The inactivation of tolC sensitizes Escherichia coli to perturbations in lipopolysaccharide transport.

The Escherichia coli outer membrane channel TolC complexes with several inner membrane efflux pumps to export compounds across the cell envelope. All components of these complexes are essential for robust efflux activity, yet E. coli is more sensitive to antimicrobial compounds when tolC is inactivated compared to the inactivation of genes encoding the inner membrane drug efflux pumps. While investigating these susceptibility differences, we identified a distinct class of inhibitors targeting the core-lipopolysaccharide translocase, MsbA. We show that tolC null mutants are sensitized to structurally unrelated MsbA inhibitors and msbA knockdown, highlighting a synthetic-sick interaction. Phenotypic profiling revealed that tolC inactivation induced cell envelope softening and increased outer membrane permeability. Overall, this work identified a chemical probe of MsbA, revealed that tolC is associated with cell envelope mechanics and integrity, and highlighted that these findings should be considered when using tolC null mutants to study efflux deficiency.

Mechanisms of cardiac collapse at high temperature in a marine teleost (Girella nigrians).

Heat-induced mortality in ectotherms may be attributed to impaired cardiac performance, specifically a collapse in maximum heart rate (fHmax), although the physiological mechanisms driving this phenomenon are still unknown. Here, we tested two proposed factors which may restrict cardiac upper thermal limits: noxious venous blood conditions and oxygen limitation. We hypothesized elevated blood [K+] (hyperkalemia) and low oxygen (hypoxia) would reduce cardiac upper thermal limits in a marine teleost (Girella nigricans), while high oxygen (hyperoxia) would increase thermal limits. We also hypothesized higher acclimation temperatures would exacerbate the harmful effects of an oxygen limitation. Using the Arrhenius breakpoint temperature test, we measured fHmax in acutely warmed fish under control (saline injected) and hyperkalemic conditions (elevated plasma [K+]) while exposed to hyperoxia (200% air saturation), normoxia (100% air saturation), or hypoxia (20% air saturation). We also measured ventricle lactate content and venous blood oxygen partial pressure (PO2) to determine if there were universal thresholds in either metric driving cardiac collapse. Elevated [K+] was not significantly correlated with any cardiac thermal tolerance metric. Hypoxia significantly reduced cardiac upper thermal limits (Arrhenius breakpoint temperature [TAB], peak fHmax, temperature of peak heart rate [TPeak], and temperature at arrhythmia [TARR]). Hyperoxia did not alter cardiac thermal limits compared to normoxia. There was no evidence of a species-wide threshold in ventricular [lactate] or venous PO2. Here, we demonstrate that oxygen limits cardiac thermal tolerance only in instances of hypoxia, but that other physiological processes are responsible for causing temperature-induced heart failure when oxygen is not limited.

Acetylcholinesterase inhibitors for autistic spectrum disorders.

BACKGROUND: Autism spectrum disorder (autism) is a neurodevelopmental condition characterised by impairments in social communication and interaction, plus restricted, repetitive patterns of behaviour and interests. Whilst some people embrace autism as part of their identity, others struggle with their difficulties, and some seek treatment. There are no current interventions that result in complete reduction of autism features. Acetylcholine is a neurotransmitter for the cholinergic system and has a role in attention, novelty seeking, and memory. Low levels of acetylcholine have been investigated as a potential contributor to autism symptomatology. Donepezil, galantamine, and rivastigmine (commonly referred to as acetylcholinesterase inhibitors) all inhibit acetylcholinesterase, and have slightly different modes of action and biological availability, so their effectiveness and side-effect profiles may vary. The effect of various acetylcholinesterase inhibitor on core autism features across the lifespan, and possible adverse effects, have not been thoroughly investigated. OBJECTIVES: To evaluate the efficacy and harms of acetylcholinesterase inhibitors for people with the core features (social interaction, communication, and restrictive and repetitive behaviours) of autism. To assess the effects of acetylcholinesterase inhibitors on non-core features of autism. SEARCH METHODS: In November 2022, we searched CENTRAL, MEDLINE, Embase, eight other databases, and two trials registers. We also searched the reference lists of included studies and relevant reviews, and contacted authors of relevant studies. SELECTION CRITERIA: Randomised controlled trials (RCTs), comparing acetylcholinesterase inhibitors (e.g. galantamine, donepezil, or rivastigmine) of varying doses, delivered orally or via transdermal patch, either as monotherapy or adjunct therapy, with placebo. People of any age, with a clinical diagnosis of autism were eligible for inclusion. DATA COLLECTION AND ANALYSIS: We used standard methodological procedures expected by Cochrane. Our primary outcomes were core features of autism and adverse effects. Secondary outcomes were language, irritability, hyperactivity, and general health and function. We used GRADE to assess certainty of evidence. MAIN RESULTS: We included two RCTs (74 participants). One study was conducted in Iran, the second in the USA, although exact location in the USA is unclear. Galantamine plus risperidone versus placebo plus risperidone One study compared the effects of galantamine plus risperidone to placebo plus risperidone (40 participants, aged 4 years to 12 years). Primary and secondary outcomes of interest were measured postintervention, using subscales of the Aberrant Behavior Checklist (score 0 to 3; higher scores = greater impairment). Very low-certainty evidence showed there was little to no difference between the two groups postintervention for social communication (mean difference (MD) -2.75, 95% confidence interval (CI) -5.88 to 0.38), and restricted and repetitive behaviour (MD -0.55, 95% CI -3.47 to 2.37). Overall autism features were not assessed. Adverse events may be higher in the galantamine plus risperidone group (75%) compared with the placebo plus risperidone group (35%): odds ratio 5.57, 95% CI 1.42 to 21.86, low-certainty evidence. No serious adverse events were reported. Low-certainty evidence showed a small difference in irritability (MD -3.50, 95% CI -6.39 to -0.61), with the galantamine plus risperidone group showing a greater decline on the irritability subscale than the placebo group postintervention. There was no evidence of a difference between the groups in hyperactivity postintervention (MD -5.20, 95% CI -10.51 to 0.11). General health and function were not assessed. Donepezil versus placebo One study compared donepezil to placebo (34 participants aged 8 years to 17 years). Primary outcomes of interest were measured postintervention, using subscales of the Modified Version of The Real Life Rating Scale (scored 0 to 3; higher scores = greater impairment). Very low-certainty evidence showed no evidence of group differences immediately postintervention in overall autism features (MD 0.07, 95% CI -0.19 to 0.33), or in the autism symptom domains of social communication (MD -0.02, 95% CI -0.34 to 0.30), and restricted and repetitive behaviours (MD 0.04, 95% CI -0.27 to 0.35). Significant adverse events leading to study withdrawal in at least one participant was implied in the data analysis section, but not explicitly reported. The evidence is very uncertain about the effect of donepezil, compared to placebo, on the secondary outcomes of interest, including irritability (MD 1.08, 95% CI -0.41 to 2.57), hyperactivity (MD 2.60, 95% CI 0.50 to 4.70), and general health and function (MD 0.03, 95% CI -0.48 to 0.54) postintervention. Across all analyses within this comparison, we judged the evidence to be very low-certainty due to high risk of bias, and very serious imprecision (results based on one small study with wide confidence intervals). The study narratively reported adverse events for the study as a whole, rather than by treatment group. AUTHORS' CONCLUSIONS: Evidence about the effectiveness of acetylcholinesterase inhibitors as a medication to improve outcomes for autistic adults is lacking, and for autistic children is very uncertain. There is a need for more evidence of improvement in outcomes of relevance to clinical care, autistic people, and their families. There are a number of ongoing studies involving acetylcholinesterase inhibitors, and future updates of this review may add to the current evidence.

Autolysin-mediated peptidoglycan hydrolysis is required for the surface display of Staphylococcus aureus cell wall-anchored proteins.

Peptidoglycan hydrolases, or autolysins, play a critical role in cell wall remodeling and degradation, facilitating bacterial growth, cell division, and cell separation. In Staphylococcus aureus, the so-called "major" autolysin, Atl, has long been associated with host adhesion; however, the molecular basis underlying this phenomenon remains understudied. To investigate, we used the type V glycopeptide antibiotic complestatin, which binds to peptidoglycan and blocks the activity of autolysins, as a chemical probe of autolysin function. We also generated a chromosomally encoded, catalytically inactive variant of the Atl enzyme. Autolysin-mediated peptidoglycan hydrolysis, in particular Atl-mediated daughter cell separation, was shown to be critical for maintaining optimal surface levels of S. aureus cell wall-anchored proteins, including the fibronectin-binding proteins (FnBPs) and protein A (Spa). As such, disrupting autolysin function reduced the affinity of S. aureus for host cell ligands, and negatively impacted early stages of bacterial colonization in a systemic model of S. aureus infection. Phenotypic studies revealed that Spa was sequestered at the septum of complestatin-treated cells, highlighting that autolysins are required to liberate Spa during cell division. In summary, we reveal the hydrolytic activities of autolysins are associated with the surface display of S. aureus cell wall-anchored proteins. We demonstrate that by blocking autolysin function, type V glycopeptide antibiotics are promising antivirulence agents for the development of strategies to control S. aureus infections.

Exploring functional interplay amongst Escherichia coli efflux pumps.

Bacterial efflux pumps exhibit functional interplay that can translate to additive or multiplicative effects on resistance to antimicrobial compounds. In diderm bacteria, two different efflux pump structural types - single-component inner membrane efflux pumps and cell envelope-spanning multicomponent systems - cooperatively export antimicrobials with cytoplasmic targets from the cell. Harnessing our recently developed efflux platform, which is built upon an extensively efflux-deficient strain of Escherichia coli, here we explore interplay amongst a panel of diverse E. coli efflux pumps. Specifically, we assessed the effect of simultaneously expressing two efflux pump-encoding genes on drug resistance, including single-component inner membrane efflux pumps (MdfA, MdtK and EmrE), tripartite complexes (AcrAB, AcrAD, MdtEF and AcrEF), and the acquired TetA(C) tetracycline resistance pump. Overall, the expression of two efflux pump-encoding genes from the same structural type did not enhance resistance levels regardless of the antimicrobial compound or efflux pump under investigation. In contrast, a combination of the tripartite efflux systems with single-component pumps sharing common substrates provided multiplicative increases to antimicrobial resistance levels. In some instances, resistance was increased beyond the product of resistance provided by the two pumps individually. In summary, the developed efflux platform enables the isolation of efflux pump function, facilitating the identification of interactions between efflux pumps.

A genetic platform to investigate the functions of bacterial drug efflux pumps.

Efflux pumps are a serious challenge for the development of antibacterial agents. Overcoming efflux requires an in-depth understanding of efflux pump functions, specificities and the development of inhibitors. However, the complexities of efflux networks have limited such studies. To address these challenges, we generated Efflux KnockOut-35 (EKO-35), a highly susceptible Escherichia coli strain lacking 35 efflux pumps. We demonstrate the use of this strain by constructing an efflux platform comprising EKO-35 strains individually producing efflux pumps forming tripartite complexes with TolC. This platform was profiled against a curated diverse compound collection, which enabled us to define physicochemical properties that contribute to transport. We also show the E. coli drug efflux network is conditionally essential for growth, and that the platform can be used to investigate efflux pump inhibitor specificities and efflux pump interplay. We believe EKO-35 and the efflux platform will have widespread application for the study of drug efflux.

Staphylococcus aureus adhesion to the host.

Staphylococcus aureus is a pathobiont capable of colonizing and infecting most tissues within the human body, resulting in a multitude of different clinical outcomes. Adhesion of S. aureus to the host is crucial for both host colonization and the establishment of infections. Underlying the pathogen's success is a complex and diverse arsenal of adhesins. In this review, we discuss the different classes of adhesins, including a consideration of the various adhesion sites throughout the body and the clinical outcomes of each infection type. The development of therapeutics targeting the S. aureus host-pathogen interaction is a relatively understudied area. Due to the increasing global threat of antimicrobial resistance, it is crucial that innovative and alternative approaches are considered. Neutralizing virulence factors, through the development of antivirulence agents, could reduce bacterial pathogenicity and the ever-increasing burden of S. aureus infections. This review provides insight into potentially efficacious adhesion-associated targets for the development of novel decolonizing and antivirulence strategies.

New generation antidepressants for depression in children and adolescents: a network meta-analysis.

BACKGROUND: Major depressive disorders have a significant impact on children and adolescents, including on educational and vocational outcomes, interpersonal relationships, and physical and mental health and well-being. There is an association between major depressive disorder and suicidal ideation, suicide attempts, and suicide. Antidepressant medication is used in moderate to severe depression; there is now a range of newer generations of these medications. OBJECTIVES: To investigate, via network meta-analysis (NMA), the comparative effectiveness and safety of different newer generation antidepressants in children and adolescents with a diagnosed major depressive disorder (MDD) in terms of depression, functioning, suicide-related outcomes and other adverse outcomes. The impact of age, treatment duration, baseline severity, and pharmaceutical industry funding was investigated on clinician-rated depression (CDRS-R) and suicide-related outcomes. SEARCH METHODS: We searched the Cochrane Common Mental Disorders Specialised Register, the Cochrane Library (Central Register of Controlled Trials (CENTRAL) and Cochrane Database of Systematic Reviews (CDSR)), together with Ovid Embase, MEDLINE and PsycINFO till March 2020. SELECTION CRITERIA: Randomised trials of six to 18 year olds of either sex and any ethnicity with clinically diagnosed major depressive disorder were included. Trials that compared the effectiveness of newer generation antidepressants with each other or with a placebo were included. Newer generation antidepressants included: selective serotonin reuptake inhibitors; selective norepinephrine reuptake inhibitors (SNRIs); norepinephrine reuptake inhibitors; norepinephrine dopamine reuptake inhibitors; norepinephrine dopamine disinhibitors (NDDIs); and tetracyclic antidepressants (TeCAs). DATA COLLECTION AND ANALYSIS: Two reviewers independently screened titles/abstracts and full texts, extracted data, and assessed risk of bias. We analysed dichotomous data as Odds Ratios (ORs), and continuous data as Mean Difference (MD) for the following outcomes: depression symptom severity (clinician rated), response or remission of depression symptoms, depression symptom severity (self-rated), functioning, suicide related outcomes and overall adverse outcomes. Random-effects network meta-analyses were conducted in a frequentist framework using multivariate meta-analysis. Certainty of evidence was assessed using Confidence in Network Meta-analysis (CINeMA). We used "informative statements" to standardise the interpretation and description of the results. MAIN RESULTS: Twenty-six studies were included. There were no data for the two primary outcomes (depressive disorder established via clinical diagnostic interview and suicide), therefore, the results comprise only secondary outcomes. Most antidepressants may be associated with a "small and unimportant" reduction in depression symptoms on the CDRS-R scale (range 17 to 113) compared with placebo (high certainty evidence: paroxetine: MD -1.43, 95% CI -3.90, 1.04; vilazodone: MD -0.84, 95% CI -3.03, 1.35; desvenlafaxine MD -0.07, 95% CI -3.51, 3.36; moderate certainty evidence: sertraline: MD -3.51, 95% CI -6.99, -0.04; fluoxetine: MD -2.84, 95% CI -4.12, -1.56; escitalopram: MD -2.62, 95% CI -5.29, 0.04; low certainty evidence: duloxetine: MD -2.70, 95% CI -5.03, -0.37; vortioxetine: MD 0.60, 95% CI -2.52, 3.72; very low certainty evidence for comparisons between other antidepressants and placebo). There were "small and unimportant" differences between most antidepressants in reduction of depression symptoms (high- or moderate-certainty evidence). Results were similar across other outcomes of benefit. In most studies risk of self-harm or suicide was an exclusion criterion for the study. Proportions of suicide-related outcomes were low for most included studies and 95% confidence intervals were wide for all comparisons. The evidence is very uncertain about the effects of mirtazapine (OR 0.50, 95% CI 0.03, 8.04), duloxetine (OR 1.15, 95% CI 0.72, 1.82), vilazodone (OR 1.01, 95% CI 0.68, 1.48), desvenlafaxine (OR 0.94, 95% CI 0.59, 1.52), citalopram (OR 1.72, 95% CI 0.76, 3.87) or vortioxetine (OR 1.58, 95% CI 0.29, 8.60) on suicide-related outcomes compared with placebo. There is low certainty evidence that escitalopram may "at least slightly" reduce odds of suicide-related outcomes compared with placebo (OR 0.89, 95% CI 0.43, 1.84). There is low certainty evidence that fluoxetine (OR 1.27, 95% CI 0.87, 1.86), paroxetine (OR 1.81, 95% CI 0.85, 3.86), sertraline (OR 3.03, 95% CI 0.60, 15.22), and venlafaxine (OR 13.84, 95% CI 1.79, 106.90) may "at least slightly" increase odds of suicide-related outcomes compared with placebo. There is moderate certainty evidence that venlafaxine probably results in an "at least slightly" increased odds of suicide-related outcomes compared with desvenlafaxine (OR 0.07, 95% CI 0.01, 0.56) and escitalopram (OR 0.06, 95% CI 0.01, 0.56). There was very low certainty evidence regarding other comparisons between antidepressants. AUTHORS' CONCLUSIONS: Overall, methodological shortcomings of the randomised trials make it difficult to interpret the findings with regard to the efficacy and safety of newer antidepressant medications. Findings suggest that most newer antidepressants may reduce depression symptoms in a small and unimportant way compared with placebo. Furthermore, there are likely to be small and unimportant differences in the reduction of depression symptoms between the majority of antidepressants. However, our findings reflect the average effects of the antidepressants, and given depression is a heterogeneous condition, some individuals may experience a greater response. Guideline developers and others making recommendations might therefore consider whether a recommendation for the use of newer generation antidepressants is warranted for some individuals in some circumstances. Our findings suggest sertraline, escitalopram, duloxetine, as well as fluoxetine (which is currently the only treatment recommended for first-line prescribing) could be considered as a first option. Children and adolescents considered at risk of suicide were frequently excluded from trials, so that we cannot be confident about the effects of these medications for these individuals. If an antidepressant is being considered for an individual, this should be done in consultation with the child/adolescent and their family/caregivers and it remains critical to ensure close monitoring of treatment effects and suicide-related outcomes (combined suicidal ideation and suicide attempt) in those treated with newer generation antidepressants, given findings that some of these medications may be associated with greater odds of these events. Consideration of psychotherapy, particularly cognitive behavioural therapy, as per guideline recommendations, remains important.

Identifying and supporting autistic preschoolers and their families.

BACKGROUND: In Australia, preschoolers are being identified and diagnosed as autistic. OBJECTIVE: The aim of this article is to describe the different paths preschool children and their families can take from identification of developmental or behavioural concerns to ongoing support, intervention and healthcare. DISCUSSION: There are many ways in which general practitioners, working alongside other professionals and with relevant services, can assist each child and family.

Will nurse leaders help eradicate 'hair racism' from nursing and health services?

AIMS: Nurse managers play key roles in creating and enforcing organisational hair policies and practices. This challenging paper will provoke discussion, debate and hopefully the dismantling of racist hair policies that disproportionately target black students and nurses. BACKGROUND: Black people have suffered from centuries of hair racism that continues today. Unfortunately, many nurse leaders underestimate the significance of this issue, while perpetuating the injustice. EVALUATION: This paper is based on research literature, media reports and authors' lived experiences regarding hair racism experienced by black people and nurses in particular. KEY ISSUES: Nurse managers often create and police organisational hair policies and dress codes. As health services pledge to eradicate racism 'in principle', ending discriminatory hair policies offers nurse managers a practical way to make this principle a reality. CONCLUSIONS: Hair racism is real and damaging for many black nurses and has no place in a modern health service. Rather than designing and policing such structural racism, nurse managers can be instrumental in ending it. IMPLICATIONS FOR NURSING MANAGEMENT: Health service hair policies targeting black nurses especially are not 'neutral'. Nurse managers can challenge this institutional discrimination, demonstrating health services' commitment to ending racism in all of its guises.

Old problems and new solutions: antibiotic alternatives in food animal production.

The antimicrobial resistance crisis is a Global Health challenge that impacts humans, animals, and the environment alike. In response to increased demands for animal protein and by-products, there has been a substantial increase in the use of antimicrobial agents in the animal industry. Indeed, they are extensively used to prevent, control, and (or) treat disease in animals. In addition to infection control, in-feed supplementation with antimicrobials became common practice for growth promotion of livestock. Unfortunately, the global overuse of antimicrobials has contributed to the emergence and spread of resistance. As such, many countries have implemented policies and approaches to eliminate the use of antimicrobials as growth promoters in food animals, which necessitates the need for alternate and One Health strategies to maintain animal health and welfare. This review summarizes the antimicrobial resistance crisis from Global Health and One Health perspectives. In addition, we outline examples of potential alternate strategies to circumvent antimicrobial use in animal husbandry practices, including antivirulence agents, bacteriophages, and nutritional measures to control bacterial pathogens. Overall, these alternate strategies require further research and development efforts, including assessment of efficacy and the associated development, manufacturing, and labor costs.

Prevalence and Age of Onset of Regression in Children with Autism Spectrum Disorder: A Systematic Review and Meta-analytical Update.

A systematic review published in 2013 reported 32% of children on the autism spectrum experience skill loss, known as autistic regression. However, the frequency varied depending on definition and measures used to capture skills. Retrospective parent report and prospective observation indicate loss of language and/or social skills, with motor skills typically unaffected. Our aim was to update the prevalence and age of onset of autistic regression through a meta-analysis of the literature to understand if there have been changes to the reported onset and prevalence since 2010. A systematic literature search was conducted using Medline, Embase, PsycINFO, and the Cochrane Library databases and included studies published from 2010 onward. Risk of bias assessment was performed on included studies. A random effects model was used to calculate the pooled prevalence and age of onset of autistic regression. Ninety-seven studies were included in the systematic review, of which 75 studies involving 33,014 participants had sufficient data for meta-analytic syntheses. The pooled proportion of autistic regression was 30% (95% confidence interval [CI]: 27-32%) but heterogeneity was high (I2 = 96.91) and did not reduce with sensitivity or subgroup analyses based on study design or clinical differences, respectively. Prevalence varied according to risk of bias (low: 27%) and definition of regression (language: 20%, language/social: 40%, mixed: 30%, and unspecified: 27%). Weighted average age of onset was 19.8 months. Findings from this meta-analysis highlight the importance of developing a standardized definition of autistic regression, and tools to measure this at multiple time points during early childhood development. LAY SUMMARY: About a third of children with Autism Spectrum Disorder experience loss of skills, which is also known as autistic regression. This paper provides an update of the rate of autistic regression in children and the age when they first experience loss of skills, based on current studies. The findings from this review contribute to our understanding of the onset patterns of autistic regression. Unfortunately, studies are not sufficiently similar, making it difficult to provide clear answers on the exact timing or type of regression seen in different children.

A Systematic Review and Synthesis of Trauma-Informed Care Within Outpatient and Counseling Health Settings for Young People.

There is growing consensus that outpatient health services for young people (aged 12-25 years) need to deliver trauma-informed care to ameliorate the effects of trauma, offer safe treatments, and avoid retraumatization. Trauma-informed care has become a familiar term for many professionals; however, its operating definition lacks clarity. MEDLINE, Embase, and PsycINFO were systematically searched to clarify what trauma-informed care is, and what it should achieve in these settings. We reviewed 3,381 unique records, of which 13 met criteria for inclusion. Content analysis identified 10 components of trauma-informed care as it has been operationalized in practice: seven of these occurred at the system-level (interagency collaboration; service provider training; safety; leadership, governance and agency processes; youth and family/carer choice in care; cultural and gender sensitivity; youth and family/carer participation), and three involved trauma-specific clinical practices (screening and assessment; psychoeducation; therapeutic interventions). There is a need for greater consensus regarding an operating definition of trauma-informed care and further research into outcomes for young people and their families/carers.

Resistance-Guided Discovery of Elfamycin Antibiotic Producers with Antigonococcal Activity.

The rise of bacterial antibiotic resistance coupled with a diminished antibiotic drug pipeline underlines the importance of developing rational strategies to discover new antimicrobials. Microbially derived natural products are the basis for most of the antibiotic arsenal available to modern medicine. Here, we demonstrate a resistance-based approach to identify producers of elfamycins, an under-explored class of natural product antibiotics that target the essential translation factor EF-Tu. Antibiotic producers carry self-resistance genes to avoid suicide. These genes are often found within the same biosynthetic gene cluster (BGC) responsible for making the antibiotic, and we exploited this trait to identify members of the kirromycin class of elfamycin producers. Genome mining of Streptomyces spp. led to the identification of three isolates that harbor kirromycin-resistant EF-Tu (EF-TuKirR) within predicted natural product BGCs. Activity-guided purification on extracts of one of the Streptomyces isolates, which was not known to produce an elfamycin, identified it as a producer of phenelfamycin B, a linear polyketide. Phenelfamycin B demonstrates impressive antibacterial activity (MIC ∼ 1 µg/mL) against multidrug-resistant Neisseria gonorrhoeae, a clinically important Gram negative pathogen. The antigonococcal activity of phenelfamycin was shown to be the result of inhibition of protein biosynthesis by binding to EF-Tu. These results indicate that a resistance-based approach of identifying elfamycin producers is translatable to other antibiotic classes that can identify new and overlooked antibiotics necessary to address the antibiotic crisis.

Development and validation of a high-throughput whole cell assay to investigate Staphylococcus aureus adhesion to host ligands.

Staphylococcus aureus adhesion to the host's skin and mucosae enables asymptomatic colonization and the establishment of infection. This process is facilitated by cell wall-anchored adhesins that bind to host ligands. Therapeutics targeting this process could provide significant clinical benefits; however, the development of anti-adhesives requires an in-depth knowledge of adhesion-associated factors and an assay amenable to high-throughput applications. Here, we describe the development of a sensitive and robust whole cell assay to enable the large-scale profiling of S. aureus adhesion to host ligands. To validate the assay, and to gain insight into cellular factors contributing to adhesion, we profiled a sequence-defined S. aureus transposon mutant library, identifying mutants with attenuated adhesion to human-derived fibronectin, keratin, and fibrinogen. Our screening approach was validated by the identification of known adhesion-related proteins, such as the housekeeping sortase responsible for covalently linking adhesins to the cell wall. In addition, we also identified genetic loci that could represent undescribed anti-adhesive targets. To compare and contrast the genetic requirements of adhesion to each host ligand, we generated a S. aureus Genetic Adhesion Network, which identified a core gene set involved in adhesion to all three host ligands, and unique genetic signatures. In summary, this assay will enable high-throughput chemical screens to identify anti-adhesives and our findings provide insight into the target space of such an approach.

Temperature sensitivity differs between heart and red muscle mitochondria in mahi-mahi (Coryphaena hippurus).

Maintaining energy balance over a wide range of temperatures is critical for an active pelagic fish species such as the mahi-mahi (Coryphaena hippurus), which can experience rapid changes in temperature during vertical migrations. Due to the profound effect of temperature on mitochondrial function, this study was designed to investigate the effects of temperature on mitochondrial respiration in permeabilized heart and red skeletal muscle (RM) fibres isolated from mahi-mahi. As RM is thought to be more anatomically isolated from rapid ambient temperature changes compared to the myocardium, it was hypothesized that heart mitochondria would be more tolerant of temperature changes through a greater ability to match respiratory capacity to an increase in temperature and to maintain coupling, when compared to RM mitochondria. Results show that heart fibres were more temperature sensitive and increased respiration rate with temperature increases to a greater degree than RM. Respiratory coupling ratios at the three assay temperatures (20, 26, and 30 °C), revealed that heart mitochondria were less coupled at a lower temperature (26 °C) compared to RM mitochondria (30 °C). In response to an in vitro acute temperature challenge, both tissues showed irreversible effects, where both heart and RM increased uncoupling whether the assay temperature was acutely changed from 20 to 30 °C or 30 to 20 °C. The findings from this study indicate that mahi-mahi heart mitochondria were more temperature sensitive compared to those from RM.

The Evolutionary Conservation of Escherichia coli Drug Efflux Pumps Supports Physiological Functions.

Bacteria harness an impressive repertoire of resistance mechanisms to evade the inhibitory action of antibiotics. One such mechanism involves efflux pump-mediated extrusion of drugs from the bacterial cell, which significantly contributes to multidrug resistance. Intriguingly, most drug efflux pumps are chromosomally encoded components of the intrinsic antibiotic resistome. In addition, in terms of xenobiotic detoxification, bacterial efflux systems often exhibit significant levels of functional redundancy. Efflux pumps are also considered to be highly conserved; however, the extent of conservation in many bacterial species has not been reported and the majority of genes that encode efflux pumps appear to be dispensable for growth. These observations, in combination with an increasing body of experimental evidence, imply alternative roles in bacterial physiology. Indeed, the ability of efflux pumps to facilitate antibiotic resistance could be a fortuitous by-product of ancient physiological functions. Using Escherichia coli as a model organism, we here evaluated the evolutionary conservation of drug efflux pumps and we provide phylogenetic analysis of the major efflux families. We show the E. coli drug efflux system has remained relatively stable and the majority (∼80%) of pumps are encoded in the core genome. This analysis further supports the importance of drug efflux pumps in E. coli physiology. In this review, we also provide an update on the roles of drug efflux pumps in the detoxification of endogenously synthesized substrates and pH homeostasis. Overall, gaining insight into drug efflux pump conservation, common evolutionary ancestors, and physiological functions could enable strategies to combat these intrinsic and ancient elements.