Evaluation of Shigella Species Azithromycin CLSI Epidemiological Cutoff Values and Macrolide Resistance Genes

Abstract

Azithromycin (AZM) has been recommended by the American Academy of Pediatrics for the treatment of shigellosis in children. In this study, 502 Shigella species isolated between 2004 and 2014 were tested for AZM epidemiological cutoff values (ECV) by disk diffusion. AZM MICs and the presence of the macrolide resistance genes [erm(A), erm(B), erm(C), ere(A), ere(B), mph(A), mph(B), mph(D), mef(A), and msr(A)] were determined for all 56 (11.1%) isolates with an AZM disk diffusion zone diameter of ≤15 mm. The distribution of AZM ECV MICs was also determined for 186 Shigella isolates with a disk zone diameter of ≥16 mm. Finally, pulsed-field gel electrophoresis (PFGE) was performed on 15 Shigella flexneri isolates with an AZM disk zone diameter of <16 mm from different years and geographic locations. Serotyping the 502 Shigella species isolates revealed that 373 (74%) were S. sonnei, 119 (24%) were S. flexneri, and 10 (2%) were S. boydii Of the 119 Shigella flexneri isolates, 48 (42%) isolates had an AZM disk diffusion zone diameter of ≤15 mm and a MIC of ≥16 µg/ml. With the exception of one isolate, all were positive for the macrolide resistance gene mph(A). S. flexneri PFGE showed four distinct patterns, with patterns I and II presenting with 92.3% genetic similarity. On the other hand, 2 (0.5%) of the 373 S. sonnei isolates had the AZM non-wild-type (NWT) ECV phenotype (those with acquired or mutational resistance), as the AZM MICs were ≥32 µg/ml and the isolates were positive for the mph(A) gene. Overall, our S. flexneri results are in concordance with the CLSI AZM ECV, but isolates with an AZM disk diffusion zone diameter between 14 and 15 mm should be carefully evaluated, as the S. flexneri AZM MIC for NWT isolates may need adjustment to 32 µg/ml. Our data on S. sonnei support that the AZM NWT ECV should be 11 mm for the disk diffusion zone diameter and ≥32 µg/ml for the MICs.

Keywords: Shigella; azithromycin; epidemiological cutoff values.

FIG 1

(A) Distribution of azithromycin MICs for Shigella species with azithromycin disk diffusion diameters of ≤15 mm. (B) Distribution of azithromycin MICs for Shigella species with azithromycin disk diffusion diameters of ≥16 mm.

FIG 2

(A) PFGE of Shigella flexneri NotI-digested chromosomal DNA along with antibiotic antimicrobial sensitivity testing patterns. In the columns headed “Antibiotics,” a gray box indicates a sensitive profile, and the absence of a gray box (white) indicates a resistant profile. The values on the tree represent the percent similarities between the isolates. (B) PFGE of Shigella flexneri XbaI-digested chromosomal DNA along with antibiotic antimicrobial sensitivity testing patterns. In the columns headed “Antibiotics,” a gray box indicates a sensitive profile, and the absence of a gray box (white) indicates a resistant profile. The values on the tree represent the percent similarities between the isolates.