Description of Methodology for Testing the Synergistic and Additive Effects of Antibiotics in Vitro

The epidemiological transformation in the early 20th century involved a shift from infectious diseases to non-infectious diseases as the leading causes of death worldwide. It became a source of hope for the ultimate victory of medicine over infections, facilitated by the discovery of many antibiotics in the 1940s and 1950s. Unfortunately, after only a few decades, due to the rapid growth of antibiotic resistance, infectious diseases have once again become one of the greatest threats to public health. Scientists such as Venkatasubramanian Ramasubramanian, president of the Clinical Infectious Disease Society of India, have been warning of a new post-antibiotic era for some time now (Sayburn 2023).

In addition to the significant rate of development of global antibiotic resistance, a critical aspect of the treatment of infections is the epidemiological situation in Poland, compared to Europe. For example, in 2021, fluoroquinolones-resistant Escherichia coli isolates accounted for 33.1% of E. coli isolates in Poland (population-weighted European average: 21.9%), carbapenem-resistant Klebsiella pneumoniae isolates for 19.5% (population-weighted European average: 11.7%), and vancomycin-resistant Enterococcus faecium isolates for 34.3% (population-weighted European average: 17.2%). More than half of the isolated K. pneumoniae strains showed multidrug resistance (MDR), which in this case meant resistance simultaneously to aminoglycosides, fluoroquinolones and third-generation cephalosporins (Żabicka and Grzegorczyk 2022). A significant difference between the degree of antibiotic resistance in Poland and Western and Northern European countries is also of great concern. In 2022, carbapenem-resistant Acinetobacter baumannii strains accounted for 76.4% of isolates in Poland, compared with only 3.5% in Germany and France and 2.7% in Sweden (European Center for Disease Prevention and Control).

The stagnation of the pharmaceutical market further hampers the effective treatment of infections. From 2017 to 2022, only 12 new antibiotics have been approved, and only two of them – vaborbactam and lefamulin – are representatives of new drug groups. A significant constraint and challenge of new antimicrobials introduction is the cost of bringing new therapeutics to market and the need for some of the latest agents to be seen as drugs of last resort. In most cases, resistance to new drugs is already reported within 2–3 years of their initial application (WHO 2021).

Although research is ongoing on new antibacterial agents and strategies, such as monoclonal antibodies or bacteriophages (WHO 2021), in light of such a dynamic development of the phenomenon of antibiotic resistance, it is necessary to search for new forms of treatment, including also these using already available antibiotics. A promising infection treatment method combines antibiotics with different mechanisms of action in therapy, including antibiotics to which bacteria were resistant in monotherapy. Their additive or synergistic action may allow them to reduce the value of their minimal inhibitory concentrations (MICs) or even break the barrier of resistance of a strain to particular drugs. Additional advantages of achieving a synergistic effect of a combined antibiotics approach include maximizing treatment effects with a reduced risk of developing de novo antibiotic resistance and possibly using lower drug doses (Garbusińska and Szliszka 2017). It is worth recalling that combined antimicrobial therapy is almost as old as antibiotic therapy itself, as evident in the history of the treatment of infections caused by Mycobacterium tuberculosis, in which the association of streptomycin with other drugs began soon after its discovery. Indeed, it was found that after the several months course of tuberculosis pharmacotherapy, the probability of developing resistance to streptomycin used in monotherapy could be as high as 100% (Brennan-Krohn and Kirby 2019a).

Several methods are currently used to study the additive and synergistic effects of antibiotics: e.g. methods based on strips impregnated with antibiotics in a concentration gradient, antimicrobials disc diffusion methods, antimicrobials micro-dilutions in agar methods, the checkerboard method, “time-kill” test, in vitro dynamic pharmacokinetic and pharmacodynamic (PK/PD) models, semi-mechanistic PK/PD models, and even in vivo animal models (Karakonstantis et al. 2022). This article characterizes and describes the step-by-step methodology of several of the methods mentioned above, which, in the authors’ opinion, have the most significant potential for use in daily laboratory and clinical practice as routine methods for testing antibiotic combinations, which in the future may contribute to improving the clinical condition of many patients suffering from infections caused by multi-drug-resistant bacterial strains.

PubMed and Google Scholar databases were used for the literature review, searching for the phrases: antibiotic resistance, antibiotic synergism, antibiotic interaction methodology, antibiotic FICI assessing, checkerboard assay, “time-kill” curves, synergism disc-based methods, synergism gradient-based methods, antibiotic gradient-based methods from the last 10 years. Among the results obtained, articles on antibiotic combinations were selected, including descriptions of the most commonly used methods for testing synergism and antibiotic adherence in substantive, clinical and/or procedural terms. Information on the epidemiology of antibiotic resistance was obtained from the Surveillance Atlas of Infectious Diseases of the European Centre for Disease Prevention and Control.

The microdilution method (so-called “checkerboard test”) is the most popular method for testing the in vitro activity of antibiotic combinations, a modification of the standard method for determining MIC in broth, a technique routinely used, for example, in assessing the MIC value of colistin. Most often, a 96-well polystyrene plate is used. It is adapted to test the simultaneous activity of two antibiotics in each well – antibiotic concentrations are placed onto the plate, creating a gradient “checkerboard” of dilutions of the two antimicrobials so that every possible combination of the two concentrations of the test drugs is evaluated within the chosen and planned concentration ranges. This method has two severe limitations. First, it is a static method in which the effect of antibiotics is assessed at a specific point in time without the possibility of being evaluated for several hours of incubation. It provides information only on the bacteriostatic effect without the possibility of testing the killing properties of drugs (in the methodology used, these two effects are indistinguishable) (Brennan-Krohn and Kirby 2019a). Limiting the number of antibiotics tested to two is also essential. With more than that, the method quickly becomes impractical, even assuming that at least one of the drugs will be tested in a relatively narrow range of concentrations (Brennan-Krohn and Kirby 2019a; Doern 2014). In addition, this method requires many reagents, and it is necessary to prepare many antibiotic dilutions.

In the described method, standardized bacterial inocula (of constant density and volume) in Mueller-Hinton broth and antibiotics (usually two from two different groups) in appropriate concentrations are placed in the wells of a polystyrene plate, the concentrations of which can be related to the concentrations achievable in the patient’s body fluids and which were obtained in serial two-fold dilutions (Doern 2014; Garbusińska and Szliszka 2017; Brennan-Krohn and Kirby 2019a). After the incubation time, bacterial growth is assessed in all of the wells of the plate. For the wells in which the bacterial growth is inhibited, the fractional inhibitory concentration index (FICI) is calculated and based on this, the antibiotic interaction is classified as antagonistic (FICI > 4), neutral (1 < FICI ≥ 4), additive (0.5 < FICI ≥ 1) or synergistic (FICI ≥ 0.5). Although authors of scientific investigation sometimes use other ranges for FICI values interpretation or do not distinguish between antibiotic addition/synergy effects, the cited classification method is the most common. The FICI for a given concentration combination at which inhibition is observed is the sum of the fractional inhibitory concentration (FIC) of both drugs, i.e. the ratio of the drug concentration in that well to its MIC. Thus, since synergism is evidenced by an FICI ≤ 0.5, and the error range for MIC testing in a standard dilution in broth is ± 1 two-fold dilution (with the error range increasing when testing drugs in combination), the definition of synergism is met if each drug in a well has a concentration of at least half that of its MIC assessed individually in a separate assay (Garbusińska and Szliszka 2017; Brennan-Krohn and Kirby 2019a).

The broth microdilution method continues to be modified and improved, such as using a bio-printer, which allows for precise micro-volume measurements and speeds up the entire procedure (Brennan-Krohn and Kirby 2019b). The procedure described below includes reagents and equipment commonly available in microbiology laboratories.

Procedure

Preparation of bacterial inoculum.

1.1

Calibrate the densitometer against a control sample with an optical density of 0.5 on the McFarland scale.

1.2.

Collect the tested strain with a sterile loop from the agar medium and place it in a solution appropriate for the method (usually saline).

1.3.

Vortex it.

1.4.

Measure of the optical density of the suspension in a densitometer.

1.5.

Optionally, add the bacterial mass or the control strain being tested using a loop to an optical density of 0.5 on the McFarland scale.

Determination of MICs of the tested antibiotics (method of choice).

Preparation of antibiotic solutions.

3.1.

Select the antibiotic to be diluted in rows 1–12, i.e. horizontal rows (antibiotic A), and another in wells A → H, i.e. vertical columns (antibiotic B); consider the appropriateness of testing one in a broader range of concentrations.

3.2.

Prepare approximately 400 μl of antibiotic A solution and approximately 350 μl of antibiotic B at a concentration four- (for A) and eight- (for B) times higher than the resistance cut-off value of a given drug for the species of the strain being tested (Brennan-Krohn et al. 2017; EUCAST 2024). Select the solvent according to the CLSI (Clinical and Laboratory Standards Institute) guidelines: “Solvents and Diluents for Preparing Stock Solutions of Antimicrobial Agents”. If the guidelines do not contain information on the tested antibiotic, use a solvent that maintains more excellent drug stability (Bellio et al. 2021).

3.2.1

It is possible to use the following pattern:

C_a – C_k = V_r

C_k – C_r = V_a

Where:

C_a – initial concentration of the antibiotic solution [mg/l],

C_k – final concentration of antibiotic solution [mg/l],

C_r – solvent concentration [mg/l],

V_r – solvent volume [μl],

V_a – initial volume of antibiotic solution [μl].

Adding V_a of the initial antibiotic solution to V_r of the solvent will provide an antibiotic solution with a concentration of C_k. C_k should have a value four times higher than the breakpoint value for resistance to a given drug for the strain of the species tested.

Preparation of a concentration checkerboard. (Fig. 1. Broth microdilution method – preparation of a concentration pattern.)

4.1.

Add 50 μl of MHB (Mueller-Hinton broth) to all wells in rows 1st to 11th and two wells in row 12th by automatic adjustable pipette. (Fig. 1A. Add Mueller-Hinton broth.)

4.2.

Add 50 μl of antibiotic A to all wells (A-H) in row 1st.

4.3.

With a multi-channel adjustable pipette, transfer 50 μl of solution from the wells of row 1st to the wells of row 2nd, from row 2nd to row 3rd, and so to row 11th. Mix the resulting solution each time with an automatic pipette (slowly aspirate and withdraw the solution several times). Transfer 50 μl of solution from wells 11A and 11B to 12A and 12B. Remove 50 μl of solution each from wells 11C-H. The well 12A will be a control for bacterial growth without antibiotics (positive control), the well 12B will serve as a procedure sterility control, and also for the remaining reagents and drugs solutions (negative control). (Fig. 1B. Prepare a series of microdilutions of the first antibiotic.)

4.4.

Prepare 8 Eppendorf tubes (1,5 ml) and label them sequentially with the letters A-H.

4.5.

Place 1 ml of MHB in all the labelled Eppendorf tubes using an automatic pipette.

4.6.

Transfer 333 μl of antibiotic B solution (with a concentration of eight times the resistance breakpoint of the applied drug for the strains of the tested species) into the Eppendorf tube A. After placing antibiotic B in MHB at a ratio of 1:3, its solution with a concentration twice higher than the resistance breakpoint concentration of the drug will be obtained.

4.7.

Transfer the volume of 333 μl from the Eppendorf tube A to the Eppendorf tube B and mix with an automatic pipette. Transfer 333 μl from the Eppendorf tube B to the Eppendorf tube C, mix and so on to the Eppendorf tube H. Remove 333 μl of solution from the Eppendorf tube H. (Fig. 1C. Prepare a series of microdilutions of the second antibiotic.)

4.8.

Pipette 50 μl of solution from the Eppendorf tube A into wells of A row (A1-A11 – excluding well A12 – serving as a positive control), from the Eppendorf tube B into all wells in B row (B1-B12), from the Eppendorf tube C into wells C1-C11, and so on. (Fig. 1D. Add antibiotics together.)

4.9.

Pipette (multi-channel pipette can be used) 50 μl of MHB into each well of the antimicrobials dilutions. Avoid adding MHB to the 12C-H wells when using the multi-channel pipette. (Fig. 1E. Re-add Mueller-Hinton broth.)

4.10.

Add 50 μl of bacterial suspension to each well with antimicrobials solutions (except 12B – as a negative control).

Incubate for 16–24 hours at 37°C.

Bacterial growth assessment.

6.1.

Assess turbidity in the wells after incubation. The absence of turbidity indicates that the antibiotics inhibit bacterial growth.

6.2.

Record/capture in which antimicrobials concentration combinations growth inhibition is observed. For these combinations, FICI calculation is necessary, as follows:

FICI = FIC_x + FIC_y

FIC_x = MIC_xc / MI^yC_x

FIC_y = MIC_yc / MIC_y

Where:

MIC_x, MIC_y – MIC of drug X or Y used alone, MIC_xc, MlC_yc – drug X or Y concentration in combination in a given well for which FICI is determined (Garbusińska i Szliszka 2017).

6.3.

Interpret the FICI values according to the criteria given above and classify the particular antimicrobials concentrations combination as synergistic, additive, neutral or antagonistic.

6.4.

If a so-called “skipped well” occurred in a row (e.g. no growth in well C8, while growth in C9, no growth in C10), the FICI for C10 was calculated to avoid misinterpretation, i.e. a false positive result (Brennan-Krohn et al. 2017).

Fig. 1.

Broth microdilution method – preparation of a concentration pattern. A

– add Mueller-Hinton broth.

B

– prepare a series of microdilutions of the first antibiotic.

C

– prepare a series of microdilutions of the second antibiotic.

D

– add antibiotics together.

E

– re-add of Mueller-Hinton broth.

The “time-kill” test is a microbiological method which provides information on both the synergistic effect of antibiotics and the kinetics of bacterial growth and activity of the bactericidal preparation (Brennan-Krohn and Kirby 2019b). The test is based on the analysis of microbial survival in prepared concentrations of two antibiotics at selected time intervals. The interaction of the two drugs is determined by comparing the CFU/ml value (colony-forming unit per ml) between the tested combination of antibiotics and the best-performing single antibiotic. The obtained results are presented on the “time-kill” curves. Synergism is established when the difference between the two trials exceeds ≥ 2_log10 (Brennan-Krohn and Kirby 2019b). A particular combination is considered bactericidal when the difference in CFU/ml between the combination of two antibiotics at the start of incubation and after 24 hours is ≥3_log10. The “time-kill” test is an alternative to the checkerboard assay (Garbusińska and Szliszka 2017).

Its decisive advantage is the ability to determine both the bacteriostatic and bactericidal action (as opposed to the checkerboard assay, which can evaluate only the bacteriostatic action). In addition, the “time-kill” test makes it possible to determine the action of a combination of antibiotics at different time points (Brennan-Krohn and Kirby 2019b). The disadvantage of the “time-kill” test is that it is more labor-intensive and time-consuming compared to the checkerboard assay. In addition, the “time-kill” test is much more expensive.

Procedure

Preparation of antibiotic solutions.

1.1.

Determination of the antimicrobial agent concentration based on its solubility and the desired final concentration. The solvent selection should follow the CLSI (Clinical and Laboratory Standards Institute) guidelines.

Initiation of pre-culture.

2.1.

Prepare a 0.5 McFarland suspension in 0.9% NaCl from an overnight culture. Adjust the bacterial concentration with a densitometer to obtain a turbidity of 0.5 on a McFarland scale.

2.2.

Add 100 μl of the bacterial suspension to 5 ml of CAMBH (Mueller-Hinton Broth, cation adjusted). Transfer a drop of the diluted suspension via a sterile inoculating loop on a blood agar plate to confirm the purity of the inoculum. The incubation of the control culture is recommended at 35°C

2.3.

Incubate the remaining suspension at 35°C for at least 3 hours until logarithmic growth is achieved.

Antimicrobial solutions.

3.1.

Add 10 ml of CAMBH to 5 glass culture tubes.

Tube 1:

Add the first antibiotic in an amount corresponding to the target antibiotic concentration.

Tube 2:

Add the second antibiotic in an amount corresponding to the target antibiotic concentration.

Tube 3:

Add the same amount of the first and the second antibiotic as in tubes 1 and 2.

Tube 4:

Growth control – do not add antibiotics.

Tube 5:

Negative control – neither antibiotic nor microorganism should be added.

Perform a series of dilutions. (Fig. 2. The “time-kill” method – a series of dilutions.)

4.1.

Prepare six 96-well plates (labelled: t₀, t₁, t₂, t₄, t₆, t₂₄) with 2 ml wells which will be used for a series of dilutions. Place 900 μl of 0.9% NaCl in rows B-H, columns 1st-5th. (Fig. 2A Prepare six 96-well plates (t₀, t₁, t₂, t₄, t₆, t₂₄) with 0.9% NaCl.)

4.2.

Transfer the culture in the logarithmic growth phase of the initial inoculum. Transfer 1 ml of suspension to a culture glass tube. To obtain a density of 1.0 McFarland use CAMBH to adjust – dilute, or add more microbial cells to concentrate the suspension.

4.3.

Add 100 μl of the suspension to tubes 1st–4th and mix gently. (Fig. 2B. Add the bacterial suspension to test tubes 1st–4th.)

4.4.

Withdraw 150 μl from each tube at time t₀ (immediately after adding the bacterial suspension) and after 1, 2, 4, 6 and 24 hours. Add portions to wells in appropriately marked rows 1 (t₀, t₁, t₂, t₄, t₆, t₂₄) of the 96-well plates. (Fig. 2C. Transfer the suspension portion to the appropriate plate a predetermined times.)

4.5.

Using a multi-channel pipette, withdraw 100 μl volume from row A and transfer it to row B. Aspirate and withdraw the solution several times to mix evenly. This action will result in a dilution of 1:10. Repeat for rows B-H. Remove 100 μl from row H. (Fig. 2D. Obtain a dilution of 1:10.)

4.6.

Using a multi-channel pipette, collect 10 μl from each well in columns 1st-3rd of all plates and transfer to appropriately labelled Mueller-Hinton agar plates for colony counting using the “drop plate” method. The labelling pattern should allow for identification of the antibiotics used, their concentrations, and the time after which a portion of the bacterial suspension was transferred to the plate (t₀-t₂₄). Allow the drop to dry completely. (Fig. 2E. Transfer a portion of the suspension to the plates using the “drop plate” method.)

4.7.

Place a 10 μl drop from the negative control to the selected plate after 24 hours to confirm procedure sterility. Invert the plate and incubate overnight under recommended conditions.

Reading the results.

5.1.

Negative control check. If any growth is observed, the results are unreliable.

5.2.

The colonies obtained should have the uniform morphology expected for the strain used.

5.3.

Identify drops with 3–30 colonies for each series of dilutions. Count the colonies in these drops and record them along with the dilution factor. If there are no drops with 3–30 colonies in the dilution series, count the last drop with more than 30 and the first with colonies.

5.4.

Calculate the CFU/ml value in the sample for each dilution series based on the number of colonies in the drop using the formula:

CFU/ml = 100 n/d

Where:

n – number of colonies,

d – dilution factor (1 for undiluted sample

–

row A, while 0,1 for the first dilution

–

row B, 0,01 for the second dilution

–

row C, etc.).

Analysis of results.

6.1.

Plot the corresponding growth curves from three cultures containing antibiotics and control growth, using time units on the x-axis and CFU/ml values on the y-axis.

6.2.

Calculate the difference between growth in the tube with no antibiotics after 24 hours and the most active single factor simultaneously (in CFU/ml). The combination is considered synergistic if the difference is ≥ 2 log₁₀. Calculate the difference in CFU/ml between the combination in the tube at 24 hours and at time 0 point. Consider the combination bactericidal if the difference is ≥ 3 log₁₀.

Fig. 2.

The “time-kill” method – a series of dilutions. A

– prepare six 96-well plates (t₀, t₁, t₂, t₄, t₆, t₂₄) with 0.9% NaCl

B

– add the bacterial suspension to test tubes 1st–4th.

C

– transfer the suspension portion to the appropriate plate at predetermined times.

D

– obtain a dilution of 1:10.

E

– transfer a portion of the suspension to the plates using the “drop plate” method.

Another method for testing antibiotic combinations, CombiANT, is not yet available in research practice or microbiological diagnostics. It was first presented in 2020, and initial studies indicate identical results of the CombiANT technique compared to the checkerboard assay (Fatsis-Kavalopoulos et al. 2020). Despite the lack of equipment and algorithms for validated results interpretation for individual bacterial species, due to the many advantages of this method and perhaps a breakthrough in the facilitation of testing antibiotic combinations, the decision was made to describe this procedure in detail in this paper.

CombiANT is a diffusion-based assay providing quantitative information on the interactions of three pairs of antibiotics. The selected antibiotics are placed in three reservoirs and then in a standard agar plate. Two fields can be distinguished. On the outside of the insert is a field where each antibiotic acts individually. Inside the antibiotic plate is a triangular field where the interaction between the individual antibiotic pairs occurs. The results are generated by an algorithm developed by the creators, calibrated to the type and rate of diffusion of each antibiotic in the chosen substrate. The extent of antibiotic interaction is determined quantitatively based on points at the edge of the inhibition zone, according to the FICI formula. The FICI values were interpreted according to clinical thresholds, where ≤ 0.5 indicates antimicrobials synergy, FICI > 0.5 and ≤ 4 indicates addition, while > 4 indicates antagonism (Tang et al. 2024).

The CombiANT method can be used without determining the strain’s susceptibility to a given antibiotic. This fact is a definitive advantage, as it reduces the waiting time for the antimicrobial sensitivity result of the tested strain. The CombiANT test has the same accuracy as the broth microdilution method but is more efficient and less complex (Fatsis-Kavalopoulos et al. 2020). An additional advantage of this method is the possibility of accurately determining the inhibitor concentration since a continuous range of antibiotic concentrations is used. In contrast, the checkerboard assay has only 2-fold antimicrobials dilutions. A significant disadvantage of this method is the need to purchase special inserts, which do not have laboratory applications for other studies. Procedure (Fig. 3. CombiANT method – the insert and experimental protocol.)

Preparation of the antibiotic insert (Fig. 3A. The insert design: antibiotic reservoirs (a-c), interaction imaging area (d)).

Fig. 3.

CombiANT method – the insert and experimental protocol. A

– the insert design: antibiotic reservoirs (a-c), interaction imaging area (d).

B

– the assay protocol: „The insert is loaded by adding 0.5 mL liquid agar (60°C) containing antibiotics into the reservoirs. The prepared inserts can be stored at 4–8°C. To activate an insert, add a second layer of 25 mL agar to enclose the insert and fill the plate, thereby permitting diffusion of the antibiotics to the agar surface and the reservoir periphery. After solidification, a bacterial cell suspension of 0.5 McFarland is inoculated on the agar surface using a sterile cotton swab and exposed to the antibiotic gradient landscape. The finished plates are incubated at 37°C, and stable zones of growth inhibition are established within 16–24 hours.” (Fatsis-Kavalopoulos et al. 2020)

The synergistic effects of antimicrobial agents in vitro can also be evaluated using methods available in most microbiological laboratories, i.e., strips impregnated with antibiotic in the concentration gradient. The following methods have been developed so far:

E-test fixed ratio method,

Before performing the methods mentioned above, the MIC values for all antibiotics tested should be determined.

Methods based on antibiotic-impregnated strips with the concentration gradient have an advantage over other methods due to their simplicity and easy access to this procedure for all laboratories. However, synergism can be tested only between two antibiotics in this way. Furthermore, the limitation of these techniques is the lack of their use against resistant isolates whose MIC values exceed the maximum antimicrobial concentration placed on a strip scale since the exact MIC value must be known a priori to assess the synergy.

E-test fixed ratio method – procedure

Prepare suspensions of the tested bacteria (density 0.5 McFarland) and inoculate on Mueller-Hinton Agar medium (MHA).

Apply a strip containing antibiotic A on the MHA.

2.1.

The area where the strip containing antibiotic A is located should be precisely marked.

Incubate a plate at room temperature for one hour to let the antibiotic diffuse from the strip into the MHA medium.

Remove the antibiotic A strip.

4.1.

Clean the strip with alcohol and leave it as a template for reading the MIC value.

4.2.

Apply the strip containing antibiotic B to the same spot.

Incubate the culture on the plate for 16–18 hours at 35°C ± 2°C (as recommended by the strip manufacturer).

Reading and interpretation of results: read the MIC values for the strips used and calculate the FICI value after the incubation (Fig. 4. Assessment of the synergistic effect of antibiotics by the method of constant coefficients.) (Laishram et al. 2017; Sreenivasan et al. 2022; Guzek 2023), which should be interpreted according to a standardized criteria.

Fig. 4.

Assessment of the synergistic effect of antibiotics by the method of constant coefficients.

Cross method – procedure

Inoculate the MHA plate with the tested bacterial suspension (density 0.5 McFarland).

Place two strips containing antibiotics A and B.

2.1.

Arrange the strips to intersect at an angle of 90° at the concentration marked as the previously determined MIC values.

Culture on a plate incubation for 16–20 hours at 35°C ± 2°C (as recommended by the strip manufacturer).

Reading and interpretation of results: read the MIC values for the strips used and calculate the FICI value after the incubation (Fig. 5. Assessment of the synergistic effect of antibiotics by cross-method.), which should be interpreted according to standardized criteria.

Fig. 5.

Assessment of the synergistic effect of antibiotics by cross-method.

Due to its high availability and ease of performing, the cross method is eagerly chosen to evaluate the combination of two antibiotics. However, the results of this procedure can differ significantly from those obtained in the “time-kill” test, which is considered the gold standard for this purpose (Nasomsong et al. 2022).

MIC:MIC ratio evaluation – procedure

Apply two strips with a concentration gradient of antibiotics A and B onto the MHA medium with a bacterial suspension plated initially.

1.1.

Mark previously obtained MIC value for each drug separately with a marker on the bottom of the plate.

Incubate the plate for one hour at room temperature to allow the antibiotics to diffuse from the strips.

Evaluation of antimicrobials synergism.

3.1.

Remove the first antibiotic strip from the culture after one hour.

3.2.

Place a new strip containing antibiotic A on the spot where the antibiotic B strip was located so that the MIC value from strip A corresponds to the MIC value for antibiotic B marked previously with the marker.

3.3.

Follow the same procedure for the second strip.

Culture on a plate incubation for 16–20 hours at 35°C ± 2°C (as recommended by the strip manufacturer).

Reading and interpretation of results: read the MIC values for the strips used and calculate the FICI value, which should be interpreted according to standardized criteria (Laishram et al. 2017; Guzek 2023). (Fig. 6. Evaluation of the synergistic effect of antibiotics by the MIC:MIC ratio evaluation.)

Fig. 6.

Evaluation of the synergistic effect of antibiotics by the MIC:MIC ratio evaluation.

According to some previous results, in comparison with gradient diffusion methods used to assess the synergy of tobramycin and ceftazidime in multidrug-resistant Pseudomonas aeruginosa, the results obtained by Okoliegbe et al. suggest that the MIC:MIC ratio method should be considered as the preferred method for antibiotic interaction studies in diagnostic practice (Okoliegbe et al. 2021).

Three of the many methods for determining antibiotic synergistic and additive activity not described in this article are worth mentioning. All of them are achievable in any basic medical diagnostic laboratory – double disc synergy test (a modification of the procedure typically used to assess the presence of drug resistance mechanisms in microorganisms), paper strip diffusion (which uses strips soaked in various antimicrobial solutions at concentrations equal to or higher than their MIC values) and the overlay inoculum susceptibility disc method.

Due to the dynamic increase in antibiotic resistance of microorganisms, mainly bacteria, and the long time required to develop and approve a new antibiotic for clinical practice, it is necessary to introduce the evaluation of the effectiveness of the already-known antibiotic combinations into routine diagnostic practice. There are many methods for testing synergistic and additive antibiotic activity, which vary in the availability of the necessary reagents and equipment, the difficulty and time of performance, as well as the reliability of the results obtained and the unambiguity of their interpretation (Papoutsaki et al. 2020; Okoliegbe et al. 2021). Depending on the study, the level of compliance with individual methods varies significantly. Differences may be related to the strain of bacteria tested, antibiotics used, or even the presence of particular resistance mechanisms. In their study of methods for detecting synergism in multidrug-resistant Gram-negative rods, Gaudereto et al. obtained compliance with the MIC: MIC method and “time-kill” ratio method at the level of 35–71% (Gaudereto et al. 2020). Therefore, a clear comparison of individual methods in a universal way for antibiotics and bacterial strains is currently beyond the capabilities of researchers due to divergent results depending on the strains, antibiotics and their combinations (Doern 2014).

The “gold standard” test remains the “time-kill” method, which provides information on bactericidal and bacteriostatic action at different time points and is a reliable reference method in comparing different procedures. However, the “time-kill” test is a time-consuming and relatively difficult method, so using strips with antimicrobials concentration gradients seems to be an attractive alternative. It is necessary to validate the results to ensure they are as reliable as possible.

In addition to routine testing of synergistic and additive activity of antibiotics, it is also necessary to develop new methods to implement in diagnostic and clinical practice. The CombiANT method seems to be a promising procedure, which may become a method of great diagnostic importance soon, considering its ability to quickly test a combination of three drugs, easy interpretation of the results and accuracy comparable to the microdilution method (Fatsis-Kavalopoulos et al. 2020).

The checkerboard assay, the most popular method for synergism testing, as a modification of the MIC determination by antimicrobials broth microdilutions method, is an intuitive and easily accessible procedure due to the broad access to the required equipment and reagents. However, in contrast to the “time-kill” method, it only provides information on the bacteriostatic effect, which is the most significant limitation that may affect further clinical management.

The most accessible, easiest and cheapest methods are those using antibiotic concentration gradient strips or discs. Although validating the methodology based on their use and introducing uniform criteria for interpretation of the results obtained is difficult, in some situations, they may be the only possible procedure. Therefore, further studies are needed to introduce them into routine diagnostic and clinical practice while obtaining results comparable to other methods. It is essential to differentiate the reliability of the results of individual procedures with gradient strips, among which the MIC:MIC ratio method seems to be the most reliable one (Okoliegbe et al. 2021).