Antibiogram results of parent isolates and standard strain:
The clinical isolates were resistant to ampicillin/sulbactam, piperacillin, ceftazidime, cefoperazone/sulbactam, cefepime, imipenem, meropenem, ciprofloxacin, tetracycline and trimethoprim/sulfamethoxazole, and had intermediate resistance to amikacin, and were susceptible to gentamicin and colistin by the VITEK2 antibiogram system. Carbapenem resistance of the clinical isolates were confirmed with MIC evaluator strips and the isolates were found resistant to imipenem and meropenem with MIC > 32 µg/mL.
The standard ATCC strain was resistant to trimethoprim/sulfamethoxazole, had intermediate resistance resistant to cefepime, and was susceptible to ampicillin/sulbactam, piperacillin, ceftazidime, cefoperazone/sulbactam, imipenem, meropenem, amikacin, gentamicin, ciprofloxacin, tetracycline and colistin by the VITEK2 antibiogram system. Imipenem and meropenem susceptibilities were also confirmed with MIC evaluator strips and it was found that MIC = 0.25 µg/mL for both carbapenems.
Colistin MIC values for the clinical isolates and ATCC strain were also determined by using a gradient test. MICs for the clinical isolates were found between 0.5 and 0.75 µg/mL and MIC for the ATCC strain was found 0.5 µg/mL. Colistin MIC values for the clinical isolates and ATCC strain were confirmed using the broth microdilution method. All were found susceptible to colistin with MIC = 0.5 µg/mL.
New MIC results of two strains during serial dilution test in the first round:
At the beginning, the colistin MICs for the selected clinical isolate (no:1) and ATCC strain were 0.75 µg/mL and 0.5 µg/mL, respectively (by the gradient test). After 48 hours incubation with 0.5x MIC colistin concentrations, the MIC values of the strains rose to 0.75 µg/mL and 1 µg/mL, respectively (step 1). After 48 hours incubation with 2x MIC colistin concentrations, the colistin MIC values of the strains rose to 1 µg/mL and 3 µg/mL, respectively (step 2). After 48 hours incubation with 32x MIC colistin concentrations, the new MIC values of the strains rose to the 32 µg/mL level for both strains (step 3). But in this step, interestingly, the clone of the clinical isolate became heteroresistant and there was another inhibition zone with MIC=2 µg/mL. Therefore, we determined two different MICs for clinical isolate in this step (Outer zone MIC=32 µg/mL, inner zone MIC=2 µg/mL) (step 3). In step 4, the MIC of the clinical isolate increased to 64 µg/mL level and no heteroresistance was observed this time. However, regarding the ATCC clone, we observed two different colony morphologies (large-mucoid and small-pale) on SBA and decided to determine the MIC values of each colony types separately. For this reason, we purified two different colony types by subculturing a single colony onto another SBA. After the purification step, we determined that the MIC of the large-mucoid colony type was 1 µg/mL, but the MIC of the small-pale colony type was 64 µg/mL. So we had another heteroresistant clone in this step. The new MIC results obtained during the serial dilution test are summarized in Table 1.
For determination of the new colistin MICs, the clones (with elevated colistin MICs) were inoculated to normal MHA plates and MHA plates containing 14 µg/mL MB powder (MBMHA) in steps 2, 3 and 4.
In step 2, the colistin MIC values of the ATCC strain and the clinical isolate were found to be 1 µg/mL and 3 µg/mL, respectively (on normal MHA). But their MIC values decreased to 0.5 µg/mL level on MBMHA.
In step 3, the MIC value of the ATCC strain was 32 µg/mL on MHA, but on MBMHA, the colistin-resistant bacteria were totally inhibited by MB and no growth was observed (Figure 1A, 1B). For the heteroresistant clinical isolate (MIC: 32, 2 µg/mL), the MIC value decreased to the 0.5 µg/mL level on MBMHA by the effect of MB (Figure 1C, 1D).
In step 4, the ATCC strain exhibited two different colony morphologies on SBA. The first colony type was small-pale, the second colony type was large-mucoid (Figure 2A). Each type of ATCC colony was investigated after being subcultured onto another SBA (Figure 3). The MIC value of the small-pale colony type was found 64 µg/mL on MHA, but on MBMHA, the colistin-resistant clones were totally inhibited by the effect of MB and no growth was observed. The MIC value of the large-mucoid colony type was found to be 1 µg/ml on MHA, but decreased to 0.5 µg/mL on MBMHA (Table 1).
In step 4, the clinical isolate colonies appeared homogeneously small-pale on SBA. The MIC value was 64 µg/mL on MHA, but on MBMHA, the colistin-resistant bacteria were totally inhibited by MB and no growth was observed. The results are summarized in Table 1.
EMB agar sensitivities of two strains with elevated MICs in the first round (Growth characteristics on EMB agar and SBA)
Subcultures of the clinical isolate and ATCC strain could grow on both SBA and EMB agar in steps 1 and 2 (in these steps all the MIC values of the clones were <= 3 µg/mL).
In step 3, we observed only small-pale colonies (MIC= 32 µg/mL) on the SBA plate of the ATCC strain. When we examined the SBA plate of the clinical isolate, we detected two types of colony (small-pale, large-mucoid). The small-pale colonies of both strains were totally inhibited on EMB agar while the large-mucoid colonies could survive on EMB agar (Table 2, Figure 2A, 2B, 2C, 2D).
In step 4, we observed only small-pale colonies (MIC= 64 µg/mL) on the SBA plate of the clinical isolate (Figure 2C) and they were totally inhibited on the EMB agar plates (Figure 2D). On the SBA plate of the ATCC strain, we detected two types of colony, which were small-pale and large-mucoid (Figure 2A). Small-pale colonies were totally inhibited by EMB while large-mucoid colonies could survive on EMB agar (Figure 2B). As a fifth step, two different colony types from SBA were also subcultured onto another SBA and EMB agar for confirmation (Figure 3). We again determined that the small-pale colony type was inhibited by EMB while the large-mucoid colony type could grow on both SBA and EMB agar. All results are summarized in Table 2.
After the induction and selection procedure of resistant clones, new MIC values of all 31 clinical isolates rose to 8 µg/mL. At the end of the EMB sensitivity/screening test, 11 out of 31 resistant clones could not grow on EMB agar (35 %) while they could grow well on SBA.