Device and method for automated antibiotic susceptibility testing of gram-negative bacteria

Abstract

An antibiotic susceptibility testing device of gram-negative bacteria, as well as a corresponding method, are discussed. The device has a temperature control unit (including a constant temperature chamber) and a contactless conductivity-based measurement system. Disposable glassy or PVC tubes are used as test vessels for AST. In the performance of AST assay, appropriate kind of liquid medium containing identical amount of target bacterial cells and target antibiotics at different concentrations are loaded into test tubes, following by incubation in the device at a setup temperature. The bacterial growth profile is monitored by collecting the differential values (ΔC) of conductivity of liquid medium, which depend on the proliferation of viable cells. Outcome of ΔC indicates whether the target bacterial cells are completely inhibited by the test antibiotic or not, enabling the user to judge the value of the minimal inhibitory concentration (MIC) simply.

Claims

1. A method for antibiotic susceptibility testing (AST) of Gram-negative bacteria with a device having a temperature control unit, a plurality of sterilized disposable test tubes, and a plurality of disposable syringe filters, the method comprising: assembling a plurality of test channels vertically in the temperature control unit, each of the plurality of test channels including an actuator electrode, a first pick-up electrode and a second pick-up electrode, coaxially fixed with a fixing plate; preparing a liquid medium containing bacterial cells to be measured; preparing a solution of target antibiotic; loading the liquid medium and the target antibiotic solution into each of the plurality of sterilized disposable test tubes, and covering an opening of the test tube with a disposable syringe filter; setting parameters of a capacitive coupled contactless conductivity detector: with an excitation amplitude of 1000 V, an excitation frequency of 1.6 MHz, a collection period of conductivity value of 1s and total collection times of 20,000 to 30,000; and respectively inserting the plurality of sterilized disposable test tubes into each of the plurality of test channels and switching on the capacitive coupled contactless conductivity detector to collect differential values (ΔCs) between a first coupling signal of capacitance and resistor (C-Rc L) and a second coupling signal of capacitance and resistor (C-Rc S).

2. The method for AST according to claim 1, wherein the Gram-negative bacteria species includes Helicobacter pylori, Escherichia coli, Shigella, Pneumobacillus, Yersinia, Acinetobacter, Legionella pneumophila, Bordetella pertussis, and Vibrio cholerae.

3. The method for AST according to claim 1, wherein the antibiotic species includes at least one of cefazolin, cefepime, cefotetan, ceftazidime, ceftriaxone, ciprofloxacin, gentamicin, levofloxacin, nitrofurantoin, cotrimoxazole, amoxicillin or clarithromycin.

4. The method for AST according to claim 1, wherein in the temperature control unit, temperature is adjusted to a desired degree over the range of 32 degrees Fahrenheit to 158 degrees Fahrenheit, with a nonidentity of 32.9 degrees Fahrenheit.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In order to more clearly illustrate the embodiments of the present invention, brief statement is provided here for the drawings. These drawings are aiming to help readers to understand some of the embodiments of the present invention. Whereas, technicians in this field can create more relevant drawings based on these drawings, rather than a creative contribution.

(2) FIG. 1 schematically shows the structure of the device in accordance with an embodiment of the present invention, wherein 1: disposable syringe filter; 2: pick-up electrode L; 3: pick-up electrode S; 4: actuator electrode A; 5: disposable test tube; 6: constant temperature chamber; 7: fixing plate.

(3) FIG. 2 schematically shows the struction of the device with a capacitively coupled contactless conductivity detector and a computer in accordance with an embodiment of the present invention.

(4) FIG. 3 schematically shows a flowchart of a method for AST of gram-negative bacteria.

DETAILED DESCRIPTION

(5) The assembly and application method of the present invention will be further stated below by means of embodiments. Apparently, the discussed embodiments are examples of the present invention, rather than all of the embodiments. Other embodiments obtained by technicians in this field without creative work are in the scope of protection.

(6) As shown in FIG. 1, an automated device for AST assay of gram-negative bacteria comprises a temperature control unit. There is a constant temperature chamber (6) in the temperature control unit. One or a row of test channels are arranged vertically in the constant temperature chamber (6). Each test channel has an actuator electrode A (4), a pick-up electrode L (2) and a pick-up electrode S (3). These three cylinder electrodes are coaxially fixed with fixing plate (7). One-end-closed disposable glassy or PVC tubes (5) are used as test vessels. These test tubes are installed into the temperature control unit through holes in the unit cover, consequently crossing actuator electrode A (4), pick-up electrode L (2) and pick-up electrode S (3) in turn. These holes in the unit cover are coaxially above the test channels. Nozzle of the test tube is stuffed with a disposable syringe filter (1). In the temperature control unit temperature can be adjusted to a desired degree over the range of 0˜70° C. The nonidentity of temperature in the unit is within 0.5° C. All the components of the temperature control unit can be purchased in electronic component store, apart from those components formed the test channels.

(7) Furthermore, the device comprises a capacitively coupled contactless conductivity detector (shown as label 8 in FIG. 2), which is controlled by a computer (shown as label 9 in FIG. 2). The capacitively coupled contactless conductivity detector (ER815) is produced by eDAQ company (Australia), as well as the relevant TERA TERM software. The actuator electrode A, pick-up electrode L and pick-up electrode S are all linked to the capacitively coupled contactless conductivity detector.

(8) In one embodiment, the distance between the pick-up electrode L (2) and pick-up electrode S (3) is 5 mm; and the distance between the pick-up electrode S (3) and the actuator electrode A (4) is 10 mm. In every test channel the three electrodes are coaxially fixed with fixing plate (7).

(9) Specifically, in one embodiment, the actuator electrode A (4), pick-up electrode L (2) and pick-up electrode S (3) are all copper cylinders in external diameter of 4.00 mm with 0.95 mm wall thickness. Their lengths are 16 mm, 16 mm and 10 mm, respectively.

(10) In one embodiment, according to the requirement of real test work conditions and the parameters of ER815 capacitively coupled contactless conductivity detector, the number of the test channel can be 1, 8, 16, 24, or 32. Namely, 1, 8, 16, 24, or 32 test channels can be arranged in the temperature control unit.

(11) In one embodiment, the disposable test tube (5) is in length of 12±2 cm, inner diameter of 2.60 mm, and outer diameter of 3.00 mm.

(12) Preferably, 0.22 μm disposable syringe filter (1) is selected.

Example 1

AST Array of Levofloxacin Against Dysentery Bacillus

(13) Step 1: According to FIG. 1, an automated device with eight test channels is set up. An HtPot50 dry incubator, which is produced by ABSON Scientific Instruments Co. (Hefei, China), is used as a temperature control unit. The used 0.22 μm disposable syringe filter is produced by Zhejiang Aijiren INC (Quzhou, China). The actuator electrode A, pick-up electrode L and pick-up electrode S are all copper cylinders in external diameter of 4.00 mm with 0.95 mm wall thickness. Their lengths are 16 mm, 16 mm and 10 mm, respectively. In every test channel the three electrodes are coaxially fixed with fixing plate. The distance between the pick-up electrode L and pick-up electrode S is 5 mm; and the distance between the pick-up electrode S and the actuator electrode A is 10 mm. All of the electrodes of the eight test channels are linked to an ER815 capacitively coupled contactless conductivity detector (eDAQ Ltd, Australia). The capacitively coupled contactless conductivity detector is controlled with TERA TERM software installed in E470c laptop computer (Legend Holdings Corporation, China).

(14) Step 2: The temperature in the constant temperature chamber (6) is set to be 36° C. with a nonidentity ≤0.5° C. by clicking the button of temperature control.

(15) Step 3: Involved hardware, containers, tools, consumables and special liquid medium for Enterobacteriaceae are sterilized with high temperature steam (121° C.), following by cooling down to room temperature in sterile vessels. In the special liquid medium the concentrations of beef powder, proteose peptone, lactose, No. 3 bile salt, sodium citrate, sodium thiosulfate and ferric citrate are 5.0, 5.0, 10.0, 8.5, 8.5, 8.5 and 1.0 g/L, respectively.

(16) Step 4: A series of levofloxacin solutions at concentrations of 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 and 12.0 g/L are prepared with ultrapure sterile water.

(17) Step 5: Eight milliliter sterilized special liquid medium for Enterobacteriaceae is transferred into a 10 ml centrifuge tube with a pipette.

(18) Step 6: Sixteen well-definite bacterial colonies are transferred from inoculating dish, on which Enterobacteriaceae is cultured for 18-20 h, into the 10 ml centrifuge tube to prepare bacterial suspension.

(19) Step 7: One-end-closed disposable glassy tubes in length of 12 cm are used as test tubes. Their inner diameter and outer diameter are 2.60 mm and of 3.00 mm, respectively. Pipetting 995 μl bacterial suspension and 5 μl levofloxacin solutions at different concetrations into each test tube (5) to make test samples. In the final test samples the concentration of the levofloxacin is 0.25, 0.50, 1.00, 2.00, 3.00, 4.00, 5.00 and 6.00 mg/1, respectively.

(20) Step 8: A disposable syringe filter (1) is stuffed into the nozzle of every test tube (5).

(21) Step 9: Starting the application software on the laptop computer loaded with TERA TERM to set the parameters of the capacitively coupled contactless conductivity detector: excitation amplitude of 1000 V, excitation frequency of 1.6 MHz, collection period of conductivity value of 1 s and total collection times of 21600.

(22) Step 10: The 8 as-prepared test tubes loaded with test samples are inserted into 8 test channels, respectively. Switching on the capacitively coupled contactless conductivity detector to collect the differential values (ΔCs) between the C-R.sub.c L and C-R.sub.c S.

(23) Step 11: At the end of incubation, the values of ΔC obtained from No. 1, No. 2 and No. 3 test tubes are equal to or larger than 18.0 μS/cm, indicating that in the liquid medium Enterobacteriaceae grow in the presence of levofloxacin at concentrations of 0.25, 0.50 and 1.00 mg/l. Whereas, the values of ΔC obtained from No. 4, No. 5, No. 6, No. 7 and No. 8 test tubes are not more than 1.0 μS/cm, indicating that the growth of Enterobacteriaceae is completely inhibited. Therefore, the MIC of this antibiotic against the resent Enterobacteriaceae strain is 2.00 mg/l.

Example 2

Characterization of the Synergetic Effect of Levofloxacin and Clarithromycin Against Helicobacter pylori

(24) Step 1 and step 2 are the same as stated in EXAMPLE 1.

(25) Step 3: Involved hardware, containers, tools, consumables and special liquid medium for Helicobacter pylori are sterilized with high temperature steam (121° C.), following by cooling down to room temperature in sterile vessels. The special liquid medium is purchased from Qingdao Hope Bio-Technology Co., Ltd (Qingdao, China).

(26) Step 4: Levofloxacin solutions at concentrations of 0.2, 0.4, 0.6 and 0.8 g/l are prepared with ultrapure sterile water. Clarithromycin solutions at concentrations of 0.2, 0.4, 0.6 and 0.8 g/l are prepared with ethyl alcohol.

(27) Step 5: Eight milliliter sterilized special liquid medium for Helicobacter pylori is transferred into a 10 ml centrifuge tube with a pipette.

(28) Step 6: Twelve well-definite bacterial colonies are transferred from inoculating dish, on which Helicobacter pylori is cultured for 18-20 h, into the 10 ml centrifuge tube to prepare bacterial suspension.

(29) Step 7: One-end-closed disposable PVC tubes in length of 12 cm are used as test tubes (5). Their inner diameter and outer diameter are 2.60 mm and of 3.00 mm, respectively. Pipetting 995 μl bacterial suspension and 5 μl levofloxacin solutions at different concentrations into No. 1, No. 2, No. 3 and No. 4 test tube (5) to make test samples. In these final test samples the concentration of the levofloxacin is 0.10, 0.20, 0.30, and 0.40 mg/1, respectively. Pipetting 990 μl bacterial suspension, 5 μl levofloxacin solutions and 5 μl clarithromycin solutions into No. 5, No. 6, No. 7 and No. 8 test tube (5) to make the other batch of test samples. In these four test samples the concentrations of the levofloxacin and clarithromycin are both at 0.10, 0.20, 0.30, and 0.40 mg/1, respectively.

(30) Step 8: A disposable syringe filter (1) is stuffed into the nozzle of every test tube (5).

(31) Step 9: Starting the application software on the laptop computer loaded with TERA TERM to set the parameters of the capacitively coupled contactless conductivity detector: excitation amplitude of 1000 V, excitation frequency of 1.6 MHz, collection period of conductivity value of 1 s and total collection times of 28800.

(32) Step 10: The 8 as-prepared test tubes (5) loaded with test samples are inserted into 8 test channels, respectively. Switching on the capacitively coupled contactless conductivity detector to collect the differential values (ΔCs) between the C-R.sub.c L and C-R.sub.c S.

(33) Step 11: At the end of incubation, the values of ΔC obtained from No. 1, No. 2, No. 3 and No. 4 test tubes are equal to or larger than 22.0 μS/cm, indicating that in the liquid medium Helicobacter pylori grow in the presence of levofloxacin at concentrations of 0.10, 0.20, 0.30, and 0.40 mg/l. The values of ΔC obtained from No. 5 and No. 6 test tubes are equal to or larger than 17.0 μS/cm, indicating that Helicobacter pylori still grow in the presence of 0.10 and 0.20 mg/l levofloxacin, even companying with the same amount of clarithromycin. Whereas, the values of ΔC obtained from No. 7 and No. 8 test tubes are not more than 1.0 μS/cm, indicating that the growth is completely inhibited. This implies that 0.30 mg/l levofloxacin together with 0.30 mg/l clarithromycin can inhibit the growth of Helicobacter pylori.

(34) Step 12: The same disposable PVC tubes in length of 12 cm are used as test tubes (5). Pipetting 995 μl bacterial suspension and 5 μl clarithromycin solutions at different concentrations into No. 1, No. 2, No. 3 and No. 4 test tube (5) to make test samples. In these final test samples the concentration of the clarithromycin is 0.10, 0.20, 0.30, and 0.40 mg/1, respectively.

(35) Step 13: A disposable syringe filter (1) is stuffed into the nozzle of every test tube (5) as stated in Step 8.

(36) Step 14: With the same approaches as stated in Step 9 and Step 10, values of ΔCs are obtained.

(37) Step 15: At the end of incubation, the values of ΔC obtained from No. 1, No. 2, No. 3 and No. 4 test tubes are equal to or larger than 14.0 μS/cm, indicating that Helicobacter pylori grow in the presence of clarithromycin at concentrations of 0.10, 0.20, 0.30, and 0.40 mg/l.

(38) Step 16: The data obtained in Step 11 and Step 15 enable the user to draw a conclusion that there is apparently synergetic effect of levofloxacin and clarithromycin against Helicobacter pylori. Neither 0.20 mg/l levofloxacin nor 0.20 mg/l clarithromycin can work alone.

(39) It is to be understood that the above embodiments are just the preferable examples. The present invention is not limited to these embodiments. Any modification, equivalent replacement, or improvement under the spirit and principle of the present invention, are under the scope of the present invention.