Drug susceptibility testing of Nocardia spp. using the disk diffusion method

Abstract

Background: Drug susceptibility testing (DST) for Nocardia spp. is essential to initiate effective antibiotic therapy. Currently, the only recommended technique is the determination of minimum inhibitory concentrations (MICs) by microdilution. This method can be tedious to perform, despite the availability of ready-to-use plates. Herein, the aim was to determine the critical inhibition diameters specific to Nocardia spp.

Methods: MICs of 134 Nocardia isolates were determined by microdilution. Interpretative categories (Susceptible/Intermediate/Resistant) were determined using Clinical and Laboratory Standards Institute breakpoints. In parallel, disk diffusion DST was performed. Receiver-operating-characteristic (ROC) curves were constructed to determine the inhibition diameter value that best discriminated between susceptible and non-susceptible strains (intermediate/resistant). The category agreement (CA), the rate of major (maj) and very major (vmj) discrepancies between microdilution and disk diffusion method was calculated.

Results: For tobramycin, the critical diameter of 19 mm (diameter ≤ 19 mm = resistant strain; diameter > 19 mm = susceptible strain) provided a CA of 98.5%, 0.0% vmj, and 2.9% maj discrepancies, reaching strictly the acceptable performance criteria defined by the U.S. Food and Drug Administration (FDA). For amikacin, the critical diameter of 25 mm (diameter ≤ 25 mm = resistant strain; diameter > 25 mm = susceptible strain) provided a CA of 98.5%, 0.0% vmj, and 1.5% maj discrepancies. For imipenem, excluding N. farcinica and N. cyriacigeorgica, the critical diameter of 29 mm (diameter ≤ 29 mm = resistant strain; diameter > 29 mm = susceptible strain), provided a CA of 98.6%, 0.0% vmj, and 0.0% maj discrepancies. Despite an estimated vmj rate 0.0%, the 95%-confident-interval exceeded the FDA criteria due to an insufficient number of amikacin/imipenem-resistant strains. For other tested antibiotics (ciprofloxacin, moxifloxacin, amoxicillin-clavulanate, ceftriaxone, cotrimoxazole, linezolid), the FDA criteria were not reached.

Conclusions: Although the FDA criteria were mostly unmet, disk diffusion DST was suitable to accurately categorize Nocardia isolates into interpretative categories for the aminoglycosides and imipenem only, excluding species N. farcinica and N. cyriacigeorgica.

Keywords: Nocardia spp.; Critical inhibition diameters; Disk diffusion; Drug susceptibility testing; Microdilution.

Fig. 1

N. farcinica (n = 28 at 48 h and 31 at 72 h), N. cyriacigeorgica (n = 27 at 48 h and 31 at 72 h) and N. brasiliensis (n = 8 at 48 h and 9 at 72 h) isolates proportion according to interpretative category S (susceptible, in green), I (intermediate, in orange), R (resistant, in red) for imipenem (in the left) and ceftriaxone (in the right) according to the incubation time (48–72 h). Minimal inhibitory concentrations were determined by broth microdilution using Sensititre™ Myco RAPMYCOI and interpretative categories were determined using Clinical Laboratory Standard Institute critical concentrations. *** p < 0.001; ** p < 0.01; NS: not significant

Fig. 2

Distribution of Nocardia isolates excluding N. farcinica and N. cyriacigeorgica (n = 72) according to the inhibition diameter (mm) measured around the imipenem disk and their minimal inhibitory concentration (MIC) for imipenem determined by broth microdilution using Sensititre™ Myco RAPMYCOI after 48 h incubation. Shades of green: strains categorized as susceptible; orange: strains categorized as intermediate; shades of red: strains categorized as resistant according to Clinical Laboratory Standard Institute critical concentrations. Bold dotted line: critical inhibition diameter according to Youden index and the point closest to the upper left-hand corner of the ROC curve (ROC01) = 28 mm (inhibition diameter ≤ 28: imipenem I or R; inhibition diameter > 28: imipenem S)

Fig. 3

Distribution of Nocardia isolates excluding N. farcinica and N. cyriacigeorgica (n = 72) according to the inhibition diameter (mm) measured around the imipenem disk and their minimal inhibitory concentration (MIC) for imipenem determined by broth microdilution using Sensititre™ Myco RAPMYCOI after 72 h incubation. Shades of green: strains categorized as susceptible; orange: strains categorized as intermediate; shades of red: strains categorized as resistant according to Clinical Laboratory Standard Institute critical concentrations. Bold dotted line: critical inhibition diameter according to Youden index and the point closest to the upper left-hand corner of the ROC curve (ROC01) = 29 mm (inhibition diameter ≤ 29: imipenem I or R; inhibition diameter > 29: imipenem S)

Fig. 4

Distribution of Nocardia isolates (n = 112) according to the inhibition diameter (mm) measured around the amoxicillin-clavulanate disk and their minimal inhibitory concentration (MIC) for amoxicillin-clavulanate determined by broth microdilution using Sensititre™ Myco RAPMYCOI after 72 h incubation. Shades of green: strains categorized as susceptible; orange: strains categorized as intermediate; shades of red: strains categorized as resistant according to Clinical Laboratory Standard Institute critical concentrations. Bold dotted line: critical inhibition diameter according to Youden index and the point closest to the upper left-hand corner of the ROC curve (ROC01) = 20 mm (inhibition diameter ≤ 20: amoxicillin-clavulanate I or R; inhibition diameter > 20: amoxicillin-clavulanate S)

Fig. 5

Distribution of Nocardia isolates (n = 112) according to the inhibition diameter (mm) measured around the amikacin disk and their minimal inhibitory concentration (MIC) for amikacin determined by broth microdilution using Sensititre™ Myco RAPMYCOI after 48 h incubation. Shades of green: strains categorized as susceptible; red: strain categorized as resistant according to Clinical Laboratory Standard Institute critical concentrations. Bold dotted line: critical inhibition diameter according to Youden index and the point closest to the upper left-hand corner of the ROC curve (ROC01) = 22 mm (inhibition diameter ≤ 22: amikacin I or R; inhibition diameter > 22: amikacin S)

Fig. 6

Distribution of Nocardia isolates (n = 134) according to the inhibition diameter (mm) measured around the amikacin disk and their minimal inhibitory concentration (MIC) for amikacin determined by broth microdilution using Sensititre™ Myco RAPMYCOI after 72 h incubation. Shades of green: strains categorized as susceptible; shades of red: strains categorized as resistant according to Clinical Laboratory Standard Institute critical concentrations. Bold dotted line: critical inhibition diameter according to Youden index and the point closest to the upper left-hand corner of the ROC curve (ROC01) = 25 mm (inhibition diameter ≤ 25: amikacin I or R; inhibition diameter > 25: amikacin S)

Fig. 7

Distribution of Nocardia isolates (n = 112) according to the inhibition diameter (mm) measured around the tobramycin disk and their minimal inhibitory concentration (MIC) for tobramycin determined by broth microdilution using Sensititre™ Myco RAPMYCOI after 48 h incubation. Shades of green: strains categorized as susceptible; orange: strains categorized as intermediate; shades of red: strains categorized as resistant according to Clinical Laboratory Standard Institute critical concentrations. Bold dotted line: critical inhibition diameter according to Youden index = 19 mm (inhibition diameter ≤ 19: tobramycin I or R; inhibition diameter > 19: tobramycin S). Solid bold line: critical inhibition diameter according to the point closest to the upper left-hand corner of the ROC curve (ROC01) = 18 mm (inhibition diameter ≤ 18: tobramycin I or R; inhibition diameter > 18: tobramycin S)

Fig. 8

Distribution of Nocardia isolates (n = 134) according to the inhibition diameter (mm) measured around the tobramycin disk and their minimal inhibitory concentration (MIC) for tobramycin determined by broth microdilution using Sensititre™ Myco RAPMYCOI after 72 h incubation. Shades of green: strains categorized as susceptible; orange: strains categorized as intermediate; shades of red: strains categorized as resistant according to Clinical Laboratory Standard Institute critical concentrations. Bold dotted line: critical inhibition diameter according to Youden index and the point closest to the upper left-hand corner of the ROC curve (ROC01) = 19 mm (inhibition diameter ≤ 19: tobramycin I or R; inhibition diameter > 19: tobramycin S)

Fig. 9

Distribution of Nocardia isolates (n = 97) according to the inhibition diameter (mm) measured around the ciprofloxacin disk and their minimal inhibitory concentration (MIC) for ciprofloxacin determined by broth microdilution using Sensititre™ Myco RAPMYCOI after 48 h incubation. Shades of green: strains categorized as susceptible; orange: strains categorized as intermediate; shades of red: strains categorized as resistant according to Clinical Laboratory Standard Institute critical concentrations. Bold dotted line: critical inhibition diameter according to Youden index and the point closest to the upper left-hand corner of the ROC curve (ROC01) = 24 mm (inhibition diameter ≤ 24: ciprofloxacin I or R; inhibition diameter > 24: ciprofloxacin S)

Fig. 10

Distribution of Nocardia isolates (n = 134) according to the inhibition diameter (mm) measured around the ciprofloxacin disk and their minimal inhibitory concentration (MIC) for ciprofloxacin determined by broth microdilution using Sensititre™ Myco RAPMYCOI after 72 h incubation. Shades of green: strains categorized as susceptible; orange: strains categorized as intermediate; shades of red: strains categorized as resistant according to Clinical Laboratory Standard Institute critical concentrations. Bold dotted line: critical inhibition diameter according to Youden index = 25 mm (inhibition diameter ≤ 25: ciprofloxacin I or R; inhibition diameter > 25: ciprofloxacin S). Solid bold line: the point closest to the upper left-hand corner of the ROC curve (ROC01) = 27 mm (inhibition diameter ≤ 27: ciprofloxacin I or R; inhibition diameter > 27: ciprofloxacin S)

Fig. 11

Distribution of Nocardia isolates (n = 93) according to the inhibition diameter (mm) measured around the moxifloxacin disk and their minimal inhibitory concentration (MIC) for moxifloxacin determined by broth microdilution using Sensititre™ Myco RAPMYCOI after 48 h incubation. Shades of green: strains categorized as susceptible; orange: strains categorized as intermediate; shades of red: strains categorized as resistant according to Clinical Laboratory Standard Institute critical concentrations. Bold dotted line: critical inhibition diameter according to Youden index and the point closest to the upper left-hand corner of the ROC curve (ROC01) = 29 mm (inhibition diameter ≤ 29: moxifloxacin I or R; inhibition diameter > 29: moxifloxacin S)

Fig. 12

Distribution of Nocardia isolates (n = 134) according to the inhibition diameter (mm) measured around the moxifloxacin disk and their minimal inhibitory concentration (MIC) for moxifloxacin determined by broth microdilution using Sensititre™ Myco RAPMYCOI after 72 h incubation. Shades of green: strains categorized as susceptible; orange: strains categorized as intermediate; shades of red: strains categorized as resistant according to Clinical Laboratory Standard Institute critical concentrations. Bold dotted line: critical inhibition diameter according to Youden index = 33 mm (inhibition diameter ≤ 33: moxifloxacin I or R; inhibition diameter > 33: moxifloxacin S). Solid bold line: the point closest to the upper left-hand corner of the ROC curve (ROC01) = 32 mm (inhibition diameter ≤ 32: moxifloxacin I or R; inhibition diameter > 32: moxifloxacin S)