in any one of claims 1 to 4, wherein the bacterial sample is heated to a first predetermined temperature of up to 70°C.

6. A method as claimed in any one of claims 1 to 5, wherein the bacterial sample is heated to a first predetermined temperature of 45 °C to 70 °C.

7. A method as claimed in any one of claims 1 to 4 wherein the bacterial sample is heated to a first predetermined temperature of 50°C to 90°C.

8. A method as claimed in any one of claims 1 to 7, wherein the bacterial sample is heated to a first predetermined temperature of 50 °C to 70 °C.

9. A method as claimed in claim 7, wherein the bacterial sample is heated to a first predetermined temperature of 70°C to 90°C.

10. A method as claimed in any one of claims 1 to 9, wherein the bacterial sample is heated to a first predetermined temperature of approximately 70°C.

11. A method as claimed in any one of claims 1 to 8, wherein the bacterial sample is heated to a first predetermined temperature of approximately 50°C.

12. A method as claimed in any one of claims 1 to 11, wherein the specified period of heating is at least 0.5 minutes.

13. A method as claimed in any one of claims 1 to 12, wherein the specified period of heating is up to 5 minutes.

14. A method as claimed in any one of claims 1 to 13, wherein the specified period of heating is from 2.5 minutes to 5 minutes.

15. A method as claimed in any one of claims 1 to 14, wherein the specified period of heating is from 2.5 minutes to 4 minutes.

16. A method as claimed in any one of claims 1 to 15, wherein the specified period of heating is approximately 3 minutes.

17. A method as claimed in any one of claims 1 to 16, wherein, after the heating period, the bacterial sample is cooled to a second predetermined temperature of room temperature or below.

18. A method as claimed in claim 17, wherein the bacterial sample is cooled to a second predetermined temperature of approximately 4°C to approximately 10°C.

19. A method as claimed in claim 17 or claim 18, wherein the bacterial sample is cooled to a second predetermined temperature of approximately 10°C.