Place Antibiotic Paper Circles to Determine Broad Spectrum β-Lactamase Enzyme.

augmentin (AMC) with cefotaxime (CTX), augmentin (AMC) with ceftazidime (CAZ), because the broad-spectrum β -lactamase enzyme of bacteria in these two regions is inhibited by the clavulanic acid inhibitor from the augmentin antibiotic paper ring, cefotaxime and ceftazidime are not destroyed, bacteria are killed, causing the sterile zone to expand here. This is called a synergistic image (champagne cork image).

Antibiotics :

Four antibiotic discs were selected to detect the production of broad-spectrum β -lactamase in the tested bacteria:

- Aztreonam (ATM - monobactam family).

- Cefotaxime (CTX) and Ceftazidime (CAZ) are two third-generation cephalosporins, destroyed by extended-spectrum β-lactamase enzymes.

- Augmentin (AMC) is an antibiotic that combines amoxicillin with a β -lactamase inhibitor, clavulanic acid.

Steps to follow:

Carry out the same steps as in the diffusion paper ring method. First, prepare bacterial broth with a concentration of 10 8 bacteria/ml, then dilute to a concentration of 10 6 bacteria/ml. Spread this bacterial broth onto the agar surface, remove the excess bacterial broth, and let the agar surface dry. Use a clean syringe to place 4 paper rings on the agar surface according to the diagram in Figure 4.

ATM

AMC

CTX

CAZ

Figure 2.2. Place antibiotic paper circles to determine extended -spectrum β -lactamase.

Note: AMC - augmentin, ATM - Aztreonam, CTX - cefotaxime, CAZ – ceftazidime.

After leaving at room temperature for 30 minutes to allow the antibiotics to diffuse evenly, place in an incubator at 35-37 0 C/18 hours with the agar plate inverted.

Evaluate the results

  - positive extended-spectrum β-lactamase (producing extended-spectrum β-lactamase) in cases where the bacterial strain is sensitive to AMC but resistant to CTX, CAZ, ATM and has a synergistic image at the border between the AMC and CTX, AMC and ATM or AMC and CAZ paper rings.

Mean:

- Bacteria producing extended-spectrum β-lactamase are inhibited by the inhibitor clavulanic acid, so they are sensitive to AMC.

- This β-lactamase enzyme has an extended spectrum and therefore affects third generation cephalosporins, so CTX, CAZ and ATM are all resistant or have reduced sensitivity (R or I).

- But the border area between the AMC and CTX or AMC and CAZ or AMC and ATM rings is inhibited by the inhibitor clavulanic acid, so the broad-spectrum β-lactamase enzyme does not affect the third generation cephalosporins, the bacteria are killed, creating an expanded image of the sterile ring. This is called the synergistic image (champagne cork image).

 The remaining cases are all read as negative extended-spectrum β-lactamase (do not produce extended-spectrum β-lactamase).

2.4.3.5. Determination of minimum inhibitory concentration of antibiotic ( MIC)

- Purpose : This technique aims to accurately determine the smallest concentration of antibiotic that inhibits the growth of a bacterial strain in a culture medium (quantitative method).

- Principle : The concentration of antibiotics increases gradually in the culture medium. When it reaches a certain concentration, it will inhibit the growth of bacteria and this can be determined with the naked eye.

- Technique [43], [77],[84], [87].

+ Mix concentrated antibiotic solution (“mother” solution)

+If using powdered antibiotics for laboratory use : Based on the activity of the antibiotics (listed on the bottle labels), it is necessary to calculate to mix the "mother" solution according to the following formula:

For example: The antibiotic chloramphenicol has an activity of 99.13%, if it is necessary to prepare 10 ml of the "mother" antibiotic solution with a concentration of 1600  g/ml, Xg of antibiotic will have to be weighed:

10ml x 1600

Xg antibiotic = 99.13 = 16,140.421  g/ml = 0.016140 g/ml

After weighing the powdered antibiotics, they will be mixed with the appropriate buffer solution. Each type of antibiotic will have different solvents and buffer solutions (depending on the manufacturer of the antibiotic). Many antibiotics can be dissolved and diluted in distilled water. Others need to be dissolved in other solutions.

special environment, then diluted in distilled water. The “mother” antibiotic solution after mixing is divided into test tubes, stored in a refrigerator at -20 0 C (except for the antibiotic nalidixic acid which is stored at room temperature). Once taken out for use, use within 24 hours.

+ Mix antibiotic concentrations

Mother solution

PBS buffer solution *

(or distilled water)

Mixing degree

Intermediate concentration

(  g/ml)

Final concentration

(  g/ml)**

2.5 ml (1280  g/ml)

2 ml

1 ml

0.5 ml

2ml 3ml

3.5 ml

7.5 ml

1/2

1/4

1/8

1/16

1280

640

320

160

128

2ml (80  g/ml)

1 ml

0.5 ml

0.5

2ml 3ml 3.5ml

7.5 ml

1/2

1/4

1/8

1/16

0.5

Maybe you are interested!

2ml (5  g/ml)

1 ml

0.5 ml

2ml 3ml 3.5ml

7.5 ml

1/2

1/4

1/8

1/16

2.5

1.25

0.64

0.32

0.25

0.125

0.064

0.032

2ml (0.32  g/ml)

1 ml

2ml 3ml

1/2

1/4

0.16

0.08

0.016

0.008

Note :

* PBS: Phosphate buffer solution used to dilute antibiotics.

** Final concentration diluted 1/10 in medium, calculated by mixing ratio 2.5 ml intermediate solution + 22.5 ml MH agar.

- Steps to take

Day 1 : Prepare strains and environment

- Strains to be tested for MIC and international standard strains are cultured in normal broth or MH broth to obtain young bacterial broth, according to the following ratio: Gram-negative bacilli: culture 0.1 ml;

- Prepare MH jelly: Weigh the jelly according to the recipe (written on the box), boil until the jelly is completely dissolved, steam the jelly at 121 0 C/15'.

- When the agar has cooled to about 40-50 0 C, use a graduated cylinder to measure 22.5 ml of agar into a conical flask containing 2.5 ml of antibiotic solution according to the concentrations in the antibiotic preparation table, shake well and pour into a cage. When the agar has solidified, store in a refrigerator at 4 - 8 0 C (can be stored for 2 weeks). Each time you do MIC, you need 2 MH medium plates without antibiotics to serve as control plates (add 2.5 ml of PBS or distilled water + 22.5 ml of MH agar medium).

Day 2 : Inoculate bacteria on the surface of MH agar plates with different antibiotic concentrations

- Cultivate pure bacteria from young bacterial broth (bacterial broth cultured overnight for 18 hours - equivalent to 10 8 bacteria/ml) or take a number of colonies from a suitable agar medium and mix them into 3 ml of PBS buffer solution (or 0.9% saline) to have a turbidity equal to that of a 0.5 Mc Farland tube - equivalent to 10 8 bacteria/ml. Continue to dilute 1/100 to have a concentration of 10 6 bacteria/ml by using a micropipette to pipet 20  l into 2 ml of PBS buffer (or 0.9% saline).

- Use a Pasteur pipette to draw about 0.5 ml of suspension (10 6 bacteria/ml) into each well of the plate, according to the diagram with the numbers of the strains recorded:

1	2	3	4
5	6	7	8	9	10
11	12	13	14	15	16
17	18	19	20	21	22
23	24	25	26	27	28
	29	30	Strain standard*	Strain standard*

* Standard strain is E.coli ATCC 25922.

- Use the 32-pin version of the nail set to dip into the well and then place it on the MH agar plate with different antibiotic concentrations.

- After placing on the agar plates, wait for the agar plates to dry (water drops at the inoculation lines penetrate the agar (about 15-20 minutes), put in an incubator at 35-37 0 C/18 hours, turn the agar plates upside down.

Day 3 : Read the results

- First, read the results on the control agar plates without antibiotics to check the purity of the bacterial strains and ensure that the bacteria do not die before and during the process. If the strains are guaranteed to be pure and growing well, then continue reading.

- Read the results of the control strains (international standard strains), compare the MIC results of these strains with the standard table (According to the American Clinical Microbiology Standards Institute - CLSI), if these results are within the limits, the allowable error is one order of magnitude smaller or one order of magnitude larger, which means that the experimental conditions have met the standard.

- Read the results, reading from the agar plate with the lowest antibiotic concentration. The MIC concentration is determined on the environmental plate where the bacteria are inhibited from growing, so the bacterial density is reduced to only 1-3 colonies growing.

- The MIC results of the strains with each antibiotic are recorded according to the sample table.

- At the lowest concentration, if no bacteria grow, the result is recorded as: less than or equal to that concentration (  ). In case bacteria still grow at the highest concentration, the result is recorded as greater than that concentration (  ).

- The MIC results of the strains will be determined according to three levels of sensitivity, based on the standard table (CLSI): susceptible (S), intermediate (I) or resistant (R).

2.4.3.6. Polymerase Chain Reaction (PCR) technique

PCR technique was performed by American Karl Mullis in 1985 [104]. Since then, this technique has been widely used and applied in many molecular biology experiments.

- Principle of PCR reaction.

The principle of PCR reaction is based on the denaturation and regeneration properties of DNA and the principle of DNA synthesis thanks to the activity of heat-resistant DNA polymerase ( Taq -polymerase) with the raw materials being 4 types of deoxyribonucleotides (dATP, dTTP, dGTP, dCTP). The enzyme DNA-polymerase catalyzes the synthesis of a new DNA strand from the template DNA strand. The reaction requires the presence of forward and reverse primers with sequences complementary to the two ends of the template DNA sequence.

Taq -polymerase is a type of DNA-polymerase (extracted from the thermotolerant bacterium Thermus aquaticus ), and primers are short DNA fragments that can pair with one end of the template strand. DNA polymerase will extend the primer to form a new fragment. The newly formed DNA fragments are then used as templates for

subsequent reactions. After each cycle, the number of DNA molecules doubles, so after n cycles the number of PCR products is 2n , enough to separate, decode or clone.

- Steps to take

PCR reaction is a series of consecutive cycles, each cycle consists of 3 steps:

- Step 1 : Is the denaturation stage . PCR reaction components generally include the following basic components: PCR buffer solution, dNTP, forward and reverse primers,

Taq -polymerase and template DNA. In this step, DNA is denatured at a temperature higher than the Tm (melting temperature) of the molecule, usually 94 0 C-95 0 C for 30-60 seconds.

- Step 2 : Annealing phase ( annealing-Ta ). The temperature must now be lowered so that the primers can pair with the template. This temperature ranges from 40-70 0 C depending on the optimal annealing temperature of the primers used and lasts for 30-60 seconds.

- Step 3 : The synthesis or extension phase . The temperature is increased to 72 0 C so that the polymerase enzymes can work best. The time depends on the length of the DNA sequence to be amplified, usually lasting from 30 minutes to several minutes.

After each cycle, the newly formed DNA strands continue to be used as templates for the synthesis of new DNA in the next cycle. The final product of the PCR product is a double-stranded DNA fragment with a length equal to the distance between the two primer gene segments and the two ends of the product are determined by the 5' ends of the two primer gene segments.

PCR products were checked on 1.2% agarose gels run with markers to compare the size of the DNA fragment of interest.

30 - 40 cycles

Each cycle consists of 3 steps.

Step 1: Denaturation 94 0 C for 1 minute

Step 2: Pair at 54 0 C for 45 seconds

Forward and reverse bait

Step 3: Extend 72 0 C for 2 minutes

(Source: Vierrstraete, 19

)

Figure 2.2. Image of PCR reaction steps

- Steps to conduct PCR with genes studied in the topic

 Isolation of DNA from bacteria by heat: a bacterial colony is cultured

After 24 hours, homogenize with 200 µl of distilled water and incubate at 100 0 C for 5 minutes, centrifuge at 1200 rpm for 5 minutes, the clear liquid containing bacterial DNA is used as a template for the PCR reaction.

 PCR components: Use PCR PreMix (Bioneer, Korea) including 1 pmol of each primer, 1.5 µl bacterial DNA, add H 2 O to a volume of 20 µl.

 PCR was performed on a Thermalcycler (Eppendorf, Germany)

with the following steps: total denaturation at 95 0 C for 5 min; 30 cycles of local denaturation at 95 0 C for 30 s, primer annealing at 50 0 C (for Quinolone and CTX-M antibiotic resistance genes) or 56 0 C (for SHV genes; TEM) for 45 s; synthesis at 72 0 C for 1 min; complete synthesis reaction at 72 0 C for 10 min and keep sample at 15 o C. PCR results were checked by 1.2% agarose gel electrophoresis with Ethidium bromide dye and viewed under ultraviolet light.

 Information on primers used to determine the presence of quinolone antibiotic resistance genes and extended-spectrum β-lactamases located on plasmids (Table 1).

 Standard DNA from Bioneer Korea.

 The positive control strain Klebsiella definitely has the genes qnr, CTX-M, SHV, TEM.

2.4.3.7. Southern Blot technique [121]: PCR-amplified DNA is opened, separated into single strands and cut with restriction enzyme nuclease S1 (specific enzyme for plasmid DNA); pulsed-field electrophoresis (PFGE) to separate the cut DNA fragments; ii) The DNA fragments are transferred to a nitrocellulose membrane;

iii) The DNA fragments on the membrane are hybridized with specific probes based on the principle of complementary DNA; iv) Immobilization and detection of the presence of the DNA-probe hybrid molecule labeled with biotin with a strong bond to avadin. Avadin mixed with alkaline phosphatase under the effect of a chromogenic substrate creates a purple color of the product.

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