In this work we have used the 5S RNA as a loading control for northern blot assays. Given that it is a ribosomal RNA we wondered whether the 5S RNA levels would be affected by either tigecycline or tetracycline exposure. As shown in Figure 4A, the 5S RNA expression levels were unaltered when the cells were challenged with AZD1390 half the MIC of tigecycline or tetracycline, and therefore it is a suitable

loading control for the northern blot assays. The four sRNAs (sYJ5, sYJ20, sYJ75 and sYJ118) that were upregulated as a response to tigecycline challenge in S. Typhimurium were also upregulated in tetracycline challenged cells (Figures 2A and 3A). This is not surprising since both tigecycline and tetracycline target the 30S ribosomal subunit. It is possible that the similar mechanisms of action of tetracycline and tigecycline trigger comparable stress-responsive pathways, which possibly include sYJ5, sYJ20, sYJ75 and sYJ118. sYJ75 has not been previously described and thus is also a novel sRNA discovered in this study. Its conservation among several species and its upregulation in S. Typhimurium upon challenge with tigecycline and tetracycline, (Figures 2A, 3A) suggest that sYJ75, combined with its conservation across different species, may represent a common denominator in the response to tigecycline

/ tetracycline exposure. Interestingly, none of the four sRNAs were found upregulated when S. Typhimurium was exposed Pregnenolone to ciprofloxacin, or when

E. coli was challenged with tigecycline (Figure 3B). When challenged with tigecycline, both S. Typhimurium and Vactosertib purchase K. pneumoniae upregulated two sRNAs, namely sYJ20 and sYJ118 (Figure 3B). Despite encoding these sequences, no upregulation was noted in E. coli cells exposed to tigecycline compared to the unexposed controls (Figure 3B). This suggests two possibilities: the first, where the tigecycline stress response involving sRNAs in E. coli is different from that in K. pneumoniae and S. Typhimurium, and the second, where the sRNAs (sYJ20 and sYJ118) may be linked to regulatory networks contributing to tigecycline resistance, i.e. RamA, only found in S. Typhimurium and K.pneumoniae but not in E. coli[40, 41]. However TargetRNA [42] predictions for sYJ20 for cognate mRNA binding partners, using default parameters, yields four mRNA sequences (Table 1). Of note, pspB and pspA which are involved in stress-response and the virulence attributes of several bacterial species [43] are potential targets of sYJ20. sYJ20-mediated control of the psp operon may explain the reduced fitness of the sroA (sYJ20) deleted Salmonella strain in a mouse infection model [44]. Table 1 TargetRNA predictions for sYJ20 Rank Gene Synonym Score Smoothened Agonist p-value sRNA start sRNA stop mRNA start mRNA stop 1 pspB STM1689 −60 0.00598756 17 28 9 −3 2 nrdI STM2806 −60 0.00598756 17 28 9 −3 3 STM0269 STM0269 −59 0.00721216 7 29 16 −4 4 pspA STM1690 −59 0.