Small trans-encoded RNAs (sRNAs) modulate the translation and decay of mRNAs in bacteria. In Gram-negative species, antisense regulation by trans-encoded sRNAs relies on the Sm-like protein Hfq. In contrast to this, Hfq is dispensable for sRNA-mediated riboregulation in the Gram-positive species studied thus far. Here, we provide evidence for Hfq-dependent translational repression in the Gram-positive human pathogen Listeria monocytogenes, which is known to encode at least 50 sRNAs. We show that the Hfq-binding sRNA LhrA controls the translation and degradation of its target mRNA by an antisense mechanism, and that Hfq facilitates the binding of LhrA to its target. The work presented here provides the first experimental evidence for Hfq-dependent riboregulation in a Gram-positive bacterium. Our findings indicate that modulation of translation by trans-encoded sRNAs may occur by both Hfq-dependent and -independent mechanisms, thus adding another layer of complexity to sRNA-mediated riboregulation in Gram-positive species.

The present study indicates that reversal of methicillin resistance by thioridazine in MRSA may be explained by a reduced transcription of mecA and blaZ, resulting in a reduced protein level of PBP2a.

The cannabinoid cannabidiol (CBD) is characterised in this study as a helper compound against resistant bacteria. CBD potentiates the effect of bacitracin (BAC) against Gram-positive bacteria (Staphylococcus species, Listeria monocytogenes, and Enterococcus faecalis) but appears ineffective against Gramnegative bacteria. CBD reduced the MIC value of BAC by at least 64-fold and the combination yielded an FIC index of 0.5 or below in most Gram-positive bacteria tested. Morphological changes in S. aureus as a result of the combination of CBD and BAC included several septa formations during cell division along with membrane irregularities. Analysis of the muropeptide composition of treated S. aureus indicated no changes in the cell wall composition. However, CBD and BAC treated bacteria did show a decreased rate of autolysis. The bacteria further showed a decreased membrane potential upon treatment with CBD; yet, they did not show any further decrease upon combination treatment. Noticeably, expression of a major cell division regulator gene, ezrA, was reduced twofold upon combination treatment emphasising the impact of the combination on cell division. Based on these observations, the combination of CBD and BAC is suggested to be a putative novel treatment in clinical settings for treatment of infections with antibiotic resistant Gram-positive bacteria. Since the discovery of penicillin in 1928 by Sir Alexander Fleming, antibiotics have saved millions of lives from fatal infections worldwide. However, with time bacteria have developed mechanisms to escape the effects of antibiotics. The amount of antibiotics used seems to be directly related to development of antibiotic resistance. Similarly, a growing number of multi drug resistant (MDR) bacteria is a result of inadequate intentions to solve the resistance problem and increasing unmet demands for new antibacterial drugs 1. With fewer antibiotics available to treat MDR bacterial infections, the possibility of entering a pre-antibiotic era is looming ahead. Alternative strategies are being explored and helper compounds, also known as antibiotic potentiators or resistant breakers, are attracting attention 2. Helper compounds are non-antibiotic compounds functioning as adjuvants for antibiotics to operate in synergy through various mechanisms including efflux pump inhibition, inhibition of enzymes, and changes in membrane permeability, all of which may contribute to increasing the efficacy of a specific antibiotic 3,4. Drugs found to contain helper compound properties are normally used for treatment of non-infectious diseases but may contain some degree of antibacterial activity itself 5. Helper compounds are usually associated with side-of-action in the central and peripheral nervous system as local anaesthetics and in psychopharmaceutic practice, where they usually block membrane associated transporter activity 5. Overuse of antibiotics is the main cause of antibiotic resistance. Therefore, by combining an antibiotic with a helper compound less antibiotic is needed in...

Subinhibitory concentrations of the neuroleptic drug thioridazine (TDZ) are well-known to enhance the killing of methicillin-resistant Staphylococcus aureus (MRSA) by β-lactam antibiotics, however, the mechanism underlying the synergy between TDZ and β-lactams is not fully understood. In the present study, we have examined the effect of a subinhibitory concentration of TDZ on antimicrobial resistance, the global transcriptome, and the cell wall composition of MRSA USA300. We show that TDZ is able to sensitize the bacteria to several classes of antimicrobials targeting the late stages of peptidoglycan (PGN) synthesis. Furthermore, our microarray analysis demonstrates that TDZ modulates the expression of genes encoding membrane and surface proteins, transporters, and enzymes involved in amino acid biosynthesis. Interestingly, resemblance between the transcriptional profile of TDZ treatment and the transcriptomic response of S. aureus to known inhibitors of cell wall synthesis suggests that TDZ disturbs PGN biosynthesis at a stage that precedes transpeptidation by penicillin-binding proteins (PBPs). In support of this notion, dramatic changes in the muropeptide profile of USA300 were observed following growth in the presence of TDZ, indicating that TDZ can interfere with the formation of the pentaglycine branches. Strikingly, the addition of glycine to the growth medium relieved the effect of TDZ on the muropeptide profile. Furthermore, exogenous glycine offered a modest protective effect against TDZ-induced β-lactam sensitivity. We propose that TDZ exposure leads to a shortage of intracellular amino acids, including glycine, which is required for the production of normal PGN precursors with pentaglycine branches, the correct substrate of S. aureus PBPs. Collectively, this work demonstrates that TDZ has a major impact on the cell wall biosynthesis pathway in S. aureus and provides new insights into how MRSA may be sensitized towards β-lactam antibiotics.