Clinical Microbiology and Antimicrobial Chemotherapy. 2024; 26(4):439-451
To develop criteria for the interpretation and quality assurance of the results of susceptibility testing of Enterobacterales and P. aeruginosa to cefepime-sulbactam.
The study included 1035 clinical Enterobacterales isolates and 407 P. aeruginosa isolated in 2023 from hospitalized patients with nosocomial and community-acquired infections. Determination of cefepime and cefepime-sulbactam MICs was performed by broth microdilution method in Mueller-Hinton II broth in accordance with ISO 20776 1:2019. Determination of MICs was performed in the concentration ranges of cefepime: 0.06–64 mg/L for Enterobacterales, 0.125–128 mg/L for P. aeruginosa, without sulbactam, as well as in the presence of a fixed sulbactam concentration (4 mg/L). Testing was performed in 25 sets simultaneously with testing of control strains – E. coli ATCC 25922, K. quasipneumoniae ATCC 700603 and P. aeruginosa ATCC 27853 (in 5 independent repeats for each strain in each set). Susceptibility to cefepime-sulbactam by disk-diffusion method was determined using Mueller-Hinton agar and disks containing a combination of cefepime and sulbactam (30 + 10 µg, Lot No. 111123002, Liofilchem S.r.l., Italy) according to the standard EUCAST method. Zones of growth inhibition (ZGI) values were determined in 3 replicates for all clinical isolates and 5 replicates for each control strain. Standard methods of descriptive statistics and Microsoft Excel charting tools were used to analyze the distributions of MIC and ZGI values. Calculation of the borderline values of ZGI for determining clinical susceptibility categories for cefepime-sulbactam was performed by constructing a negative linear regression reflecting the relationship between log2(MIC) and ZGI values, and determining the ZGI values corresponding to the borderline values of cefepime MIC established by EUCAST.
For the two most common Enterobacterales species (E. coli and K. pneumoniae) close values of regression coefficients and good negative correlation of MIC and ZGI values were established (R2 > 0.8). Subanalysis of the data on the distribution of MIC and ZGI values for other Enterobacterales species showed a low correlation (R2 = 0.4947) between these parameters in species producing intrinsic AmpC cephalosporinases: C. freundii, E. cloacae complex spp., H. alvei, K. aerogenes, M. morganii, Providencia spp. and Serratia spp. (isolates belonging to these species were excluded from further analysis). The final sample for calculating cefepime-sulbactam ZGI breakpoints for Enterobacterales included 890 isolates (2670 comparisons) belonging to 9 species: C. koseri, E. coli, K. oxytoca, K. pneumoniae, P. mirabilis, P. penneri, P. vulgaris, R. ornithinolytica and S. enterica. The R2 value of the linear regression was 0.8912. The calculated breakpoints for ZGI: S ≥ 26 mm, R < 22 mm. Values of 22–25 mm coinciding with the clinical susceptibility category “I” was also marked as “zone of technical uncertainty – ZTN” according to the EUCAST definition, because it includes a significant number of isolates that can be categorized differently when determining sensitivity by DDM and on the basis of MIC estimation. For P. aeruginosa, the R2 value of the linear regression was 0.8131; the calculated breakpoint for ZGI was R < 25 mm; the zone of technical uncertainty was 24–27 mm. For control strains: ZGI values of K. quasipneumoniae ATCC 700603 differed withing 4 mm (range: 25–28; mean: 27.6; median: 28 mg/L); E. coli ATCC 25922 – withing 2 mm (range: 33–35; mean: 34.7; median: 35 mg/l); P. aeruginosa ATCC 27853 – withing 2 mm (range: 31–33; mean: 31.9; median: 32 mg/l).
The developed criteria for interpretation and quality assurance of the results of susceptibility testing of Enterobacterales and P. aeruginosa to cefepime-sulbactam can be recommended for approval and inclusion in the Russian recommendations “Determination of sensitivity of microorganisms to antimicrobial agents”.