COL(R) Acinetobacter baumannii sRNA Signatures: Computational Comparative Identification and Biological Targets

Multidrug-Resistant (MDR) and Extensively Drug Resistant (XDR) Acinetobacter baumannii (Ab) represent a serious cause of healthcare-associated infections worldwide. Currently, the available treatment options are very restricted and colistin-based therapies are last-line treatments of these infections, even though colistin resistant (COL(R)) Ab have rarely been isolated yet. In bacteria, small non-coding RNAs (sRNAs) have been implicated in regulatory pathways of different biological functions, however, no knowledge exists about the sRNA role on the biological adaptation in COL(R) Ab. Our study investigated two Italian XDR isogenic colistin-susceptible/resistant (COL(S/R)) Ab strain-pairs to discover new sRNA signatures. Comparative sRNA transcriptome (sRNAome) analyses were carried out by Illumina RNA-seq using both a Tru-Seq and a Short Insert library, whilst Ab ATCC 17978 and ACICU Reference Genome assembly, mapping, annotation and statistically significant differential expression (q-value ≤ 0.01) of the raw reads were performed by the Rockhopper tool. A computational filtering, sorting only similarly statistically significant differentially expressed (DE) sRNAs mapping on the same gene in both COL(R) Ab isolates was conducted. COL(R) vs. COL(S) sRNAome, analyzed integrating the DE sRNAs obtained from the two different libraries, revealed some statistically significant DE sRNAs in COL(R) Ab. In detail, we found: (i) two different under-expressed cis-acting sRNAs (AbsRNA(1) and AbsRNA(2)) mapping in antisense orientation the 16S rRNA gene A1S_r01, (ii) one under-expressed cis-acting sRNA (AbsRNA(3)) targeting the A1S_2505 gene (hypothetical protein), (iii) one under-expressed microRNA-size small RNA fragment (AbsRNA(4)) and its pre-microAbsRNA(4) targeting the A1S_0501 gene (hypothetical protein), (iv) as well as an over-expressed microRNA-size small RNA fragment (AbsRNA(5)) and its pre-microAbsRNA(5) targeting the A1S_3097 gene (signal peptide). Custom TaqMan(®) probe-based real-time qPCRs validated the expression pattern of the selected sRNA candidates shown by RNA-seq. Furthermore, analysis on sRNA ΔA1S_r01, ΔA1S_2505 as well as the over-expressed A1S_3097 mutants revealed no effects on colistin resistance. Our study, for the first time, found the sRNAome signatures of clinical COL(R) Ab with a computational prediction of their targets related to protein synthesis, host-microbe interaction and other different biological functions, including biofilm production, cell-cycle control, virulence, and antibiotic-resistance.