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

The SuperCAZ/AVI medium was developed for screening ceftazidime-avibactam (CZA) resistance among Gram-negative bacteria (Enterobacterales and Pseudomonas aeruginosa). It was evaluated using 50 CZA-susceptible and 42 CZA-resistant Gram-negative isolates. Its sensitivity and specificity of detection were 100%. Excellent performance of the medium was also observed by testing spiked stools, with the lower limit of detection ranging from 101 to 102 CFU/ml.


RESULTS
A total of 92 isolates of worldwide origin were included in this study to evaluate the performance of the SuperCAZ/AVI medium. The ␤-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 ϳ1.5 ϫ 10 8 CFU/ml), serial 10-fold dilutions were made in 0.85% saline solution, and 100-l 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 l 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 ϫ 10 3 CFU/ml, i.e., the CZA-resistant isolates recovered on SuperCAZ/AVI medium plates at Ͻ1 ϫ 10 3 CFU/ml were considered positive, while the CZA-susceptible isolates grown using an inoculum of Ն1 ϫ 10 3 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 ϫ 10 3 CFU/ml (1 ϫ 10 1 to 1 ϫ 10 2 CFU/ml) ( Table 2). In contrast, growth of Enterobacter cloacae the CZA-susceptible isolates was possible only when an inoculum of Ͼ10 3 CFU/ml was used (the lower limit of detection was above the cutoff value of 10 3 CFU/ml), giving rise to 100% sensitivity and specificity. Spiked stools were also tested with the same representative collection of CZAresistant and -susceptible Gram-negative bacteria (n ϭ 92) using this selective culture medium. Spiked fecal samples were made by adding 100 l of serial 10-fold bacterial dilutions to 900 l 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 l) of the spiked stool suspension were inoculated onto the SuperCAZ/AVI medium. Aliquots (100 l) 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 10 1 to 10 2 CFU/ml, whereas the lower limit of detection for the CZA-susceptible strains was above the cutoff value, at Ն10 6 CFU/ml ( Table 2). Sensitivity and specificity were determined using the same cutoff value, set at 10 3 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.

DISCUSSION
The SuperCAZ/AVI medium constitutes an adequate screening medium for the detection of CZA-resistant bacteria regardless of their resistance mechanisms. This SuperCAZ/AVI medium may be used for the screening of patients potentially colonized with CZA-resistant strains in order to rapidly implement infection control measures aimed at limiting their spread. This medium is also adequate for epidemiological surveys aiming to evaluate the prevalence of CZA-resistant Gram-negative bacteria in a given population. Further clinical evaluation of the proposed medium in daily clinical practice is needed. It may be useful for rapid identification of outbreaks of CZA-resistant strains, such as those reported in the United States (12) and Italy (13).