Novel inhibitors of the Staphylococcus aureus quorum sensing system

The recent rise of antibiotic resistant micro-organisms has led to an increased demand for the production of novel and effective antimicrobial therapies. Staphylococcus aureus is a Gram positive human pathogen that some clinical isolate has evolved resistance to all currently available ‘front line’ antibiotics. In this study a series of small molecules, both natural and synthetic, were assessed for their ability to inhibit S. aureus virulence factor production, in the hope to identify novel molecules that inhibit S. aureus pathogenicity. Such molecules have the advantage over classical antibiotics in that they do not generate a selective pressure that can cause the evolution of drug resistant strains. IQS (Integrated Quorum Sensing Signal, 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde), is a secondary metabolite produced by Pseudomonas aeruginosa that has been suggested to function as a quorum sensing signal molecule. Here, IQS was found to inhibit S. aureus exotoxin production. In an attempt to improve potency, a small library of structurally diverse IQS analogues was synthesised. Two analogues were shown to have increased potency relative to natural IQS. Interestingly, data suggest that the active analogues, which are structurally distinct, have a different mechanism of action. The use of both in vivo reporters and biochemical analysis with purified AgrA, the response regulator that transduces the S. aureus QS signal, suggested that the lead analogue (IQS3) binds to and disrupts the DNA binding of AgrA, thus inhibiting QS by S. aureus. RNAseq data support the biochemical analysis in that the expression of virulence genes by S. aureus treated with the lead analogue mirrors that an S. aureus QS mutant. Finally, the cytotoxicity of our lead analogues were assessed. Overall, data suggest that our lead IQS analogue is a good ‘starter’ molecule for further modification in the hope to increase anti-virulence properties whilst reducing cytotoxicity

The obligate intracellular bacterium Orientia tsutsugamushi differentiates into a developmentally distinct extracellular state

Orientia tsutsugamushi (Ot) is an obligate intracellular bacterium in the family Rickettsiaceae that causes scrub typhus, a severe mite-borne human disease. Its mechanism of cell exit is unusual amongst Rickettsiaceae, as Ot buds off the surface of infected cells enveloped in plasma membrane. Here, we show that Ot bacteria that have budded out of host cells are in a distinct developmental stage compared with intracellular bacteria. We refer to these two stages as intracellular and extracellular bacteria (IB and EB, respectively). These two forms differ in physical properties: IB is both round and elongated, and EB is round. Additionally, IB has higher levels of peptidoglycan and is physically robust compared with EB. The two bacterial forms differentially express proteins involved in bacterial physiology and host-pathogen interactions, specifically those involved in bacterial dormancy and stress response, and outer membrane autotransporter proteins ScaA and ScaC. Whilst both populations are infectious, entry of IB Ot is sensitive to inhibitors of both clathrin-mediated endocytosis and macropinocytosis, whereas entry of EB Ot is only sensitive to a macropinocytosis inhibitor. Our identification and detailed characterization of two developmental forms of Ot significantly advances our understanding of the intracellular lifecycle of an important human pathogen

The utility of an AMR dictionary as an educational tool to improve public understanding of antimicrobial resistance

Background: Communicating about antimicrobial resistance (AMR) to the public is challenging.   Methods: We developed a dictionary of terms commonly used to communicate about AMR. For each term, we developed learning points to explain AMR and related concepts in plain language.  We conducted a pilot evaluation in 374 high school students in Ubon Ratchathani, Thailand. In three 50-minute sessions, students were asked to answer five true/false questions using a paper-based questionnaire. The first session assessed their understanding of AMR at baseline, the second after searching the internet, and the third after the provision of the printed AMR dictionary and its web address.    Results: We developed the AMR dictionary as a web-based application (www.amrdictionary.net). The Thai version of the AMR dictionary included 35 terms and associated learning points, seven figures displaying posters promoting AMR awareness in Thailand, and 66 recommended online videos. In the pretest, the proportion of correct responses to each question ranged from 10% to 57%; 10% of the students correctly answered that antibiotics cannot kill viruses and 57% correctly answered that unnecessary use of antibiotics makes them ineffective. After the internet searches, the proportions of correct answers increased, ranging from 62% to 89% (all p&lt;0.001). After providing the AMR dictionary, the proportions of correct answers increased further, ranging from 79% to 89% for three questions (p&lt;0.001), and did not change for one question (p=0.15). Correct responses as to whether taking antibiotics often has side-effects such as diarrhoea reduced from 85% to 74% (p&lt;0.001). The dictionary was revised based on the findings and comments received. Conclusions: Understanding of AMR among Thai high school students is limited. The AMR dictionary can be a useful supportive tool to increase awareness and improve understanding of AMR. Our findings support the need to evaluate the effectiveness of communication tools in the real-world setting.</ns3:p

Novel inhibitors of the Staphylococcus aureus quorum sensing system

The recent rise of antibiotic resistant micro-organisms has led to an increased demand for the production of novel and effective antimicrobial therapies. Staphylococcus aureus is a Gram positive human pathogen that some clinical isolate has evolved resistance to all currently available ‘front line’ antibiotics. In this study a series of small molecules, both natural and synthetic, were assessed for their ability to inhibit S. aureus virulence factor production, in the hope to identify novel molecules that inhibit S. aureus pathogenicity. Such molecules have the advantage over classical antibiotics in that they do not generate a selective pressure that can cause the evolution of drug resistant strains. IQS (Integrated Quorum Sensing Signal, 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde), is a secondary metabolite produced by Pseudomonas aeruginosa that has been suggested to function as a quorum sensing signal molecule. Here, IQS was found to inhibit S. aureus exotoxin production. In an attempt to improve potency, a small library of structurally diverse IQS analogues was synthesised. Two analogues were shown to have increased potency relative to natural IQS. Interestingly, data suggest that the active analogues, which are structurally distinct, have a different mechanism of action. The use of both in vivo reporters and biochemical analysis with purified AgrA, the response regulator that transduces the S. aureus QS signal, suggested that the lead analogue (IQS3) binds to and disrupts the DNA binding of AgrA, thus inhibiting QS by S. aureus. RNAseq data support the biochemical analysis in that the expression of virulence genes by S. aureus treated with the lead analogue mirrors that an S. aureus QS mutant. Finally, the cytotoxicity of our lead analogues were assessed. Overall, data suggest that our lead IQS analogue is a good ‘starter’ molecule for further modification in the hope to increase anti-virulence properties whilst reducing cytotoxicity