Our study for the first time investigated the presence of hetero- and adaptive resistance to polymyxin B in unrelated OXA-23-producing CRAB isolates. Additionally, the stability of these phenomena was evaluated in two distinct conditions. Since the susceptibility breakpoint for polymyxin B according to CLSI is 2 mg/L , real hetero-resistance for polymyxin B was not found in any isolate, differently from previous studies with colistin [9, 16, 17]. However, the presence of “higher MIC” subpopulations, within the susceptibility range, was detected in 90% of tested isolates, including at least one isolate representative of each of the 15 clones. These “higher MIC” subpopulations presented MICs 2- to at least 4-fold dilutions higher than the original population.
The presence of adaptive resistance to polymyxin B was shown in 55% of 22 tested isolates (present in 7 of 15 clones), all demonstrating high-level resistance to polymyxin B (MIC = 64 mg/L). Although some molecular mechanisms of adaptive resistance to polymyxins, such as mutations in pmr CAB and lpxA gene in A. baumannii[18, 19] and PhoP-PhoQ, PmrA-PmrB and recently ParR-ParS in P. aeruginosa, have been characterized, the presence of this resistance phenotype has not been systematically evaluated. Thus, our study further suggests that adaptative resistance might be most common than possibly expected, at least in CRAB, since approximately half of tested clones showed such adaptive phenotype. Indeed, the frequency might be even higher if the agar plate with the lowest polymyxin B concentration had <0.25 mg/L of the drug. Although seven isolates with MIC ≤0.125 mg/L still have growth on these plates, these concentrations may have inhibited the growth of other eight isolates.
The present study also showed that the MIC of the “higher MIC” subpopulations remained stable after 4-days into antimicrobial-free medium, but returns to the MIC of the original population after storage at −80°C, suggesting that it might involve some molecular basis also associated with an unstable phenotype. As expected, since without the drug-sustaining effect the adaptive resistance is unstable, the MICs of resistant isolates selected in the adaptive resistance experiment decreased 1- to 2-fold dilutions after serial passage into antimicrobial-free medium and all tested isolates returns to the baseline level after the storage at −80°C.
Only one isolate that has presented adaptive resistance has not presented “higher MIC” subpopulation in PAP. It belongs to the clone A, which has other three isolates tested in both experiments, all showing the presence of both phenomena. It is also interesting that these latter three isolates were identical by typing while the former showed 92% of similarity with these latter ones (data not shown). Another isolate has neither presented “higher MIC” subpopulation nor adaptive resistance and belongs to a clone with two representative isolates among the 80 CRAB typed in this study.
Unfortunately, we were not able to determine the molecular determinants of these phenotypes in this study. We also could not determine if the absence of real hetero-resistance (i.e. presence of subpopulations with MICs higher than the susceptibility breakpoint) was a specific characteristic of polymyxin B, and would occur with colistin, or “higher MIC” subpopulations within the susceptibility range was only detected, instead of subpopulations with “resistance MICs” because the baseline MIC of half of the tested isolates were very low (≤0.125 mg/L).
In summary, our study showed that the presence of “higher MIC” subpopulations in CRAB isolates was extremely common. Additionally, high-level adaptive resistance was also very frequent. The clinical significance of each phenomenon should be further investigated, since both may potentially affect the outcomes of patients on therapy with polymyxins.