Bacterial isolates and antimicrobial susceptibility testing

95 A. baumannii isolates were randomly selected from the collection of the Swiss National Reference Center of Emerging Antibiotic Resistance (NARA). Isolates from this collection were previously sent to the reference center for investigation of their carbapenemase content. The main β-lactamase gene as a source of the multidrug resistance pattern of all isolates had been previously characterized, and it is shown on Table 1.

Table 1 Results of the rapid FDC NP test for detection of cefiderocol susceptibility/resistance in Acinetobacter baumannii isolates

The BMD reference method was performed to determine the minimum inhibitory concentration (MIC) of all isolates following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines [14]. Results were interpreted according to the pharmacokinetic/pharmacodynamic (PK/PD) breakpoints from EUCAST (susceptible ≤ 2 mg/L; resistant > 2 mg/L) [15, 16]. BMD was considered the gold standard when comparing to the results obtained with the rapid FDC Acinetobacter baumannii NP test. The reference strain A. baumannii CIP 70.10 (ATCC 15151) was used as quality control for both techniques, and the reference Pseudomonas aeruginosa (ATCC 27853) strain was also used for BMD quality control.

Optimizing rapid FDC Acinetobacter baumannii NP test

The rapid test is based on the reduction of resazurin (a viability colorant) to resorufin product by bacterial viable cells, thus detecting bacterial growth in the presence of a fixed concentration of FDC. Bacterial growth is visually detected by a color change from blue (resazurin) to violet or pink (resorufin).

In order to reach optimal conditions of the test, several different parameters were tested using the FDC-susceptible A. baumannii CIP 70.10 and one resistant isolate with a MIC ≥ 128 mg/L. These parameters comprised variable FDC concentrations (8, 16, 24.8, 32, 38.4, 46.2, 64, and 128 mg/L), variable bacterial inoculum concentrations (0.5 of the McFarland’s scale, 1/5, 1/10, 1/15, 1/20, 1/25 of the 1.0 McFarland’s scale), different bacterial inoculum volumes (20 and 50 µL), different reagent solution volume (150 and 180 µL), and variable incubation times (4 h, 4h15, 4h30, 4h45, and 5 h).

The optimal conditions were FDC final concentration at 38.4 mg/L, inoculum concentration of 1/20 of the 1.0 McFarland’s scale, bacterial inoculum volume of 20 µL, reagent solution volume of 180 µL, and 4h30-4h45 of incubation time.

Preparation of the rapid FDC Acinetobacter baumannii NP solution

The solution was prepared with iron-depleted Mueller–Hinton broth (ID-MHB; chelex 100 resin, Bio-Rad, Marnes-la-Coquette, France; MHB, AxonLab, Baden, Switzerland) [14] supplemented or not with FDC (Shionogi, Osaka, Japan) at a concentration of 42.67 mg/L targeting to achieve a final fixed concentration of 38.4 mg/L in a 200 µL final volume in each microplate’s well. The solution can be stored at − 80 °C for 2 weeks.

Bacterial inoculum

Overnight cultures were grown on UriSelect 4 (Bio-Rad, Marnes-la-Coquette, France). After that, a 1.0 McFarland bacterial inoculum was prepared by adding bacterial colonies in 5 mL sterile NaCl (0.85%) and then diluting 1/20 (50 µL of inoculum in 950 µL of NaCl) before inoculation into the microplates in a range from 15 min to 1 h after preparation [15].

Tray inoculation

A 96-well polystyrene microplate (round base, with lid, sterile; Sarstedt, Nuembrecht, Germany) was used to perform the rapid FDC Acinetobacter baumannii NP test. The bacterial suspension was inoculated in two independent wells, with and without FDC. The steps to perform the test were as follows: (1) 180 µL of FDC-free solution was added to wells A1-A4; (2) 180 µL of FDC at a concentration of 42.67 mg/L was added to wells B1-B4; (3) 20 µL (1/20 of the 1.0 McFarland scale) of A. baumannii CIP 70.10 (negative control) was added to wells A1 and B1; (4) 20 µL of a FDC-resistant isolate (positive control) was added to wells A2 and B2; (5) 20 µL of a tested isolate was added to wells A3 and B3; and (6) 20 µL of NaCl 0.85% was added to wells A4 and B4 (Fig. 1). After preparing the microplate for the test and before inoculating the bacterial suspensions, the rapid FDC Acinetobacter baumannii NP solution was pre-warmed at 37 °C for 15–30 min before use to prevent growth delay and therefore a delayed color change.

Fig. 1

The rapid FDC Acinetobacter baumannii NP test. Column A presents the solution free of FDC. Column B presents the solution with FDC (38.4 mg/L). Reference strain A. baumannii CIP 70.10 (ATCC 15151) was inoculated in A1 and B1; FDC-resistant isolate (positive control) was inoculated in A2 and B2; tested isolate (resistant to FDC) that grew in both absence and presence of FDC was inoculated in A3 and B3; and NaCl 0.85% was inoculated in A4 and B4 as control of contamination and possible spontaneous color change. Bacterial growth is evidenced by a color change of the medium from blue to pink or violet

Tray incubation and reading

The tray was not sealed and the test was incubated for 3 h at 35 ± 2 °C without shaking. After that time, 20 µL of resazurin reagent PrestoBlue (Thermo Fisher Scientific, Cleveland, OH, USA) was added to each well [(concentration of 10% (vol/vol)] and the test was incubated again. Reading was performed visually by checking the tray for no spontaneous color change after 4 h and then every 15 min until reaching a total of 4h45 h of incubation. The test was considered positive when the isolate grew (color change from blue to pink or violet) in the presence of FDC, and negative when no growth was observed (no color change) in the presence of FDC. Figure 1 provides a comprehensive illustration of the visual interpretation of the rapid FDC Acinetobacter baumannii NP test.

To assure optimal quality control of the test and consequently validate the rapid FDC Acinetobacter baumannii NP test, the following conditions had to be reached; (1) blue-to-pink/violet color change observed, confirming the bacterial growth and viability for all isolates in wells without FDC (A1-A3); (2) no color change observed (remaining blue) in wells after adding NaCl 0.85%, confirming the absence of contamination (A4 and B4); (3) blue to pink/violet color changes observed in the wells where the positive control and the tested isolate were added (A2 and B2; A3 and B3); and (4) no color change observed with the reference strain A. baumannii CIP 70.10 in the well with FDC (negative control) (B1). See Fig. 1 for some examples.

Data analysis

Discrepancies between the rapid FDC Acinetobacter baumannii NP test and the BMD standard reference method were determined and classified, if present, as very major errors (VME) and major errors (ME) as previously described [17, 18]. Sensitivity, specificity, accuracy, and precision parameters were also calculated to evaluate the performance of the test proposed. Results were blindly read and interpreted independently by two laboratory members.