Acinetobacter baumannii`nin kolistin maruziyetine uyumu: İn vivo ve in vitro deneyimler

Acinetobacter baumannii (A.baumannii) is one of the emerging pathogens which causes severe infections with high mortality. Multidrug-resistant (MDR) and colistin-resistant A.baumannii spread worldwide especially in healthcare centers. A.baumannii has two genetic mechanisms of colistin resistance: Complete loss of LPS as a result of mutations in lpx genes and point mutations in pmrCAB operon. Both mechanisms cause structural changes and eventually prevent colistin binding to cell wall. However, triggering factors and evolutionary mechanisms of colistin resistance are still not clearly understood. In this study, we aimed to mimic induction of colistin resistance by colistin exposure, follow-up the progression of resistance and compare in vitro results with in vivo resistance identified in an isolate from a patient. A 35-years old female patient admitted in VKV American Hospital (Istanbul) intensive care unit between February and March 2016 was chosen for the study. A total of four colistin susceptible and one colistin resistant A.baumannii isolates were collected. Patient's clinical data and outcome were recorded. Daily serial passages of susceptible isolates in presence of colistin at 1 mg/L concentration were performed to mimic development of colistin resistance in vitro by colistin exposure. Minimum inhibitory concentrations (MICs) were measured by broth microdilution. The colistin-resistant isolate and certain generations of in vitro experiment were chosen for molecular analyses. The pmrCAB complete operon and lpxA, lpxC, and lpxD genes were sequenced. The expression levels of pmrC, pmrA, and pmrB were studied by qRT-PCR. The patient's data and the results of molecular tests were compared. The pmrA, pmrB and pmrC genes were 1.6, 1.74 and 1.72 times overexpressed in colistin resistant clinical isolate than susceptible one. Colistin resistance was identified in the patient at 25th day of the colistin therapy. In 38 serial passages of four susceptible isolates, colistin MIC values were above breakpoint level of 2 mg/L after first passage. At the 26th generation of experiment, pmrC expressions of all isolates reached to a peak level (2.11; 29.65-fold). Moreover, in 2 of the four isolates pmrA (1.97 and 8.54-fold) and pmrB (2.31 and 11.24-fold) expressions were at the highest level in the same generation. We also detected multiple insertions in pmrA, pmrB, pmrC, lpxA, lpxC and lpxD genes. In conclusion, upregulation of pmrC, pmrA, and pmrB in combination with insertions in these genes and lpx genes may trigger development of colistin resistance. The highest pmrCAB expressions after 26th day of exposure and isolation of resistant strain from the patient at 25th day of colistin therapy suggested us that long term therapy is required for development of colistin resistance in A.baumannii. Therefore, duration of colistin use and combined therapy options should be considered and controlled properly during colistin therapy.