Host-induced cell wall remodeling impairs opsonophagocytosis of Staphylococcus aureus by neutrophils.

The bacterial pathogen Staphylococcus aureus responds to the host environment by increasing the thickness of its cell wall. However, the impact of cell wall thickening on susceptibility to host defenses is unclear. Using bacteria incubated in human serum, we show that host-induced increases in cell wall thickness led to a reduction in the exposure of bound antibody and complement and a corresponding reduction in phagocytosis and killing by neutrophils. The exposure of opsonins bound to protein antigens or lipoteichoic acid (LTA) was most significantly reduced, while opsonization by IgG against wall teichoic acid or peptidoglycan was largely unaffected. Partial digestion of accumulated cell wall using the enzyme lysostaphin restored opsonin exposure and promoted phagocytosis and killing. Concordantly, the antibiotic fosfomycin inhibited cell wall remodeling and maintained the full susceptibility of S. aureus to opsonophagocytic killing by neutrophils. These findings reveal that host-induced changes to the S. aureus cell wall reduce the ability of the immune system to detect and kill this pathogen through reduced exposure of protein- and LTA-bound opsonins. IMPORTANCE: Understanding how bacteria adapt to the host environment is critical in determining fundamental mechanisms of immune evasion, pathogenesis, and the identification of targets for new therapeutic approaches. Previous work demonstrated that Staphylococcus aureus remodels its cell envelope in response to host factors and we hypothesized that this may affect recognition by antibodies and thus killing by immune cells. As expected, incubation of S. aureus in human serum resulted in rapid binding of antibodies. However, as bacteria adapted to the serum, the increase in cell wall thickness resulted in a significant reduction in exposure of bound antibodies. This reduced antibody exposure, in turn, led to reduced killing by human neutrophils. Importantly, while antibodies bound to some cell surface structures became obscured, this was not the case for those bound to wall teichoic acid, which may have important implications for vaccine design.

Integrated Analysis of Patient Networks and Plasmid Genomes to Investigate a Regional, Multispecies Outbreak of Carbapenemase-Producing Enterobacterales Carrying Both blaIMP and mcr-9 Genes.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) are challenging in healthcare, with resistance to multiple classes of antibiotics. This study describes the emergence of imipenemase (IMP)-encoding CPE among diverse Enterobacterales species between 2016 and 2019 across a London regional network. METHODS: We performed a network analysis of patient pathways, using electronic health records, to identify contacts between IMP-encoding CPE-positive patients. Genomes of IMP-encoding CPE isolates were overlaid with patient contacts to imply potential transmission events. RESULTS: Genomic analysis of 84 Enterobacterales isolates revealed diverse species (predominantly Klebsiella spp, Enterobacter spp, and Escherichia coli); 86% (72 of 84) harbored an IncHI2 plasmid carrying blaIMP and colistin resistance gene mcr-9 (68 of 72). Phylogenetic analysis of IncHI2 plasmids identified 3 lineages showing significant association with patient contacts and movements between 4 hospital sites and across medical specialties, which was missed in initial investigations. CONCLUSIONS: Combined, our patient network and plasmid analyses demonstrate an interspecies, plasmid-mediated outbreak of blaIMPCPE, which remained unidentified during standard investigations. With DNA sequencing and multimodal data incorporation, the outbreak investigation approach proposed here provides a framework for real-time identification of key factors causing pathogen spread. Plasmid-level outbreak analysis reveals that resistance spread may be wider than suspected, allowing more interventions to stop transmission within hospital networks.SummaryThis was an investigation, using integrated pathway networks and genomics methods, of the emergence of imipenemase-encoding carbapenemase-producing Enterobacterales among diverse Enterobacterales species between 2016 and 2019 in patients across a London regional hospital network, which was missed on routine investigations.

Development of an inhibitor of the mutagenic SOS response that suppresses the evolution of quinolone antibiotic resistance.

Antimicrobial resistance (AMR) is a growing threat to health globally, with the potential to render numerous medical procedures so dangerous as to be impractical. There is therefore an urgent need for new molecules that function through novel mechanisms of action to combat AMR. The bacterial DNA-repair and SOS-response pathways promote survival of pathogens in infection settings and also activate hypermutation and resistance mechanisms, making these pathways attractive targets for new therapeutics. Small molecules, such as IMP-1700, potentiate DNA damage and inhibit the SOS response in methicillin-resistant S. aureus; however, understanding of the structure-activity relationship (SAR) of this series is lacking. We report here the first comprehensive SAR study of the IMP-1700 scaffold, identifying key pharmacophoric groups and delivering the most potent analogue reported to date, OXF-077. Furthermore, we demonstrate that as a potent inhibitor of the mutagenic SOS response, OXF-077 suppresses the rate of ciprofloxacin resistance emergence in S. aureus. This work supports SOS-response inhibitors as a novel means to combat AMR, and delivers OXF-077 as a tool molecule for future development.

Explaining empirical dynamic modelling using verbal, graphical and mathematical approaches.

Empirical dynamic modelling (EDM) is becoming an increasingly popular method for understanding the dynamics of ecosystems. It has been applied to laboratory, terrestrial, freshwater and marine systems, used to forecast natural populations and has addressed fundamental ecological questions. Despite its increasing use, we have not found full explanations of EDM in the ecological literature, limiting understanding and reproducibility. Here we expand upon existing work by providing a detailed introduction to EDM. We use three progressively more complex approaches. A short verbal explanation of EDM is then explicitly demonstrated by graphically working through a simple example. We then introduce a full mathematical description of the steps involved. Conceptually, EDM translates a time series of data into a path through a multi-dimensional space, whose axes are lagged values of the time series. A time step is chosen from which to make a prediction. The state of the system at that time step corresponds to a 'focal point' in the multi-dimensional space. The set (called the library) of candidate nearest neighbours to the focal point is constructed, to determine the nearest neighbours that are then used to make the prediction. Our mathematical explanation explicitly documents which points in the multi-dimensional space should not be considered as focal points. We suggest a new option for excluding points from the library that may be useful for short-term time series that are often found in ecology. We focus on the core simplex and S-map algorithms of EDM. Our new R package, pbsEDM, enhances understanding (by outputting intermediate calculations), reproduces our results and can be applied to new data. Our work improves the clarity of the inner workings of EDM, a prerequisite for EDM to reach its full potential in ecology and have wide uptake in the provision of advice to managers of natural resources.

A monthly gridded burned area database of national wildland fire data.

We assembled the first gridded burned area (BA) database of national wildfire data (ONFIRE), a comprehensive and integrated resource for researchers, non-government organisations, and government agencies analysing wildfires in various regions of the Earth. We extracted and harmonised records from different regions and sources using open and reproducible methods, providing data in a common framework for the whole period available (starting from 1950 in Australia, 1959 in Canada, 1985 in Chile, 1980 in Europe, and 1984 in the United States) up to 2021 on a common 1° × 1° grid. The data originate from national agencies (often, ground mapping), thus representing the best local expert knowledge. Key opportunities and limits in using this dataset are discussed as well as possible future expansions of this open-source approach that should be explored. This dataset complements existing gridded BA data based on remote sensing and offers a valuable opportunity to better understand and assess fire regime changes, and their drivers, in these regions. The ONFIRE database can be freely accessed at https://zenodo.org/record/8289245 .

The perception of time is slowed in response to exercise, an effect not further compounded by competitors: behavioral implications for exercise and health.

INTRODUCTION: The theory of relativity postulates that time is relative to context and exercise seems such a situation. The purpose of this study was to examine whether situational factors such as perceived exertion and the introduction of an opponent influence competitors' perception of time. METHODS: Thirty-three recreationally active adults (F = 16; M = 17) performed three standardized 4-km cycling trials in a randomized order. Velotron 3D software was used to create a visual, virtual environment representing (1) a solo time trial (FAM and SO), (2) a time trial with a passive opponent avatar (PO), and (3) a time trial with an opponent avatar and participant instruction to actively finish the trial before the opponent (AO). Participants were asked to estimate a 30-s time period using a standardized protocol for reproducibility before exercise at 500 m, 1500 m, 2500 m, and post exercise. Rate of perceived exertion (RPE) was measured throughout the trials. RESULTS: Exercise trials revealed that time was perceived to run "slow" compared to chronological time during exercise compared to resting and post-exercise measurements (p < 0.001). There was no difference between exercise conditions (SO, PO, and AO) or time points (500 m, 1500 m, and 2500 m). RPE increased throughout the trials. CONCLUSION: The results of this study demonstrate for the first time that exercise both with and without the influence of opponents influences time perception. This finding has important implications for healthy exercise choices and also for optimal performance. Independent of RPE, time was perceived to move slower during exercise, underpinning inaccurate pacing and decision-making across physical activities.

Integrated analysis of patient networks and plasmid genomes reveals a regional, multi-species outbreak of carbapenemase-producing Enterobacterales carrying both blaIMP and mcr-9 genes.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) are challenging in healthcare, with resistance to multiple classes of antibiotics. This study describes the emergence of IMP-encoding CPE amongst diverse Enterobacterales species between 2016 and 2019 across a London regional network. METHODS: We performed a network analysis of patient pathways, using electronic health records, to identify contacts between IMP-encoding CPE positive patients. Genomes of IMP-encoding CPE isolates were overlayed with patient contacts to imply potential transmission events. RESULTS: Genomic analysis of 84 Enterobacterales isolates revealed diverse species (predominantly Klebsiella spp, Enterobacter spp, E. coli); 86% (72/84) harboured an IncHI2 plasmid carrying blaIMP and colistin resistance gene mcr-9 (68/72). Phylogenetic analysis of IncHI2 plasmids identified three lineages showing significant association with patient contacts and movements between four hospital sites and across medical specialities, which was missed on initial investigations. CONCLUSIONS: Combined, our patient network and plasmid analyses demonstrate an interspecies, plasmid-mediated outbreak of blaIMPCPE, which remained unidentified during standard investigations. With DNA sequencing and multi-modal data incorporation, the outbreak investigation approach proposed here provides a framework for real-time identification of key factors causing pathogen spread. Plasmid-level outbreak analysis reveals that resistance spread may be wider than suspected, allowing more interventions to stop transmission within hospital networks.

Extended Total Temporomandibular Joint Replacement - A Feasible Option for Functional and Aesthetic Reconstruction of Mandibular Defects Involving the Temporomandibular Joint.

Study design: Retrospective case series. Objective: Alloplastic temporomandibular joint replacement has been established as a standard technique for end- stage temporomandibular (TMJ) pathologies. Joint replacement when there are extensive mandibular defects remains a challenging clinical problem. Custom-made extended temporomandibular joint replacement is a feasible option but there is limited information about this emerging technique. Methods: Included were all patients undergoing extended TMJ-replacements (TMJe), all operatrions were carried out by the senior author. Surgical technique was either single stage or two stage protocol. Surgical details and pitfalls and outcome of more than 2 years follow-up with reference to thirteen including twelve patients were recorded. Results: The most common diagnosis was ameloblastoma of the mandibular ramus. Single stage or two stagge regime were carried out depending on resection requirements and involvement of teeth. Improved mouth opening of more than 30mm was achieved in 10 of 12 patients. One patient with previous TMJ replacement reported temporary weakness of the facial nerve, which resolved after 10 months. Conclusions: The authors suggest a simplified anatomically based single-stage or two-stage regime, with both regimes achieved excellent anatomic reconstruction, facial appearance and function with low surgical morbidity. Custom-made extended temporomandibular joint protheses appear an advanced and reliable solution for reconstruction of combined complex mandibular defects including the temporomandibular joint. If surgical clearance of the pathology can be achieved, a single-stage regime is favoured.

The influence of sex difference on behavior and adult hippocampal neurogenesis in C57BL/6 mice.

Animal models have been used extensively in in vivo studies, especially within the biomedical field. Traditionally, single-sex studies, mostly males, are used to avoid any potential confounding variation caused by sex difference and the female estrous cycle. Historically, female animal subjects are believed to exhibit higher variability, and this could increase the statistical power needed to test a hypothesis. This study sets out to evaluate whether a sex difference does exist in mouse behavior, and whether female mice featured higher variability. We assessed the sensorimotor skills, anxiety-like behavior, depression-like behavior, and cognitive abilities of mice through a series of commonly used behavioral tests. Except for the stronger grip force and lower tactile sensory sensitivity detected in male mice, there was no significant difference between males and females in other tests. Furthermore, immunolabeling of neurogenesis markers suggested no significant difference between sexes in adult hippocampal neurogenesis. Within group variances were equivalent; females did not exhibit higher variability than males. However, the overall negative results could be due to the limitation of small sample size. In conclusion, our study provides evidence that sex difference in mice does not significantly influence these commonly used behavioral tests nor adult neurogenesis under basal conditions. We suggest that female mice could also be considered for test inclusion in future experiment design.

Frozen (capsulitis) of shoulder Treated by LED Red light: A case report.

Shoulder capsulitis is a painful condition in which movement of the shoulder becomes limited. Objective: The aim, based on study of a single case, was to evaluate subjective and objective short-term effects of radiation of the shoulder joint using LED red light (630nm, 37.5 Jcm over 500s) in a case of chronic shoulder capsulitis. The case was a 96-year-old man with painful and restricted movement of the right shoulder joint. A record was made of parameters before the start of treatment using the Oxford Shoulder Score [1] and repeated following 12 treatment sessions in a four-week period. Results indicate improvement in a number of parameters and almost pain free essential movement without use of NSAI gel. On the basis of findings, the method warrants a pilot study to test reproducibility and to establish optimal number of treatments. Treatment was to two areas.

IS1-related large-scale deletion of chromosomal regions harbouring the oxygen-insensitive nitroreductase gene nfsB causes nitrofurantoin heteroresistance in Escherichia coli.

Nitrofurantoin is a broad-spectrum first-line antimicrobial used for managing uncomplicated urinary tract infection (UTI). Loss-of-function mutations in chromosomal genes nfsA, nfsB and ribE of Escherichia coli are known to reduce nitrofurantoin susceptibility. Here, we report the discovery of nitrofurantoin heteroresistance in E. coli clinical isolates and a novel genetic mechanism associated with this phenomenon. Subpopulations with lower nitrofurantoin susceptibility than major populations (hereafter, nitrofurantoin-resistant subpopulations) in two E. coli blood isolates (previously whole-genome sequenced) were identified using population analysis profiling. Each isolate was known to have a loss-of-function mutation in nfsA. From each isolate, four nitrofurantoin-resistant isolates were derived at a nitrofurantoin concentration of 32 mg l-1, and a comparator isolate was obtained without any nitrofurantoin exposure. Genomes of derived isolates were sequenced on Illumina and Nanopore MinION systems. Genetic variation between isolates was determined based on genome assemblies and read mapping. Nitrofurantoin minimum inhibitory concentrations (MICs) of both blood isolates were 64 mg l-1, with MICs of major nitrofurantoin-susceptible populations varying from 4 to 8 mg l-1. Two to 99 c.f.u. per million demonstrated growth at the nitrofurantoin concentration of 32 mg l-1, which is distinct from that of a homogeneously susceptible or resistant isolate. Derived nitrofurantoin-resistant isolates had 11-66 kb deletions in chromosomal regions harbouring nfsB, and all deletions were immediately adjacent to IS1-family insertion sequences. Our findings demonstrate that the IS1-associated large-scale genetic deletion is a hitherto unrecognized mechanism of nitrofurantoin heteroresistance and could compromise UTI management. Further, frequencies of resistant subpopulations from nitrofurantoin-heteroresistant isolates may challenge conventional nitrofurantoin susceptibility testing in clinical settings.

Symbiotic Firmicutes establish mutualism with the host via innate tolerance and resistance to control systemic immunity.

The intestinal microbiota regulates immunity across organ systems. Which symbionts control systemic immunity, the mechanisms they use, and how they avoid widespread inflammatory damage are unclear. We uncover host tolerance and resistance mechanisms that allow Firmicutes from the human microbiota to control systemic immunity without inducing immunopathology. Intestinal processing releases Firmicute glycoconjugates that disseminate, resulting in release of cytokine IL-34 that stimulates macrophages and enhances defenses against pneumonia, sepsis, and meningitis. Despite systemic penetration of Firmicutes, immune homeostasis is maintained through feedback control whereby IL-34-mediated mTORC1 activation in macrophages clears polymeric glycoconjugates from peripheral tissues. Smaller glycoconjugates evading this clearance mechanism are tolerated through sequestration by albumin, which acts as an inflammatory buffer constraining their immunological impact. Without these resistance and tolerance mechanisms, Firmicutes drive catastrophic organ damage and cachexia via IL-1ß. This reveals how Firmicutes are safely assimilated into systemic immunity to protect against infection without threatening host viability.

The interaction between land-use change and fire regimes, directly and indirectly, affects the urban avian assemblages of Darwin, Australia.

The interaction between environmental stressors may be a greater threat to biota than any individual ecological threat on its own. Land-use change and inappropriate fire regimes are known to pose great challenges to biodiversity conservation worldwide. Despite much research being conducted into their singular impacts on ecosystems, very few have investigated how their interaction may be affecting the biota of a region. We used data from surveys in 1998/2000 and 2019/2020 to compare the feeding guild assemblages of bird communities in different habitats within the greater Darwin region. By compiling two sets of spatial data, land-use change, and fire history mapping, we were able to investigate their interaction and impact on the avian assemblages in the Darwin urban area. Using Generalized Linear Mixed Models (GLMM) we found that an increase in urbanization significantly affected fire occurrence across study sites. Furthermore, we found that the interaction between land-use change and fire regimes had a significant effect on species that primarily feed on fruit. We conclude that while an increase in urbanization did not directly affect the avian assemblages, the impact of land-use change on the fire regimes indirectly impacted urban bird community structures.

Incentivizing sustainable fire management in Australia's northern arid spinifex grasslands.

Fire management across Australia's fire-prone 1.2 M km2 northern savannas region has been transformed over the past decade supported by the inception of Australia's national regulated emissions reduction market in 2012. Today, incentivised fire management is undertaken over a quarter of that entire region, providing a range of socio-cultural, environmental, and economic benefits, including for remote Indigenous (Aboriginal and Torres Strait Islander) communities and enterprises. Building on those advances, here we explore the emissions abatement potential for expanding incentivised fire management opportunities to include a contiguous fire-prone region, extending to monsoonal but annually lower (<600 mm) and more variable rainfall conditions, supporting predominantly shrubby spinifex (Triodia) hummock grasslands characteristic of much of Australia's deserts and semi-arid rangelands. Adapting a standard methodological approach applied previously for assessing savanna emissions parameters, we first describe fire regime and associated climatic attributes for a proposed ∼850,000 km2 lower rainfall (600-350 mm MAR) focal region. Second, based on regional field assessments of seasonal fuel accumulation, combustion, burnt area patchiness, and accountable methane and nitrous oxide Emission Factor parameters, we find that significant emissions abatement is feasible for regional hummock grasslands. This applies specifically for more frequently burnt sites under higher rainfall conditions if substantial early dry season prescribed fire management is undertaken resulting in marked reduction in late dry season wildfires. The proposed Northern Arid Zone (NAZ) focal envelope is substantially under Indigenous land ownership and management, and in addition to reducing emissions impacts associated with recurrent extensive wildfires, development of commercial landscape-scale fire management opportunities would significantly support social, cultural and biodiversity management aspirations as promoted by Indigenous landowners. Combined with existing regulated savanna fire management regions, inclusion of the NAZ under existing legislated abatement methodologies would effectively provide incentivised fire management covering a quarter of Australia's landmass. This could complement an allied (non-carbon) accredited method valuing combined social, cultural and biodiversity outcomes from enhanced fire management of hummock grasslands. Although the management approach has potential application to other international fire-prone savanna grasslands, caution is required to ensure that such practice does not result in irreversible woody encroachment and undesirable habitat change.

Lipopolysaccharide as an antibiotic target.

Gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii are amongst the highest priority drug-resistant pathogens, for which new antibiotics are urgently needed. Whilst antibiotic drug development is inherently challenging, this is particularly true for Gram-negative bacteria due to the presence of the outer membrane, a highly selective permeability barrier that prevents the ingress of several classes of antibiotic. This selectivity is largely due to an outer leaflet composed of the glycolipid lipopolysaccharide (LPS), which is essential for the viability of almost all Gram-negative bacteria. This essentiality, coupled with the conservation of the synthetic pathway across species and recent breakthroughs in our understanding of transport and membrane homeostasis has made LPS an attractive target for novel antibiotic drug development. Several different targets have been explored and small molecules developed that show promising activity in vitro. However, these endeavours have met limited success in clinical testing and the polymyxins, discovered more than 70 years ago, remain the only LPS-targeting drugs to enter the clinic thus far. In this review, we will discuss efforts to develop therapeutic inhibitors of LPS synthesis and transport and the reasons for limited success, and explore new developments in understanding polymyxin mode of action and the identification of new analogues with reduced toxicity and enhanced activity.

Phenotypic variation in the molt characteristics of a seasonal coat color-changing species reveals limited resilience to climate change.

The snowshoe hare (Lepus americanus) possesses a broad suite of adaptations to winter, including a seasonal coat color molt. Recently, climate change has been implicated in the range contraction of snowshoe hares along the southern range boundary. With shortening snow season duration, snowshoe hares are experiencing increased camouflage mismatch with their environment reducing survival. Phenological variation of hare molt at regional scales could facilitate local adaptation in the face of climate change, but the level of variation, especially along the southern range boundary, is unknown. Using a network of trail cameras and historical museum specimens, we (1) developed contemporary and historical molt phenology curves in the Upper Great Lakes region, USA, (2) calculated molt rate and variability in and among populations, and (3) quantified the relationship of molt characteristics to environmental conditions for snowshoe hares across North America. We found that snowshoe hares across the region exhibited similar fall and spring molt phenologies, rates and variation. Yet, an insular island population of hares on Isle Royale National Park, MI, completed their molt a week earlier in the fall and initiated molt almost 2 weeks later in the spring as well as exhibited slower rates of molting in the fall season compared to the mainland. Over the last 100 years, snowshoe hares across the region have not shifted in fall molt timing; though contemporary spring molt appears to have advanced by 17 days (~ 4 days per decade) compared to historical molt phenology. Our research indicates that some variation in molt phenology exists for snowshoe hares in the Upper Great Lakes region, but whether this variation is enough to offset the consequences of climate change remains to be seen.

XerC Is Required for the Repair of Antibiotic- and Immune-Mediated DNA Damage in Staphylococcus aureus.

To survive in the host environment, pathogenic bacteria need to be able to repair DNA damage caused by both antibiotics and the immune system. The SOS response is a key bacterial pathway to repair DNA double-strand breaks and may therefore be a good target for novel therapeutics to sensitize bacteria to antibiotics and the immune response. However, the genes required for the SOS response in Staphylococcus aureus have not been fully established. Therefore, we carried out a screen of mutants involved in various DNA repair pathways to understand which were required for induction of the SOS response. This led to the identification of 16 genes that may play a role in SOS response induction and, of these, 3 that affected the susceptibility of S. aureus to ciprofloxacin. Further characterization revealed that, in addition to ciprofloxacin, loss of the tyrosine recombinase XerC increased the susceptibility of S. aureus to various classes of antibiotics, as well as to host immune defenses. Therefore, the inhibition of XerC may be a viable therapeutic approach to sensitize S. aureus to both antibiotics and the immune response.

Gating kinetics and pharmacological properties of small-conductance Ca2+-activated potassium channels.

Small-conductance (SK) calcium-activated potassium channels are a promising treatment target in atrial fibrillation. However, the functional properties that differentiate SK inhibitors remain poorly understood. The objective of this study was to determine how two unrelated SK channel inhibitors, apamin and AP14145, impact SK channel function in excised inside-out single-channel recordings. Surprisingly, both apamin and AP14145 exert much of their inhibition by inducing a class of very-long-lived channel closures (apamin: τc,vl = 11.8 ± 7.1 s, and AP14145: τc,vl = 10.3 ± 7.2 s), which were never observed under control conditions. Both inhibitors also induced changes to the three closed and two open durations typical of normal SK channel gating. AP14145 shifted the open duration distribution to favor longer open durations, whereas apamin did not alter open-state kinetics. AP14145 also prolonged the two shortest channel closed durations (AP14145: τc,s = 3.50 ± 0.81 ms, and τc,i = 32.0 ± 6.76 ms versus control: τc,s = 1.59 ± 0.19 ms, and τc,i = 13.5 ± 1.17 ms), thus slowing overall gating kinetics within bursts of channel activity. In contrast, apamin accelerated intraburst gating kinetics by shortening the two shortest closed durations (τc,s = 0.75 ± 0.10 ms and τc,i = 5.08 ± 0.49 ms) and inducing periods of flickery activity. Finally, AP14145 introduced a unique form of inhibition by decreasing unitary current amplitude. SK channels exhibited two clearly distinguishable amplitudes (control: Ahigh = 0.76 ± 0.03 pA, and Alow = 0.54 ± 0.03 pA). AP14145 both reduced the fraction of patches exhibiting the higher amplitude (AP14145: 4/9 patches versus control: 16/16 patches) and reduced the mean low amplitude (0.38 ± 0.03 pA). Here, we have demonstrated that both inhibitors introduce very long channel closures but that each also exhibits unique effects on other components of SK gating kinetics and unitary current. The combination of these effects is likely to be critical for understanding the functional differences of each inhibitor in the context of cyclical Ca2+-dependent channel activation in vivo.

Growth Arrest of Staphylococcus aureus Induces Daptomycin Tolerance via Cell Wall Remodelling.

Almost all bactericidal drugs require bacterial replication and/or metabolic activity for their killing activity. When these processes are inhibited by bacteriostatic antibiotics, bacterial killing is significantly reduced. One notable exception is the lipopeptide antibiotic daptomycin, which has been reported to efficiently kill growth-arrested bacteria. However, these studies employed only short periods of growth arrest (<1 h), which may not fully represent the duration of growth arrest that can occur in vivo. We found that a growth inhibitory concentration of the protein synthesis inhibitor tetracycline led to a time-dependent induction of daptomycin tolerance in S. aureus, with an approximately 100,000-fold increase in survival after 16 h of growth arrest, relative to exponential-phase bacteria. Daptomycin tolerance required glucose and was associated with increased production of the cell wall polymers peptidoglycan and wall-teichoic acids. However, while the accumulation of peptidoglycan was required for daptomycin tolerance, only a low abundance of wall teichoic acid was necessary. Therefore, whereas tolerance to most antibiotics occurs passively due to a lack of metabolic activity and/or replication, daptomycin tolerance arises via active cell wall remodelling. IMPORTANCE Understanding why antibiotics sometimes fail to cure infections is fundamental to improving treatment outcomes. This is a major challenge when it comes to Staphylococcus aureus because this pathogen causes several different chronic or recurrent infections. Previous work has shown that a lack of replication, as often occurs during infection, makes bacteria tolerant of most bactericidal antibiotics. However, one antibiotic that has been reported to kill nonreplicating bacteria is daptomycin. In this work, we show that the growth arrest of S. aureus does in fact lead to daptomycin tolerance, but it requires time, nutrients, and biosynthetic pathways, making it distinct from other types of antibiotic tolerance that occur in nonreplicating bacteria.