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Staphylococcus aureus colonizes patients with atopic dermatitis (AD) and exacerbates disease by promoting inflammation. The present study investigated the safety and mechanisms of action of Staphylococcus hominis A9 ( Sh A9), a bacterium isolated from healthy human skin, as a topical therapy for AD. Sh A9 killed S. aureus on the skin of mice and inhibited expression of a toxin from S. aureus ( psm α) that promotes inflammation. A first-in-human, phase 1, double-blinded, randomized 1-week trial of topical Sh A9 or vehicle on the forearm skin of 54 adults with S. aureus -positive AD (NCT03151148) met its primary endpoint of safety, and participants receiving Sh A9 had fewer adverse events associated with AD. Eczema severity was not significantly different when evaluated in all participants treated with Sh A9 but a significant decrease in S. aureus and increased Sh A9 DNA were seen and met secondary endpoints. Some S. aureus strains on participants were not directly killed by Sh A9, but expression of mRNA for psm α was inhibited in all strains. Improvement in local eczema severity was suggested by post-hoc analysis of participants with S. aureus directly killed by Sh A9. These observations demonstrate the safety and potential benefits of bacteriotherapy for AD. First-in-human test of topical application of a commensal bacterium on skin of individuals with atopic dermatitis reduces colonization by proinflammatory Staphylococcus aureus .

A key aspect of bacterial survival is the ability to evolve while migrating across spatially varying environmental challenges. Laboratory experiments, however, often study evolution in well-mixed systems. Here, we introduce an experimental device, the microbial evolution and growth arena (MEGA)-plate, in which bacteria spread and evolved on a large antibiotic landscape (120×60 centimeters) that allowed visual observation of mutation and selection in a migrating bacterial front. While resistance increased consistently, multiple coexisting lineages diversified both phenotypically and genotypically. Analyzing mutants at and behind the propagating front, we found that evolution is not always led by the most resistant mutants; highly resistant mutants may be trapped behind more sensitive lineages. The MEGA-plate provides a versatile platform for studying microbial adaption and directly visualizing evolutionary dynamics.

Bacterial persister cells are phenotypic variants that exhibit a transient non-growing state and antibiotic tolerance. Here, we provide in vitro evidence of Staphylococcus aureus persisters within infected host cells. We show that the bacteria surviving antibiotic treatment within host cells are persisters, displaying biphasic killing and reaching a uniformly non-responsive, non-dividing state when followed at the single-cell level. This phenotype is stable but reversible upon antibiotic removal. Intracellular S. aureus persisters remain metabolically active but display an altered transcriptomic profile consistent with activation of stress responses, including the stringent response as well as cell wall stress, SOS and heat shock responses. These changes are associated with multidrug tolerance after exposure to a single antibiotic. We hypothesize that intracellular S. aureus persisters may constitute a reservoir for relapsing infection and could contribute to therapeutic failures. Bacterial persister cells exhibit a transient non-growing state and antibiotic tolerance. Here, Peyrusson et al. provide evidence of metabolically active Staphylococcus aureus persisters within infected host cells exposed to antibiotics and analyse transcriptomic alterations associated with persistence.

Microbial biocontainment is an essential goal for engineering safe, next-generation living therapeutics. However, the genetic stability of biocontainment circuits, including kill switches, is a challenge that must be addressed. Kill switches are among the most difficult circuits to maintain due to the strong selection pressure they impart, leading to high potential for evolution of escape mutant populations. Here we engineer two CRISPR-based kill switches in the probiotic Escherichia coli Nissle 1917, a single-input chemical-responsive switch and a 2-input chemical- and temperature-responsive switch. We employ parallel strategies to address kill switch stability, including functional redundancy within the circuit, modulation of the SOS response, antibiotic-independent plasmid maintenance, and provision of intra-niche competition by a closely related strain. We demonstrate that strains harboring either kill switch can be selectively and efficiently killed inside the murine gut, while strains harboring the 2-input switch are additionally killed upon excretion. Leveraging redundant strategies, we demonstrate robust biocontainment of our kill switch strains and provide a template for future kill switch development. Biocontainment is a key to developing safe genetically-engineered microbes (GEMs). Here the authors demonstrate genetically stable CRISPR-based kill switches that control GEMs’ viability in animal hosts, enabling their safe biomedical applications.

New drugs, but also shorter, better-tolerated regimens are needed to tackle the high global burden of tuberculosis complicated by drug resistance and retroviral disease. We investigated new multiple-agent combinations over the first 14 days of treatment to assess their suitability for future development. In this prospective, randomised, early bactericidal activity (EBA) study, treatment-naive, drug-susceptible patients with uncomplicated pulmonary tuberculosis were admitted to hospitals in Cape Town, South Africa, between Oct 7, 2010, and Aug 19, 2011. Patients were randomised centrally by computer-generated randomisation sequence to receive bedaquiline, bedaquiline-pyrazinamide, PA-824-pyrazinamide, bedaquiline-PA-824, PA-824-moxifloxacin-pyrazinamide, or unmasked standard antituberculosis treatment as positive control. The primary outcome was the 14-day EBA assessed in a central laboratory from the daily fall in colony forming units (CFU) of M tuberculosis per mL of sputum in daily overnight sputum collections. Bilinear regression curves were fitted for each group separately and groups compared with ANOVA for ranks, followed by pair-wise comparisons adjusted for multiplicity. Clinical staff were partially masked but laboratory personnel were fully masked. This study is registered, NCT01215851. The mean 14-day EBA of PA-824-moxifloxacin-pyrazinamide (n=13; 0·233 [SD 0·128]) was significantly higher than that of bedaquiline (14; 0·061 [0·068]), bedaquiline-pyrazinamide (15; 0·131 [0·102]), bedaquiline-PA-824 (14; 0·114 [0·050]), but not PA-824-pyrazinamide (14; 0·154 [0·040]), and comparable with that of standard treatment (ten; 0·140 [0·094]). Treatments were well tolerated and appeared safe. One patient on PA-824-moxifloxacin-pyrazinamide was withdrawn because of corrected QT interval changes exceeding criteria prespecified in the protocol. PA-824-moxifloxacin-pyrazinamide is potentially suitable for treating drug-sensitive and multidrug-resistant tuberculosis. Multiagent EBA studies can contribute to reducing the time needed to develop new antituberculosis regimens. The Global Alliance for TB Drug Development (TB Alliance).

The photocatalytic properties of titanium dioxide are well known and have many applications including the removal of organic contaminants and production of self-cleaning glass. There is an increasing interest in the application of the photocatalytic properties of TiO2 for disinfection of surfaces, air and water. Reviews of the applications of photocatalysis in disinfection (Gamage and Zhang 2010; Chong et al., Wat Res 44(10):2997–3027, 2010) and of modelling of TiO2 action have recently been published (Dalrymple et al. , Appl Catal B 98(1–2):27–38, 2010). In this review, we give an overview of the effects of photoactivated TiO2 on microorganisms. The activity has been shown to be capable of killing a wide range of Gram-negative and Gram-positive bacteria, filamentous and unicellular fungi, algae, protozoa, mammalian viruses and bacteriophage. Resting stages, particularly bacterial endospores, fungal spores and protozoan cysts, are generally more resistant than the vegetative forms, possibly due to the increased cell wall thickness. The killing mechanism involves degradation of the cell wall and cytoplasmic membrane due to the production of reactive oxygen species such as hydroxyl radicals and hydrogen peroxide. This initially leads to leakage of cellular contents then cell lysis and may be followed by complete mineralisation of the organism. Killing is most efficient when there is close contact between the organisms and the TiO2 catalyst. The killing activity is enhanced by the presence of other antimicrobial agents such as Cu and Ag.

The success of endodontic treatment depends on the thorough removal of microorganisms from the root canal system. The search for new ways to eliminate the microorganisms is therefore justified. Nd:YAP is a laser that uses yttrium aluminum perovskite, doped with neodymium crystal, as active laser medium. We used the Nd:YAP laser in an in vitro experiment to evaluate the bactericidal effect of three parameters of Nd:YAP laser-activated irrigation on biofilms of Enterococcus faecalis in root canals. The canals of 45 extracted human single-root teeth were prepared on a #35 Mtwo instrument and contaminated with E. faecalis for 14 days. Forty infected single-root teeth were then randomly divided into four groups according to the irrigation agitation protocols as follows: 5.25% sodium hypochlorite (NaOCl), Nd:YAP laser (180 mJ) + NaOCl, Nd:YAP laser (280 mJ) + NaOCl, and Nd:YAP laser (360 mJ) + NaOCl. The remaining bacteria were counted immediately using the cell count method. Teeth were firstly spilt and one half examined by scanning electron microscopy (SEM). The other half involved examination of bacterial colonization in dentinal tubules using confocal laser scanning microscopy (CLSM). Nd:YAP laser (280 mJ) + NaOCl and Nd:YAP laser (360 mJ) + NaOCl completely removed the E. faecalis biofilms from the root canal walls and made it the cleanest among the treatment groups. Bacterial reductions in the treatment groups for dentinal tubules are presented in a descending order as follows: Nd:YAP laser (360 mJ) (53.7%), Nd:YAP laser (280 mJ) (51.5%) > Nd:YAP laser (180 mJ) (45.3%) > 5.25% NaOCl (31.9%) > control (19.3%) (p < 0.05). Nd:YAP laser of 280 mJ and 360 mJ showed effective bactericidal effect in removing E. faecalis biofilm from the root canal walls and dentinal tubules.

Introns are ubiquitous features of all eukaryotic cells. Introns need to be removed from nascent messenger RNA through the process of splicing to produce functional proteins. Here we show that the physical presence of introns in the genome promotes cell survival under starvation conditions. A systematic deletion set of all known introns in budding yeast genes indicates that, in most cases, cells with an intron deletion are impaired when nutrients are depleted. This effect of introns on growth is not linked to the expression of the host gene, and was reproduced even when translation of the host mRNA was blocked. Transcriptomic and genetic analyses indicate that introns promote resistance to starvation by enhancing the repression of ribosomal protein genes that are downstream of the nutrient-sensing TORC1 and PKA pathways. Our results reveal functions of introns that may help to explain their evolutionary preservation in genes, and uncover regulatory mechanisms of cell adaptations to starvation. Transcriptomic and genetic analyses of a deletion set of all known introns in genes of the budding yeast Saccharomyces cerevisiae indicate that introns promote resistance to starvation.

Persister cells, rare phenotypic variants that survive normally lethal levels of antibiotics, present a major barrier to clearing bacterial infections 1 . However, understanding the precise physiological state and genetic basis of persister formation has been a longstanding challenge. Here we generated a high-resolution single-cell 2 RNA atlas of Escherichia coli growth transitions, which revealed that persisters from diverse genetic and physiological models converge to transcriptional states that are distinct from standard growth phases and instead exhibit a dominant signature of translational deficiency. We then used ultra-dense CRISPR interference 3 to determine how every E. coli gene contributes to persister formation across genetic models. Among critical genes with large effects, we found lon , which encodes a highly conserved protease 4 , and yqgE , a poorly characterized gene whose product strongly modulates the duration of post-starvation dormancy and persistence. Our work reveals key physiologic and genetic factors that underlie starvation-triggered persistence, a critical step towards targeting persisters in recalcitrant bacterial infections. Single-cell transcriptome analyses of growth phases in Escherichia coli identify a distinct transcriptional state occupied by antibiotic-tolerant persisters in which lon and yqgE have major roles.

Aciduric bacteria that can survive in extremely acidic conditions (pH < 4.0) are challenging to the current antimicrobial approaches, including antibiotics and photodynamic bacteria inactivation (PDI). Here, we communicate a photosensitizer design concept of halogenation of fluorescein for extremely acidic PDI. Upon halogenation, the well-known spirocyclization that controls the absorption of fluorescein shifts to the acidic pH range. Meanwhile, the heavy atom effect of halogens boosts the generation of singlet oxygen. Accordingly, several photosensitizers that could work at even pH < 2.0 were discovered for a broad band of aciduric bacteria families, with half maximal inhibitory concentrations (IC 50 ) lower than 1.1 μM. Since one of the discovered photosensitizers is an FDA-approved food additive (2’,4’,5’,7’-tetraiodofluorescein, TIF), successful bacteria growth inhibition in acidic beverages was demonstrated, with greatly extended shelf life from 2 days to ~15 days. Besides, the in vivo PDI of Candidiasis with TIF under extremely acidic condition was also demonstrated. Photodynamic inactivation is a common antibacterial method but often fails in extremely acidic environments (pH< 4). Here, the authors demonstrate halogenation of fluorescein for the development of a photodynamic agent which works in extremely acidic environments and demonstrate sterilisation applications in fruit juice and in vivo.