The MIC-inhibition zone diameter distributions for CAZ, MEM, AMC and DOX against B. pseudomallei are displayed (Fig. 1). The mode and distributions of DD zone diameters and BMD MIC are 28, 6–32 mm and 4, 1–128 μg/ml (CAZ); 26, 17–32 mm and 1, 0.25–4 μg/ml (MEM); 25, 20–28 mm and 8, 4–16 μg/ml (AMC); and 28 and 29, 24–33 mm and 2, 0.5–8 μg/ml (DOX) (Fig. 1 and Tables 1 and 2). EUCAST recommends the use of susceptible, increased exposure (SI) instead of intermediate-resistant (I) to indicate higher dose antibiotics required for the isolates resides between susceptible (S) and resistant (R) category [7]. In this study, (S), (SI) and (I) were used to indicate non-resistant isolates. DD produced good categorical agreements to differentiate antibiotic-resistant and non-resistant isolates, exhibiting concordance of 100% with BMD for CAZ and DOX, 98.6% for MEM and 97.2% for AMC. Pearson’s correlations between DD and BMD were 0.69 and 0.45 (p < 0.01) for CAZ and MEM, respectively. Sixty-two AMC-intermediate-resistant isolates (16 μg/ml) based on CLSI MIC cutoff were termed as non-resistant and within the (SI) range (22–49 mm) except four isolates interpreted as R by DD at 20–21 mm (resistant, < 22 mm). The MIC50 was 4 μg/ml, 1 μg/ml, 8 μg/ml and 2 μg/ml and MIC90 was 4 μg/ml, 2 μg/ml, 16 μg/ml and 2 μg/ml for CAZ, MEM, AMC and DOX, respectively.

Antibiotic MIC-inhibition zone diameter distributions of B. pseudomallei for A ceftazidime, B meropenem, C amoxicillin-clavulanate and D doxycycline. Corresponding MIC values are shown through the colouring of bars. Red bar indicates resistant (R) which correspond to CLSI M45 MIC breakpoints for B. pseudomallei except meropenem by EUCAST MIC breakpoint

In this study, 19 of 143 B. pseudomallei isolates do not grow on ASH. The most frequently observed colony morphotypes were types VI, III and IV each at > 20% with smooth surface in the centre of colony (Table 3). Further investigation on the antibiotic susceptibility profile showed that reduced antibiotic susceptibility for AMC was observed in all colony morphotypes except type V with highest frequency in type VI. CAZ-R, CAZ-I and MEM-R were observed in each of types VII, I and IV, respectively. Among 135 patients’ samples, an additional eight different B. pseudomallei colony morphotypes were observed on ASH from eight patients’ samples. The isolates were cultured from IMRS54 (A and B), IMRS71 (A and B), IMRS101 (M and W), IMRS103 (A and B), IMRS131 (P and D), IMRS166 (P and D), IMRS187 (A and B) and IMRS195 (A and B) (Table S1). Among the eight samples, combination of colony morphotypes III-VI were most commonly observed (37.5%) followed by III-IV (25%), II-VI (25%) and IV-VI (12.5%). All showed similar antibiotic susceptibility pattern between both colony morphotypes. The details of each colony morphotype and antibiotic susceptibility are described in Table S1.