A Selective Culture Medium for Screening Ceftazidime-Avibactam Resistance in Enterobacterales and Pseudomonas aeruginosa

RESULTS

A total of 92 isolates of worldwide origin were included in this study to evaluate the performance of the SuperCAZ/AVI medium. The β-lactamase contents of all strains were characterized at the molecular level by PCR and sequencing or, for some isolates, by whole-genome sequencing (Table 2). A total of 50 strains were susceptible to CZA (40 Enterobacterales, including Enterobacter cloacae, K. pneumoniae, and Escherichia coli, and 10 P. aeruginosa strains), and 42 were resistant to CZA (20 Enterobacterales, including E. cloacae, K. pneumoniae, and E. coli, and 22 P. aeruginosa strains) (Table 2).

Starting with an optical density of a 0.5 McFarland standard (an inoculum of ∼1.5 × 10⁸ CFU/ml), serial 10-fold dilutions were made in 0.85% saline solution, and 100-μl aliquots of each dilution were plated onto the SuperCAZ/AVI medium. To quantify the viable bacteria in each dilution step, tryptic soy agar plates were inoculated concomitantly with 100 μl of each suspension and were incubated overnight at 37°C. Viable colonies were counted the following day. When no growth was observed after 18 h, incubation was extended up to 48 h in order to definitely assess the negativity of the culture. The lower limit of detection for the strains tested was determined using the SuperCAZ/AVI medium.

The sensitivity and specificity cutoff values for the detection of CZA-resistant Enterobacterales and P. aeruginosa were set at 1 × 10³ CFU/ml, i.e., the CZA-resistant isolates recovered on SuperCAZ/AVI medium plates at <1 × 10³ CFU/ml were considered positive, while the CZA-susceptible isolates grown using an inoculum of ≥1 × 10³ CFU/ml were considered negative (10). All the CZA-resistant isolates could be recovered within 24 h on SuperCAZ/AVI medium plates by using an inoculum below the cutoff value of 1 × 10³ CFU/ml (1 × 10¹ to 1 × 10² CFU/ml) (Table 2). In contrast, growth of the CZA-susceptible isolates was possible only when an inoculum of >10³ CFU/ml was used (the lower limit of detection was above the cutoff value of 10³ CFU/ml), giving rise to 100% sensitivity and specificity.

Spiked stools were also tested with the same representative collection of CZA-resistant and -susceptible Gram-negative bacteria (n = 92) using this selective culture medium. Spiked fecal samples were made by adding 100 μl of serial 10-fold bacterial dilutions to 900 μl of a stool suspension. Stool suspensions were obtained by suspending 6 g of freshly pooled feces from healthy volunteers in 60 ml of distilled water as described previously (10). Aliquots (100 μl) of the spiked stool suspension were inoculated onto the SuperCAZ/AVI medium. Aliquots (100 μl) of stool suspensions with no bacteria added were plated onto the SuperCAZ/AVI medium as negative controls. The lower limit of detection was below the cutoff value for all CZA-resistant strains with which stools were spiked, ranging from 10¹ to 10² CFU/ml, whereas the lower limit of detection for the CZA-susceptible strains was above the cutoff value, at ≥10⁶ CFU/ml (Table 2). Sensitivity and specificity were determined using the same cutoff value, set at 10³ CFU/ml (10). Again, the sensitivity and specificity of the SuperCAZ/AVI medium for isolating CZA-resistant isolates were both 100%.

To assess the storage stability of the SuperCAZ/AVI medium, Candida albicans and Staphylococcus aureus strains, as well as the CZA-susceptible E. coli ATCC 25955 reference strain, were subcultured daily onto the SuperCAZ/AVI medium from a single batch of medium stored at 4°C. No growth was observed consistently for at least a 7-day period.