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 (AbsRNA₁ and AbsRNA₂) mapping in antisense orientation the 16S rRNA gene A1S_r01, (ii) one under-expressed cis-acting sRNA (AbsRNA₃) targeting the A1S_2505 gene (hypothetical protein), (iii) one under-expressed microRNA-size small RNA fragment (AbsRNA₄) and its pre-microAbsRNA₄ targeting the A1S_0501 gene (hypothetical protein), (iv) as well as an over-expressed microRNA-size small RNA fragment (AbsRNA₅) and its pre-microAbsRNA₅ 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.