Steroid Drugs Inhibit Bacterial Respiratory Oxidases and Are Lethal Toward Methicillin-Resistant Staphylococcus aureus.

BACKGROUND: Cytochrome bd complexes are respiratory oxidases found exclusively in prokaryotes that are important during infection for numerous bacterial pathogens. METHODS: In silico docking was employed to screen approved drugs for their ability to bind to the quinol site of Escherichia coli cytochrome bd-I. Respiratory inhibition was assessed with oxygen electrodes using membranes isolated from E. coli and methicillin-resistant Staphylococcus aureus strains expressing single respiratory oxidases (ie, cytochromes bd, bo', or aa3). Growth/viability assays were used to measure bacteriostatic and bactericidal effects. RESULTS: The steroid drugs ethinylestradiol and quinestrol inhibited E. coli bd-I activity with median inhibitory concentration (IC50) values of 47 ± 28.9 µg/mL (158 ± 97.2 µM) and 0.2 ± 0.04 µg/mL (0.5 ± 0.1 µM), respectively. Quinestrol inhibited growth of an E. coli "bd-I only" strain with an IC50 of 0.06 ± 0.02 µg/mL (0.2 ± 0.07 µM). Growth of an S. aureus "bd only" strain was inhibited by quinestrol with an IC50 of 2.2 ± 0.43 µg/mL (6.0 ± 1.2 µM). Quinestrol exhibited potent bactericidal effects against S. aureus but not E. coli. CONCLUSIONS: Quinestrol inhibits cytochrome bd in E. coli and S. aureus membranes and inhibits the growth of both species, yet is only bactericidal toward S. aureus.

Suicides in Aboriginal and non-Aboriginal people following hospital admission for suicidal ideation and self-harm: A retrospective cohort data linkage study from the Northern Territory.

PURPOSE: This study aimed to explore risk factors for suicide in Aboriginal and non-Aboriginal people following hospital admission for suicidal ideation and self-harm in the Northern Territory, Australia to help clarify opportunities for improved care and intervention for these population groups. METHODS: Individuals with at least one hospital admission involving suicidal ideation and/or self-harm between 1 July 2001 and 31 December 2013 were retrospectively recruited and followed up using linked mortality records to 31 December 2014. Survival analyses stratified by Indigenous status identified socio-demographic and clinical characteristics from index hospital admissions associated with suicide. RESULTS: Just over half of the 4391 cohort members identified as Aboriginal (n = 2304; 52.4%). By 2014, 281 deaths were observed comprising 68 suicides, representing a 2.6% and 2.0% probability of suicide for Aboriginal and non-Aboriginal people, respectively. After adjusting for other characteristics, a higher risk of suicide was associated with male sex (Aboriginal adjusted hazard ratio: 4.14; 95% confidence interval: [1.76, 9.75]; non-Aboriginal adjusted hazard ratio: 5.96; 95% confidence interval: [1.98, 17.88]) and repeat hospital admissions involving self-harm (Aboriginal adjusted hazard ratio: 1.37; 95% confidence interval: [1.21, 1.55]; non-Aboriginal adjusted hazard ratio: 1.29; 95% confidence interval: [1.10, 1.51]). Severe mental disorders were associated with a four times higher risk of suicide (adjusted hazard ratio: 4.23; 95% confidence interval: [1.93, 9.27]) in Aboriginal people only. CONCLUSION: The findings highlight non-clinical risk factors for suicide that suggest the need for comprehensive psychosocial assessment tailored to Aboriginal and non-Aboriginal people hospitalised with suicidal ideation or self-harm. Implementing appropriate management and aftercare within a broader public health framework is needed to support recovery and reduce long-term suicide risk in the community, especially for Aboriginal people and males.

The polyene antifungal candicidin is selectively packaged into membrane vesicles in Streptomyces S4.

In recent years, much attention has been focused on the biogenesis, engineering and utilisation of outer membrane vesicles (OMVs) in Gram-negative bacteria in a range of environments and niches. While the precise mechanism of biogenesis is unknown, it is focused on the modification of the Gram-negative cell wall to facilitate blebbing at sites of weakness in and around the characteristically thin peptidoglycan layer within the periplasm. Here, we investigate the biogenesis of membrane vesicles (MVs) in the Gram-positive organism Streptomyces albus S4 (Seipke et al. J Bacteriol 193:4270-4271, 2011 and Fazal et al. Antonie Van Leeuwenhoek 113:511-520, 2020). The S. albus S4 strain is an antifungal (candicidin and antimycin) producing organism that was isolated from attine ants (Barke et al. BMC Biol 8:109, 2010). The biogenesis and characterisation of S. albus S4 MVs is demonstrated using the wild-type (WT) and mutant strains ΔantC (no antimycin production) ΔfscC (no candicidin production) and ΔantC ΔfscC (produces neither antimycin nor candicidin). Here, we have shown that the S. albus S4 strain produces MVs and that these are comprised of both specific protein profiles and secondary metabolites, with a clear demonstration of the ability to selectively package one antifungal (candicidin) but not the other (antimycin).

Proton motive force underpins respiration-mediated potentiation of aminoglycoside lethality in pathogenic Escherichia coli.

It is well known that loss of aerobic respiration in Gram-negative bacteria can diminish the efficacy of a variety of bactericidal antibiotics, which has lead to subsequent demonstrations that the formation of reactive oxygen species (ROS) and the proton motive force (PMF) can both play a role in antibiotic toxicity. The susceptibility of Gram-negative bacteria to aminoglycoside antibiotics, particularly gentamicin, has previously been linked to both the production of ROS and the rate of antibiotic uptake that is mediated by the PMF, although the relative contributions of ROS and PMF to aminoglycoside toxicity has remained poorly understood. Herein, gentamicin was shown to elicit a very modest increase in ROS levels in an aerobically grown Escherichia coli clinical isolate. The well-characterised uncoupler 2,4-dinitrophenol (DNP) was used to disrupt the PMF, which resulted in a significant decrease in gentamicin lethality towards E. coli. DNP did not significantly alter respiratory oxygen consumption, supporting the hypothesis that this uncoupler does not increase ROS production via elevated respiratory oxidase activity. These observations support the hypothesis that maintenance of PMF rather than induction of ROS production underpins the mechanism for how the respiratory chain potentiates the toxicity of aminoglycosides. This was further supported by the demonstration that the uncoupler DNP elicits a dramatic decrease in gentamicin lethality under anaerobic conditions. Together, these data strongly suggest that maintenance of the PMF is the dominant mechanism for the respiratory chain in potentiating the toxic effects of aminoglycosides.

Adapting an evidence-supported optimization program for mental health and sport performance in collegiate athletes to fit youth from ethnic/racial minority and low-income neighborhoods: A National Institutes of Health stage model feasibility study.

The current study addresses the need to empirically develop effective mental health interventions for youth from ethnic/racial minority and low-income neighborhoods. Using Stage Model evaluation methods supported by the National Institutes of Health in the US to address underutilization of mental healthcare among racial/ethnic minority youth, this feasibility study demonstrates empirical adaptation of an innovative sport-specific psychological intervention for use in youth from ethnic/racial minority and low-income neighborhoods. An international group of professionals familiar with sport performance and mental health intervention serving the target population experientially examined the adapted intervention protocols in workshops and provided feedback. Survey results indicated the professionals found the intervention components were easy to administer and likely to be safe, enjoyable, engaging and efficacious for youth mental health and sport performance. The protocols were revised based on feedback from these professionals and the intervention was examined in a case trial involving an Asian American youth who evidenced Social Anxiety Disorder. Case study results indicated the intervention could be implemented with integrity, and severity of psychiatric symptoms and factors interfering with sport performance decreased after intervention implementation. The participant's relationships with family, coaches and teammates were also improved.

Nitric oxide (NO) elicits aminoglycoside tolerance in Escherichia coli but antibiotic resistance gene carriage and NO sensitivity have not co-evolved.

The spread of multidrug-resistance in Gram-negative bacterial pathogens presents a major clinical challenge, and new approaches are required to combat these organisms. Nitric oxide (NO) is a well-known antimicrobial that is produced by the immune system in response to infection, and numerous studies have demonstrated that NO is a respiratory inhibitor with both bacteriostatic and bactericidal properties. However, given that loss of aerobic respiratory complexes is known to diminish antibiotic efficacy, it was hypothesised that the potent respiratory inhibitor NO would elicit similar effects. Indeed, the current work demonstrates that pre-exposure to NO-releasers elicits a > tenfold increase in IC50 for gentamicin against pathogenic E. coli (i.e. a huge decrease in lethality). It was therefore hypothesised that hyper-sensitivity to NO may have arisen in bacterial pathogens and that this trait could promote the acquisition of antibiotic-resistance mechanisms through enabling cells to persist in the presence of toxic levels of antibiotic. To test this hypothesis, genomics and microbiological approaches were used to screen a collection of E. coli clinical isolates for antibiotic susceptibility and NO tolerance, although the data did not support a correlation between increased carriage of antibiotic resistance genes and NO tolerance. However, the current work has important implications for how antibiotic susceptibility might be measured in future (i.e. ± NO) and underlines the evolutionary advantage for bacterial pathogens to maintain tolerance to toxic levels of NO.

Developing best practice guidelines for the psychosocial assessment of Aboriginal and Torres Strait Islander people presenting to hospital with self-harm and suicidal thoughts.

OBJECTIVE: To develop guidelines for the culturally responsive psychosocial assessment of Aboriginal and Torres Strait Islander people presenting to hospital with self-harm and suicidal thoughts. METHOD: The Delphi method was used to establish expert consensus. A systematic search and review of relevant research literature, existing guidelines and grey literature was undertaken to develop a 286-item questionnaire. The questionnaire contained best practice statements to guide clinicians undertaking psychosocial assessment of Aboriginal and Torres Strait Islander people presenting to hospital with self-harm and suicidal thoughts. An expert panel comprising 28 individuals with clinical, community-based and lived experience in Aboriginal and Torres Strait Islander mental health and/or suicide prevention were recruited and independently rated the items over three rounds. Statements endorsed as essential or important by 90% or more of the expert panel were then synthesised into recommendations for the best practice guideline document. RESULTS: A total of 226 statements across all relevant areas of clinical practice were endorsed. No statements covering the use of structured assessment tools were endorsed. The endorsed statements informed the development of a set of underlying principles of culturally competent practice and recommendations for processes of effective and appropriate engagement; risks, needs and strengths to be assessed; formulation of psychosocial assessment; and recommendations specific to children and young people. CONCLUSION: The guidelines are based on recommendations endorsed across a range of expertise to address an important gap in the evidence-base for clinically effective and culturally responsive assessment of self-harm and suicidal thoughts by Aboriginal and Torres Strait Islander people in hospital settings. Further work is needed to develop an implementation strategy and evaluate the recommendations in practice.

Adaptation of the Ages and Stages Questionnaire for Remote Aboriginal Australia.

A key challenge to providing quality developmental care in remote Aboriginal primary health care (PHC) centers has been the absence of culturally appropriate developmental screening instruments. This study focused on the cross-cultural adaptation of the Ages and Stages Questionnaires, 3rd edition (ASQ-3), with careful attention to language and culture. We aimed to adapt the ASQ-3 for use with remote dwelling Australian Aboriginal children, and to investigate the cultural appropriateness and feasibility of the adapted ASQ-3 for use in this context. We undertook a qualitative study in two remote Australian Aboriginal communities, using a six-step collaborative adaptation process. Aboriginal Health Workers (AHWs) were trained to use the adapted ASQ-3, and follow-up interviews examined participants' views of the cultural acceptability and usefulness of the adapted instrument. The adapted ASQ-3 was found to have high face validity and to be culturally acceptable and relevant to parents, AHWs, and early childhood development experts.

Trends in hospital admissions involving suicidal behaviour in the Northern Territory, 2001-2013.

OBJECTIVE: To investigate trends in hospital admissions involving suicidal behaviour in the Northern Territory (NT) resident population over the period 2001-2013. METHODS: Estimates of age-standardised rates and average changes in the annual rate of hospital admissions involving suicidal behaviour were calculated by socio-demographic characteristics and types of suicidal behaviour. RESULTS: Overall rates for Indigenous admissions were 2.7 times higher than non-Indigenous admissions and had increased by almost twice as much. While male and female rates of admission were similar for both Indigenous and non-Indigenous residents, the average annual change in rates was greater for Indigenous females (13.4%) compared to males (8.8%) and for non-Indigenous males (7.7%) compared to females (5.2%). Younger and middle-aged Indigenous admissions experienced increasing rates of admissions, whilst trends were similar across age groups for non-Indigenous admissions. Admissions with a diagnosis of suicidal ideation increased the most across all groups. Trends in intentional self-harm admissions differed according to Indigenous status and sex. CONCLUSIONS: There have been substantial increases in hospital admissions involving suicidal behaviour in the NT, most markedly for Indigenous residents. Indigenous females and youth appear to be at increasing risk. The steep increase in suicidal ideation across all groups warrants further investigation.

Challenges in monitoring the development of young children in remote Aboriginal health services: clinical audit findings and recommendations for improving practice.

INTRODUCTION: Early detection of developmental difficulties is universally considered a necessary public health measure, with routine developmental monitoring an important function of primary healthcare services. This study aimed to describe the developmental monitoring practice in two remote Australian Aboriginal primary healthcare services and to identify gaps in the delivery of developmental monitoring services. METHODS: A cross-sectional baseline medical record audit of all resident children aged less than 5 years in two remote Aboriginal health centres in the Northern Territory (NT) in Australia was undertaken between December 2010 and November 2011. RESULTS: A total of 151 medical records were audited, 80 in Community A and 71 in Community B. Developmental checks were more likely among children who attended services more regularly. In Community A, 63 (79%) medical records had some evidence of a developmental check and in Community B there were 42 (59%) medical records with such evidence. However, there was little indication of how assessments were undertaken: only one record noted the use of a formal developmental screening measure. In Community A, 16 (16%) records documented parent report and 20 (20%) documented staff observations, while in Community B, the numbers were 2 (3%) and 11 (19%), respectively. The overall recorded prevalence of developmental difficulties was 21% in Community A and 6% in Community B. CONCLUSIONS: This is the first study to describe the quality of developmental monitoring practice in remote Australian Aboriginal health services. The audit findings suggest the need for a systems-wide approach to the delivery and recording of developmental monitoring services. This will require routine training of remote Aboriginal health workers and remote area nurses in developmental monitoring practice including the use of a culturally appropriate, structured developmental screening measure.

The Effect of a Short-Term Occupational Therapy Intervention on the Participation and Personal Factors of Preschoolers with Developmental Disabilities.

BACKGROUND: Preschoolers with developmental disabilities are referred to occupational therapy due to their decreased participation in daily life occupations. The purpose of this study was to evaluate the improvement in preschoolers' participation and sensory-motor abilities following an occupational therapy intervention. MATERIALS AND METHODS: A prospective cohort study of 38 preschoolers and their parents was conducted using an interrupted time-series design, including assessments at three time points: base line (upon referral to an occupational therapy assessment), pre-intervention, and post-intervention after 9-12 sessions of occupational therapy interventions. Children were evaluated with the Developmental Test of Visual-Motor Integration, as well as the balance and fine motor precision sub tests of the Bruininks-Oseretsky Test of Motor Proficiency. Parents completed the Children's Participation Questionnaire and the Child Performance Skills Questionnaire. Each intervention session was documented by the therapists using the Documentation of Occupational Therapy Session Intervention form. RESULTS: Significant improvement in children's sensory-motor abilities were found in balance, visual integration, and fine motor precision post-intervention. There were also improvements in the measures of diversity, children's independence, and parental satisfaction. CONCLUSIONS: A short-term occupational therapy intervention applied to preschoolers with developmental disabilities is effective in improving their sensory-motor abilities, as well as in promoting their participation in daily activities.

CO(2) acts as a signalling molecule in populations of the fungal pathogen Candida albicans.

When colonising host-niches or non-animated medical devices, individual cells of the fungal pathogen Candida albicans expand into significant biomasses. Here we show that within such biomasses, fungal metabolically generated CO(2) acts as a communication molecule promoting the switch from yeast to filamentous growth essential for C. albicans pathology. We find that CO(2)-mediated intra-colony signalling involves the adenylyl cyclase protein (Cyr1p), a multi-sensor recently found to coordinate fungal responses to serum and bacterial peptidoglycan. We further identify Lys 1373 as essential for CO(2)/bicarbonate regulation of Cyr1p. Disruption of the CO(2)/bicarbonate receptor-site interferes selectively with C. albicans filamentation within fungal biomasses. Comparisons between the Drosophila melanogaster infection model and the mouse model of disseminated candidiasis, suggest that metabolic CO(2) sensing may be important for initial colonisation and epithelial invasion. Our results reveal the existence of a gaseous Candida signalling pathway and its molecular mechanism and provide insights into an evolutionary conserved CO(2)-signalling system.

Folic acid supplementation increases survival and modulates high risk HPV-induced phenotypes in oral squamous cell carcinoma cells and correlates with p53 mRNA transcriptional down-regulation.

BACKGROUND: Although the primary risk factors for developing oral cancers are well understood, less is known about the relationship among the secondary factors that may modulate the progression of oral cancers, such as high-risk human papillomavirus (HPV) infection and folic acid (FA) supplementation. This study examined high-risk HPV and FA supplementation effects, both singly and in combination, to modulate the proliferative phenotypes of the oral cancer cell lines CAL27, SCC25 and SCC15. RESULTS: Using a comprehensive series of integrated in vitro assays, distinct effects of HPV infection and FA supplementation were observed. Both high-risk HPV strains 16 and 18 induced robust growth-stimulating effects in CAL27 and normal HGF-1 cells, although strain-specific responses were observed in SCC25 and SCC15 cells. Differential effects were also observed with FA administration, which significantly altered the growth rate of the oral cancer cell lines CAL27, SCC15, and SCC25, but not HGF-1 cells. Unlike HPV, FA administration induced broad, general increases in cell viability among all cell lines that were associated with p53 mRNA transcriptional down-regulation. None of these cell lines were found to harbor the common C677T mutation in methylenetetrahydrofolate reductase (MTHFR), which can reduce FA availability and may increase oral cancer risk. CONCLUSION: Increased FA utilization and DNA hypermethylation are common features of oral cancers, and in these cell lines, specifically. The results of this study provide further evidence that FA antimetabolites, such as Fluorouracil (f5U or 5-FU) and Raltitrexed, may be alternative therapies for tumors resistant to other therapies. Moreover, since the incidence of oral HPV infection has been increasing, and can influence oral cancer growth, the relationship between FA bioavailability and concomitant HPV infection must be elucidated. This study is among the first pre-clinical studies to evaluate FA- and HPV-induced effects in oral cancers, both separately and in combination, which provides additional rationale for clinical screening of HPV infection prior to treatment.

Culture, context and therapeutic processes: delivering a parent-child intervention in a remote Aboriginal community.

OBJECTIVE: Little is written about the process of delivering mainstream, evidence-based therapeutic interventions for Aboriginal children and families in remote communities. Patterns of interaction between parents and children and expectations about parenting and professional roles and responsibilities vary across cultural contexts. This can be a challenging experience for professionals accustomed to work in urban settings. Language is only a part of cultural difference, and the outsider in a therapeutic group in an Aboriginal community is outside not only in language but also in access to community relationships and a place within those relationships. METHOD: This paper uses examples from Let's Start, a therapeutic parent-child intervention to describe the impact of distance, culture and relationships in a remote Aboriginal community, on the therapeutic framework, group processes and relationships. RESULTS: Cultural and contextual factors influence communication, relationships and group processes in a therapeutic group program for children and parents in a remote Aboriginal community. Group leaders from within and from outside the community, are likely to have complementary skills. CONCLUSIONS: Cultural and contextual factors influence communication, relationships and group processes in a therapeutic group program for children and parents in a remote Aboriginal community. Group leaders from within and from outside the community, are likely to have complementary skills. Program adaptation, evaluation and staff training and support need to take these factors into account to ensure cultural accessibility without loss of therapeutic fidelity and efficacy.

Validity and reliability of resiliency measures trialled for the evaluation of a preventative Resilience-promoting social-emotional curriculum for remote Aboriginal school students.

PURPOSE: We aimed to test the reliability and validity of two brief measures of resilience adopted for the evaluation of a preventative social-emotional curriculum implemented for Aboriginal middle school students from socially disadvantaged remote communities in Australia's Northern Territory. The questionnaires chosen were intended to measure psychological resilience and socio-cultural resilience as complementary dimensions of the capacity to cope in circumstances of significant life stress and risk of self-harm. METHODS: Confirmatory factor analysis (CFA) was conducted to assess construct validity of the 10-item Connor-Davidson Resilience Scale (CD-RISC-10), a measure of psychological resilience, and the 12-item Child and Youth Resilience Measure (CYRM-12), a measure of socio-cultural resilience, with a sample of 520 students. Associations between resilience and psychological distress and emotional and behavioural difficulty were analysed in relation to life stressors to assess criterion validity of the scales. RESULTS: CFA provided support for the validity of the respective constructs. There was good fit for both scales. However, assessment of criterion validity of the scales suggested that the adapted measure of socio-cultural resilience (CYRM-12NT) showed higher reliability and a clearer indication of predictive validity than the measure of psychological resilience (CD-RISC-10). CONCLUSIONS: The CYRM-12NT appears to be a more useful measure of resilience among Aboriginal youth exposed to significant life stress and disadvantage. However, both measures may require further development to enhance their validity and utility among potentially at-risk adolescents in socially, culturally and linguistically diverse remote Aboriginal communities.

Deletion of glyceraldehyde-3-phosphate dehydrogenase (gapN) in Clostridium saccharoperbutylacetonicum N1-4(HMT) using CLEAVE™ increases the ATP pool and accelerates solvent production.

The development and advent of mutagenesis tools for solventogenic clostridial species in recent years has allowed for the increased refinement of industrially relevant strains. In this study we have utilised CLEAVE™, a CRISPR/Cas genome editing system developed by Green Biologics Ltd., to engineer a strain of Clostridium saccharoperbutylacetonicum N1-4(HMT) with potentially useful solvents titres and energy metabolism. As one of two enzymes responsible for the conversion of glyceraldehyde-3-phosphate (GAP) to 3-phosphoglyceric acid in glycolysis, it was hypothesised that deletion of gapN would increase ATP and NADH production that could in turn improve solvent production. Herein, whole genome sequencing has been used to evaluate CLEAVE™ and the successful knockout of gapN, demonstrating a clean knockout with no other detectable variations from the wild type sequence. Elevated solvent levels were detected during the first 24 h of batch fermentation, indicating an earlier shift to solventogenesis. A 2.4-fold increase in ATP concentration was observed, and quantitation of NAD(P)H derivatives revealed a more reducing cytoplasm for the gapN strain. These findings expand our understanding of clostridium carbon metabolism and report a new approach to optimising biofuel production.

Passive control of quorum sensing: prevention of Pseudomonas aeruginosa biofilm formation by imprinted polymers.

Here we present the first molecular imprinted polymer (MIP) that is able to attenuate the biofilm formation of the opportunistic human pathogen Pseudomonas aeruginosa through specific sequestration of its signal molecule N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C(12)-AHL). The MIP was rationally designed using computational modeling, and its capacity and specificity and that of a corresponding blank polymer toward signal molecule of P. aeruginosa (3-oxo-C(12)-AHL) and its analogue were tested. The biofilm formation in the presence of polymers and without polymers was studied using scanning confocal laser microscopy. Staining with crystal violet dye was used for the quantification of the biofilm formation. A significant reduction of the biofilm growth was observed in the presence of MIP (>80%), which was superior to that of the resin prepared without template, which showed a reduction of 40% in comparison with biofilm, which was grown without polymer addition. It was shown that 3-oxo-C(12)-AHL-specific MIP prevented the development of quorum-sensing-controlled phenotypes (in this case, biofilm formation) from being up-regulated. The developed MIP could be considered as a new tool for the elimination of life-threatening infections in a multitude of practical applications; it could, for example, be grafted on the surface of medical devices such as catheters and lenses, be a component of paints, or be used as a wound adsorbent.

Reciprocal Packaging of the Main Structural Proteins of Type 1 Fimbriae and Flagella in the Outer Membrane Vesicles of "Wild Type" Escherichia coli Strains.

Fundamental aspects of outer membrane vesicle (OMV) biogenesis and the engineering of producer strains have been major research foci for many in recent years. The focus of this study was OMV production in a variety of Escherichia coli strains including wild type (WT) (K12 and BW25113), mutants (from the Keio collection) and proprietary [BL21 and BL21 (DE3)] strains. The present study investigated the proteome and prospective mechanism that underpinned the key finding that the dominant protein present in E. coli K-12 WT OMVs was fimbrial protein monomer (FimA) (a polymerizable protein which is the key structural monomer from which Type 1 fimbriae are made). However, mutations in genes involved in fimbriae biosynthesis (ΔfimA, B, C, and F) resulted in the packaging of flagella protein monomer (FliC) (the major structural protein of flagella) into OMVs instead of FimA. Other mutations (ΔfimE, G, H, I, and ΔlrhA-a transcriptional regulator of fimbriation and flagella biosynthesis) lead to the packaging of both FimA and Flagellin into the OMVs. In the majority of instances shown within this research, the production of OMVs is considered in K-12 WT strains where structural appendages including fimbriae or flagella are temporally co-expressed throughout the growth curve as shown previously in the literature. The hypothesis, proposed and supported within the present paper, is that the vesicular packaging of the major FimA is reciprocally regulated with the major FliC in E. coli K-12 OMVs but this is abrogated in a range of mutated, non-WT E. coli strains. We also demonstrate, that a protein of interest (GFP) can be targeted to OMVs in an E. coli K-12 strain by protein fusion with FimA and that this causes normal packaging to be disrupted. The findings and underlying implications for host interactions and use in biotechnology are discussed.

Parallel bioreactor system for accessible and reproducible anaerobic culture.

When working with anaerobic bacteria it is important to have the capability to perform parallel bioreactor growth experiments that are both controllable and reproducible, although capital and consumables costs for commercially available systems are often prohibitively high. Hence, a three-vessel parallel bioreactor system was designed and constructed that has the capabilities for batch and fed batch processes and can also be set up for continuous culture at a fraction of the cost of commercial systems. This system carries over many of the same functionalities of those systems with a higher price point of entry, including in-line monitoring of temperature, pH, and redox poise. To validate the performance of this system Clostridium saccharoperbutylacetonicum was grown under conditions that promote ABE fermentation, an established industrial process used to produce the solvents acetone, butanol and ethanol. Measurements of cell density, pH, and redox poise all confirmed reproducible culture conditions for these parallel vessels, and solvent quantitation via GCMS verified consistent metabolic activities for the separate cultures. In future, this system will be of interest to researchers that require high performance parallel fermentation platforms but where commercial systems are not accessible.

Repurposing in vitro approaches for screening anti-parasitic drugs against the brain-eating amoeba Naegleria fowleri.

Naegleria fowleri is both a pathogenic and a free-living microbial eukaryote, responsible for the development of primary amoebic meningoencephalitis (PAM) in humans. PAM is a rapid, severe and fatal underestimated infectious disease, which has been reported in countries with warmer climates. The major drawbacks with PAM are the lack of effective therapies and delay in diagnosis. The current frontline treatment presents a low rate of recovery (5%) and severe adverse effects. For example, many drug candidates lack efficacy, since they do not effectively cross the blood-brain-barrier. Consequently, more effective drugs are urgently needed. Herein, we report a new in vitro method suitable for medium- and high-throughput drug discovery assays, using the closely related Naegleria gruberi as a model. We have subsequently used this method to screen a library of 1175 Food and Drug Administration-approved drugs. As a result, we present three drugs (camptothecin, pyrimethamine, and terbinafine) that can be repurposed, and are anticipated to readily cross the blood-brain-barrier with activity against Naegleria species in therapeutically achievable concentrations. Successively, we integrated several in vitro assays that resulted in identifying fast-acting and high amoebicidal drugs. In conclusion, we present a new approach for the identification of anti-Naegleria drugs along with three potential drug candidates for further development for the treatment of PAM.