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No preschool teacher starts off the year aiming for a chaotic classroom filled with squabbling students. However, if they do not have a purposeful plan, that is likely what they will end up with. Small changes can make huge differences, and a year started off right can lead to a joyful preschool experience for the precious children in our care. This article will present a plan for engineering the classroom for peace. This evidence-based, developmentally appropriate plan will focus on strategies to prepare the physical environment, set the tone, and be available.

Legionella is the causative agent of Legionnaires’ disease, and its prevalence in potable water is a significant public health issue. Water stagnation within buildings increases the risk of Legionella. However, there are limited studies investigating how stagnation arising through intermittent usage affects Legionella proliferation and the studies that are available do not consider viable but non culturable (VBNC) Legionella. This study used a model plumbing system to examine how intermittent water stagnation affects both VBNC and culturable Legionella. The model plumbing system contained a water tank supplying two biofilm reactors. The model was initially left stagnant for ≈5 months (147 days), after which one reactor was flushed daily, and the other weekly. Biofilm coupons, and water samples were collected for analysis at days 0, 14 and 28. These samples were analysed for culturable and VBNC Legionella, free-living amoebae, and heterotrophic bacteria. After 28 days, once-a-day flushing significantly (p

Abstract Introduction Oral administration of zeaxanthin (Zx) 20 mg daily in patients with unilateral neovascular age-related macular degeneration (nAMD) treated with triple therapy (photodynamic therapy/intravitreal bevacizumab/intravitreal dexamethasone) reduced fellow-eye 2-year nAMD incidence from 23 to 6% (p = 0.02) in a prior clinical trial. We questioned the long-term benefit and thus analyzed case–control 5-year patient data of trial participants and additional participants with 5-year follow-up, also performing cost-utility and cost–benefit analyses. Methods Consecutive, unilateral nAMD patient outcomes for those taking 20 mg Zx supplementation orally for ≥ 5 years were compared with the Comparison of AMD Treatments Trials (CATT) 5-year historical controls for fellow-eye nAMD conversion. Eleven-year mean life expectancy, cost-utility and cost–benefit models were undertaken employing a 3% discount rate and 2020 US real dollars. Results Among 227 consecutive patients with nAMD/Zx-supplementation, 202 (90%) had 5-year follow-up. The fellow-eye nAMD 5-year conversion incidence using a Kaplan–Meier cumulative event estimate was 22% (49/227), versus 48% (167/348) with CATT control data (p

In this article, we explore how tourism in Xinjiang is politically weaponised. Commodifying Uyghur cultural heritage for tourism allows the Chinese Communist Party (CCP) to insist it is not committing cultural genocide, but actually “conserving” Uyghur culture. This directly bears on the CCP’s internment of Muslim minorities in “re-education” camps, ostensibly to target Islamic “extremism.” We explore how tourism to Xinjiang is presented as a “success” of the camps and conscripted into the “Sinicisation” of the region and the secularising of minorities’ cultures. Places and practices are deconstructed as cultural heritage, and reconstructed to provide tourists with “exotic” experiences of “wonderful Xinjiang.” This transforms the “tourist gaze” into a “testimonial” one: tourists to Xinjiang are made into witnesses that “Xinjiang is beautiful” and Uyghurs are “happy.” In this, touristic development and tourists themselves are key agents in the CCP’s territorialisation of Xinjiang, the sinicisation of Uyghur culture, and the legitimation of the violence of the camps.

Hospital water systems are a significant source of Legionella, resulting in the potentially fatal Legionnaires’ disease. One of the biggest challenges for Legionella management within these systems is that under unfavorable conditions Legionella transforms itself into a viable but non culturable (VBNC) state that cannot be detected using the standard methods. This study used a novel method (flow cytometry-cell sorting and qPCR [VFC+qPCR] assay) concurrently with the standard detection methods to examine the effect of temporary water stagnation, on Legionella spp. and microbial communities present in a hospital water system. Water samples were also analyzed for amoebae using culture and Vermamoeba vermiformis and Acanthamoeba specific qPCR. The water temperature, number and duration of water flow events for the hand basins and showers sampled was measured using the Enware Smart Flow® monitoring system. qPCR analysis demonstrated that 21.8% samples were positive for Legionella spp., 21% for L. pneumophila, 40.9% for V. vermiformis and 4.2% for Acanthamoeba. All samples that were Legionella spp. positive using qPCR (22%) were also positive for VBNC Legionella spp.; however, only 2.5% of samples were positive for culturable Legionella spp. 18.1% of the samples were positive for free-living amoebae (FLA) using culture. All samples positive for Legionella spp. were also positive for FLA. Samples with a high heterotrophic plate count (HPC ≥ 5 × 103 CFU/L) were also significantly associated with high concentrations of Legionella spp. DNA, VBNC Legionella spp./L. pneumophila (p < 0.01) and V. vermiformis (p < 0.05). Temporary water stagnation arising through intermittent usage (< 2 hours of usage per month) significantly (p < 0.01) increased the amount of Legionella spp. DNA, VBNC Legionella spp./L. pneumophila, and V. vermiformis; however, it did not significantly impact the HPC load. In contrast to stagnation, no relationship was observed between the microbes and water temperature. In conclusion, Legionella spp. (DNA and VBNC) was associated with V. vermiformis, heterotrophic bacteria, and stagnation occurring through intermittent usage. This is the first study to monitor VBNC Legionella spp. within a hospital water system. The high percentage of false negative Legionella spp. results provided by the culture method supports the use of either qPCR or VFC+qPCR to monitor Legionella spp. contamination within hospital water systems.

Legionella pneumophila is a waterborne pathogen and, as the causative agent of Legionnaires’ disease, a significant public health concern. Exposure to environmental stresses, and disinfection treatments, promotes the formation of resistant and potentially infectious viable but non-culturable (VBNC) Legionella. The management of engineered water systems to prevent Legionnaires’ disease is hindered by the presence of VBNC Legionella that cannot be detected using the standard culture (ISO11731:2017-05) and quantitative polymerase reaction (ISO/TS12869:2019) methods. This study describes a novel method to quantify VBNC Legionella from environmental water samples using a “viability based flow cytometry-cell sorting and qPCR” (VFC + qPCR) assay. This protocol was then validated by quantifying the VBNC Legionella genomic load from hospital water samples. The VBNC cells were unable to be cultured on Buffered Charcoal Yeast Extract (BCYE) agar; however, their viability was confirmed through their ATP activity and ability to infect amoeba hosts. Subsequently, an assessment of the ISO11731:2017-05 pre-treatment procedure demonstrated that acid or heat treatment cause underestimation of alive Legionella population. Our results showed that these pre-treatment procedures induce culturable cells to enter a VBNC state. This may explain the observed insensitivity and lack of reproducibility often observed with the Legionella culture method. This study represents the first time that flow cytometry-cell sorting in conjunction with a qPCR assay has been used as a rapid and direct method to quantify VBNC Legionella from environmental sources. This will significantly improve future research evaluating Legionella risk management approaches for the control of Legionnaires’ disease.

This reflective piece is about my journey as an international PhD candidate from the UK to Aotearoa (New Zealand). I speak about my search to try and find a sense of belonging as an outsider and some of the challenges in doing community-focused research as an early career researcher from elsewhere. This piece includes key challenges of building networks, collaboration and overcoming the death of my father, half-way through my PhD. I speak about a toolkit of strength and resilience I had to create and now use in the support of my students and peers as a lecturer in education during the pandemic. This piece is about finding kindness and compassion in myself and receiving it from others to help with isolation in difficult times. Keywords: Reflection, Resilience, Compassion, Kindness, Networks, Community, Isolation How to cite this article: Dencer-Brown, A.M. 2020. From isolation to cross-cultural collaboration: My international PhD journey as tō iwi. Scholarship of Teaching and Learning in the South. 4(2): 228-234. https://doi.org/10.36615/sotls.v4i2.142. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

ABSTRACT Antimicrobial resistance in Neisseria gonorrhoeae has reached an alarming level, severely impacting the effective treatment of gonorrhea. Belonging to the resistance-nodulation-cell division (RND) superfamily of efflux transporters, the MtrD membrane protein of N. gonorrhoeae provides resistance to a broad range of antimicrobial compounds. A unique feature of MtrD is an 11-residue sequence (from N917 to P927 [N917-P927]) that connects transmembrane helices (TMS) 9 and 10; this sequence is not present in homologous RND proteins. This study explores the structural and functional roles of the N917-P927 region by means of mutant analysis and molecular dynamics simulations. We show that N917-P927 plays a key role in modulating substrate access to the binding cleft and influences the overall orientation of the protein within the inner membrane necessary for optimal functioning. Removal of N917-P927 significantly reduced MtrD-mediated resistance to a range of antimicrobials and mutations of three single amino acids impacted MtrD-mediated multidrug resistance. Furthermore, molecular dynamics simulations showed deletion of N917-P927 in MtrD may dysregulate access of the substrate to the binding cleft and closure of the substrate-binding pocket during the transport cycle. These findings indicate that N917-P927 is a key region for interacting with the inner membrane, conceivably influencing substrate capture from the membrane-periplasm interface and thus is essential for full multidrug resistance capacity of MtrD. IMPORTANCE The historical sexually transmitted infection gonorrhea continues to be a major public health concern with an estimated global annual incidence of 86.9 million cases. N. gonorrhoeae has been identified by the World Health Organization as one of the 12 antimicrobial-resistant bacterial species that poses the greatest risk to human health. As the major efflux pump in gonococci, the MtrD transporter contributes to the cell envelope barrier in this organism and pumps antimicrobials from the periplasm and inner membrane, resulting in resistance. This study demonstrates that a unique region of the MtrD protein that connects TMS 9 and TMS 10 forms a structure that may interact with the inner membrane positioning TMS 9 and stabilizing the protein facilitating substrate capture from the inner membrane-periplasm interface. Analysis of mutants of this region identified that it was essential for MtrD-mediated multidrug resistance. Characterization of the structure and function of this unique local region of MtrD has implications for drug efflux mechanisms used by related proteins and is important knowledge for development of antibiotics that bypass efflux.

Plant-derived pharmacological agents have been used extensively to dissect the structure–function relationships of mammalian GABA receptors and ion channels. Picrotoxin is a non-competitive antagonist of mammalian GABAA receptors. Here, we report that picrotoxin inhibits the anion (malate) efflux mediated by wheat (Triticum aestivum) ALMT1 but has no effect on GABA transport. The EC50 for inhibition was 0.14 nM and 0.18 nM when the ALMTs were expressed in tobacco BY2 cells and in Xenopus oocytes, respectively. Patch clamping of the oocyte plasma membrane expressing wheat ALMT1 showed that picrotoxin inhibited malate currents from both sides of the membrane. These results demonstrate that picrotoxin inhibits anion efflux effectively and can be used as a new inhibitor to study the ion fluxes mediated by ALMT proteins that allow either GABA or anion transport.

Herpes simplex virus encephalitis (HSE) is the most common cause of sporadic viral encephalitis, and despite targeted antiviral therapy, outcomes remain poor. Although the innate immune system is critical for restricting herpes simplex virus type I (HSV-1) in the brain, there is evidence that prolonged neuroinflammation contributes to HSE pathogenesis. In this study, we investigated the contribution of inflammasomes to disease pathogenesis in a murine model of HSE. Inflammasomes are signaling platforms that activate the pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18. We found that mice deficient in the inflammasome adaptor protein, apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), had significantly improved survival and lower levels of IL-1β and IL-18 in the brain. Importantly, this difference in survival was independent of viral replication in the central nervous system (CNS). We found that microglia, the resident macrophages of the CNS, are the primary mediators of the ASC-dependent inflammasome response during infection. Using in vitro glial infections and a murine HSE model, we demonstrate that inflammasome activation contributes to the expression of chemokine (C-C motif) ligand 6 (CCL6), a leukocyte chemoattractant. The lower concentration of CCL6 in the brains of ASC-/- mice correlated with lower numbers of infiltrating macrophages during infection. Together, these data suggest that inflammasomes contribute to pathogenic inflammation in HSE and provide a mechanistic link between glial inflammasome activation and leukocyte infiltration. The contribution of inflammasomes to survival was independent of viral replication in our study, suggesting a promising new target in combating harmful inflammation in HSE.

Acinetobacter baumannii is an opportunistic human pathogen responsible for numerous severe nosocomial infections. Genome analysis on the A. baumannii clinical isolate 04117201 revealed the presence of 13 two-component signal transduction systems (TCS). Of these, we examined the putative TCS named here as StkSR. The stkR response regulator was deleted via homologous recombination and its progeny, ΔstkR, was phenotypically characterized. Antibiogram analyses of ΔstkR cells revealed a two-fold increase in resistance to the clinically relevant polymyxins, colistin and polymyxin B, compared to wildtype. PAGE-separation of silver stained purified lipooligosaccharide isolated from ΔstkR and wildtype cells ruled out the complete loss of lipooligosaccharide as the mechanism of colistin resistance identified for ΔstkR. Hydrophobicity analysis identified a phenotypical change of the bacterial cells when exposed to colistin. Transcriptional profiling revealed a significant up-regulation of the pmrCAB operon in ΔstkR compared to the parent, associating these two TCS and colistin resistance. These results reveal that there are multiple levels of regulation affecting colistin resistance; the suggested ‘cross-talk’ between the StkSR and PmrAB two-component systems highlights the complexity of these systems.

Legionella is an opportunistic waterborne pathogen associated with Legionnaires' disease and Pontiac fever. Despite improved public awareness, the incidence of Legionella associated infections has been increasing. Aerosols generated from engineered potable water systems are a demonstrated cause of both nosocomial and community-acquired legionellosis. The ecology of Legionella in these systems is complex with multiple factors impacting their colonization and persistence. Flow dynamics has been identified as an important factor and stagnation in cooling towers is an accepted risk for increased Legionella growth; however, less is known about the impact of flow dynamic on Legionella in potable water systems. This is especially complex due to the inherent intermittent and variable usage observed within outlets of a potable water system. This systematic literature review examines the role of fluid dynamics and stagnation on the colonization and growth of Legionella in potable water systems. Twenty two of 24 identified studies show a positive association between stagnation zones and increased colonization of Legionella. These zones included dead legs, dead ends, storage tanks, and obstructed water flow (such as intermittent usage or flow restriction). Prolonged stagnation in building plumbing systems also deteriorates the quality of thermally or chemically treated potable water. This stimulates the colonization of Legionella established biofilms. Such biofilms are intrinsically resistant to disinfection procedures and accelerate the rate of decay of chemical disinfectants. Sub-lethal doses of disinfectants and the presence of protozoan hosts in stationary water promote generation of viable but non-culturable Legionella cells. This results in false negatives in surveillance methods that use culture methodology. In conclusion, elimination of temporal and permanent stagnation points can improve the quality of potable water, efficacy of disinfectants, and reduce the risk of legionellosis. Current guidelines and water safety plans recognize the risks associated with permanent stagnation point (dead ends and dead legs); however, there is a need for greater emphasis on controlling temporal stagnation arising from intermittent usage.

The increasing emergence of antimicrobial resistance in staphylococcal bacteria is a major health threat worldwide due to significant morbidity and mortality resulting from their associated hospital- or community-acquired infections. Dramatic decrease in the discovery of new antibiotics from the pharmaceutical industry coupled with increased use of sanitisers and disinfectants due to the ongoing COVID-19 pandemic can further aggravate the problem of antimicrobial resistance. Staphylococci utilise multiple mechanisms to circumvent the effects of antimicrobials. One of these resistance mechanisms is the export of antimicrobial agents through the activity of membrane-embedded multidrug efflux pump proteins. The use of efflux pump inhibitors in combination with currently approved antimicrobials is a promising strategy to potentiate their clinical efficacy against resistant strains of staphylococci, and simultaneously reduce the selection of resistant mutants. This review presents an overview of the current knowledge of staphylococcal efflux pumps, discusses their clinical impact, and summarises compounds found in the last decade from plant and synthetic origin that have the potential to be used as adjuvants to antibiotic therapy against multidrug resistant staphylococci. Critically, future high-resolution structures of staphylococcal efflux pumps could aid in design and development of safer, more target-specific and highly potent efflux pump inhibitors to progress into clinical use.

ABSTRACT A key mechanism that Neisseria gonorrhoeae uses to achieve multidrug resistance is the expulsion of structurally different antimicrobials by the MtrD multidrug efflux protein. MtrD resembles the homologous Escherichia coli AcrB efflux protein with several common structural features, including an open cleft containing putative access and deep binding pockets proposed to interact with substrates. A highly discriminating N. gonorrhoeae strain, with the MtrD and NorM multidrug efflux pumps inactivated, was constructed and used to confirm and extend the substrate profile of MtrD to include 14 new compounds. The structural basis of substrate interactions with MtrD was interrogated by a combination of long-timescale molecular dynamics simulations and docking studies together with site-directed mutagenesis of selected residues. Of the MtrD mutants generated, only one (S611A) retained a wild-type (WT) resistance profile, while others (F136A, F176A, I605A, F610A, F612C, and F623C) showed reduced resistance to different antimicrobial compounds. Docking studies of eight MtrD substrates confirmed that many of the mutated residues play important nonspecific roles in binding to these substrates. Long-timescale molecular dynamics simulations of MtrD with its substrate progesterone showed the spontaneous binding of the substrate to the access pocket of the binding cleft and its subsequent penetration into the deep binding pocket, allowing the permeation pathway for a substrate through this important resistance mechanism to be identified. These findings provide a detailed picture of the interaction of MtrD with substrates that can be used as a basis for rational antibiotic and inhibitor design. IMPORTANCE With over 78 million new infections globally each year, gonorrhea remains a frustratingly common infection. Continuous development and spread of antimicrobial-resistant strains of Neisseria gonorrhoeae, the causative agent of gonorrhea, have posed a serious threat to public health. One of the mechanisms in N. gonorrhoeae involved in resistance to multiple drugs is performed by the MtrD multidrug resistance efflux pump. This study demonstrated that the MtrD pump has a broader substrate specificity than previously proposed and identified a cluster of residues important for drug binding and translocation. Additionally, a permeation pathway for the MtrD substrate progesterone actively moving through the protein was determined, revealing key interactions within the putative MtrD drug binding pockets. Identification of functionally important residues and substrate-protein interactions of the MtrD protein is crucial to develop future strategies for the treatment of multidrug-resistant gonorrhea.

Batrachochytrium dendrobatidis and B. salamandrivorans are important amphibian pathogens responsible for morbidity and mortality in free-ranging and captive frogs, salamanders, and caecilians. While B. dendrobatidis has a widespread global distribution, B. salamandrivorans has only been detected in amphibians in Asia and Europe. Although molecular detection methods for these fungi are well-characterized, differentiation of the morphologically similar organisms in the tissues of affected amphibians is incredibly difficult. Moreover, an accurate tool to identify and differentiate Batrachochytrium in affected amphibian tissues is essential for a specific diagnosis of the causative agent in chytridiomycosis cases. To address this need, an automated dual-plex chromogenic RNAScope®in situ hybridization (ISH) assay was developed and characterized for simultaneous detection and differentiation of B. dendrobatidis and B. salamandrivorans. The assay, utilizing double Z target probe pairs designed to hybridize to 28S rRNA sequences, was specific for the identification of both organisms in culture and in formalin-fixed paraffin-embedded amphibian tissues. The assay successfully identified organisms in tissue samples from five salamander and one frog species preserved in formalin for up to 364 days and was sensitive for the detection of Batrachochytrium in animals with qPCR loads as low as 1.1 × 102 zoospores/microliter. ISH staining of B. salamandrivorans also highlighted the infection of dermal cutaneous glands, a feature not observed in amphibian B. dendrobatidis cases and which may play an important role in B. salamandrivorans pathogenesis in salamanders. The developed ISH assay will benefit both amphibian chytridiomycosis surveillance projects and pathogenesis studies by providing a reliable tool for Batrachochytrium differentiation in tissues.

ABSTRACT Insertion sequences (IS) are fundamental mediators of genome plasticity with the potential to generate phenotypic variation with significant evolutionary outcomes. Here, a recently active miniature inverted-repeat transposon element (MITE) was identified in a derivative of Acinetobacter baumannii ATCC 17978 after being subjected to stress conditions. Transposition of the novel element led to the disruption of the hns gene, resulting in a characteristic hypermotile phenotype. DNA identity shared between the terminal inverted repeats of this MITE and coresident ISAba12 elements, together with the generation of 9-bp target site duplications, provides strong evidence that ISAba12 elements were responsible for mobilization of the MITE (designated MITEAba12) within this strain. A wider genome-level survey identified MITEAba12 in 30 additional Acinetobacter genomes at various frequencies and one Moraxella osloensis genome. Ninety MITEAba12 copies could be identified, of which 40% had target site duplications, indicating recent transposition events. Elements ranged between 111 and 114 bp; 90% were 113 bp in length. Using the MITEAba12 consensus sequence, putative outward-facing Escherichia coli σ70 promoter sequences in both orientations were identified. The identification of transcripts originating from the promoter in one direction supports the proposal that the element can influence neighboring host gene transcription. The location of MITEAba12 varied significantly between and within genomes, preferentially integrating into AT-rich regions. Additionally, a copy of MITEAba12 was identified in a novel 8.5-kb composite transposon, Tn6645, in the M. osloensis CCUG 350 chromosome. Overall, this study shows that MITEAba12 is the most abundant nonautonomous element currently found in Acinetobacter. IMPORTANCE One of the most important weapons in the armory of Acinetobacter is its impressive genetic plasticity, facilitating rapid genetic mutations and rearrangements as well as integration of foreign determinants carried by mobile genetic elements. Of these, IS are considered one of the key forces shaping bacterial genomes and ultimately evolution. We report the identification of a novel nonautonomous IS-derived element present in multiple bacterial species from the Moraxellaceae family and its recent translocation into the hns locus in the A. baumannii ATCC 17978 genome. The latter finding adds new knowledge to only a limited number of documented examples of MITEs in the literature and underscores the plastic nature of the hns locus in A. baumannii. MITEAba12, and its predicted parent(s), may be a source of substantial adaptive evolution within environmental and clinically relevant bacterial pathogens and, thus, have broad implications for niche-specific adaptation.

The Gram-negative opportunistic bacterium Acinetobacter baumannii is a significant cause of hospital-borne infections worldwide. Alarmingly, the rapid development of antimicrobial resistance coupled with the remarkable ability of isolates to persist on surfaces for extended periods of time has led to infiltration of A. baumannii into our healthcare environments. A major virulence determinant of A. baumannii is the presence of a capsule that surrounds the bacterial surface. This capsule is comprised of tightly packed repeating polysaccharide units which forms a barrier around the bacterial cell wall, providing protection from environmental pressures including desiccation and disinfection regimes as well as host immune responses such as serum complement. Additionally, capsule has been shown to confer resistance to a range of clinically relevant antimicrobial compounds. Distressingly, treatment options for A. baumannii infections are becoming increasingly limited, and the urgency to develop effective infection control strategies and therapies to combat infections is apparent. An increased understanding of the contribution of capsule to the pathobiology of A. baumannii is required to determine its feasibility as a target for new strategies to combat drug resistant infections. Significant variation in capsular polysaccharide structures between A. baumannii isolates has been identified, with over 100 distinct capsule types, incorporating a vast variety of sugars. This review examines the studies undertaken to elucidate capsule diversity and advance our understanding of the role of capsule in A. baumannii pathogenesis.

The misuse and overuse of antibiotics have led to the emergence of multidrug-resistant microorganisms, which decreases the chance of treating those infected with existing antibiotics. This resistance calls for the search of new antimicrobials from prolific producers of novel natural products including marine sponges. Many of the novel active compounds reported from sponges have originated from their microbial symbionts. Therefore, this study aims to screen for bioactive metabolites from bacteria isolated from sponges. Twelve sponge samples were collected from South Australian marine environments and grown on seven isolation media under four incubation conditions; a total of 1234 bacterial isolates were obtained. Of these, 169 bacteria were tested in media optimized for production of antimicrobial metabolites and screened against eleven human pathogens. Seventy bacteria were found to be active against at least one test bacterial or fungal pathogen, while 37% of the tested bacteria showed activity against Staphylococcus aureus including methicillin-resistant strains and antifungal activity was produced by 21% the isolates. A potential novel active compound was purified possessing inhibitory activity against S. aureus. Using 16S rRNA, the strain was identified as Streptomyces sp. Our study highlights that the marine sponges of South Australia are a rich source of abundant and diverse bacteria producing metabolites with antimicrobial activities against human pathogenic bacteria and fungi.

Background: Infection with methicillin-resistant staphylococci (MRS) can be life-threatening in humans and its presence in animals is a cause for public health concern. The aim of this study was to measure the prevalence of MRS in captive and free-ranging wallabies over a 16-month period in South Australia, Australia. Materials and methods: Eighty-nine purified staphylococcal isolates recovered from 98 captive and free-ranging wallabies' anterior nasal swabs were used in this study. All isolates were tested for the presence of the mecA, mecA1, and mecC genes. Multiplex PCR-directed SCCmec-typing, ccrB-typing, and determination of the minimal inhibitory concentration of oxacillin were performed on mec-positive isolates. Results and discussion: In total, 11 non-Staphylococcus aureus MRS were isolated from 7 out of 98 animals, corresponding to a 7.1% carriage rate. The SCCmec types I, III, and V were identified by multiplex PCR and sequencing of the ccrB gene. This is the first report of MRS carriage in both captive and free-ranging wallabies in Australia. These data demonstrate a low prevalence of MRS and no association between wallaby captivity status and MRS carriage could be assigned. These animals may act as a reservoir for the exchange of genetic elements between staphylococci. Furthermore, the mecA genes of animal isolates were identical to that found in human MRS strains and thus the possibility of zoonotic transfer must be considered.

The budding yeast Candida albicans is one of the most significant fungal pathogens worldwide. It proliferates in two distinct cell types: blastopores and filaments. Only cells that are able to transform from one cell type into the other are virulent in mouse disease models. Programmed cell death is a controlled form of cell suicide that occurs when C. albicans cells are exposed to fungicidal drugs like amphotericin B and caspofungin, and to other stressful conditions. We now provide evidence that suggests that programmed cell death is cell-type specific in yeast: Filamentous C. albicans cells are more resistant to amphotericin B- and caspofungin-induced programmed cell death than their blastospore counterparts. Finally, our genetic data suggests that this phenomenon is mediated by a protective mechanism involving the yeast metacaspase, MCA1.

Pheochromocytomas (PCs) are tumors arising from the chromaffin cells of the adrenal glands and are the most common tumors of the adrenal medulla in animals. In people, these are highly correlated to inherited gene mutations in the succinate dehydrogenase (SDH) pathway; however, to date, little work has been done on the genetic basis of these tumors in animals. In humans, immunohistochemistry has proven valuable as a screening technique for SDH mutations. Human PCs that lack succinate dehydrogenase B (SDHB) immunoreactivity have a high rate of mutation in the SDH family of genes, while human PCs lacking succinate dehydrogenase A (SDHA) immunoreactivity have mutations in the SDHA gene. To determine if these results are similar for dogs, we performed SDHA and SDHB immunohistochemistry on 35 canine formalin-fixed, paraffin-embedded (FFPE) PCs. Interestingly, there was a loss of immunoreactivity for both SDHA and SDHB in four samples (11%), suggesting a mutation in SDHx including SDHA. An additional 25 (71%) lacked immunoreactivity for SDHB, while retaining SDHA immunoreactivity. These data suggest that 29 out of the 35 (82%) may have an SDH family mutation other than SDHA. Further work is needed to determine if canine SDH immunohistochemistry on PCs correlates to genetic mutations that are similar to human PCs.

Healthcare-associated infections (HAIs) are one of the most common patient complications, affecting 7% of patients in developed countries each year. The rise of antimicrobial resistant (AMR) bacteria has been identified as one of the biggest global health challenges, resulting in an estimated 23,000 deaths in the US annually. Environmental reservoirs for AMR bacteria such as bed rails, light switches and doorknobs have been identified in the past and addressed with infection prevention guidelines. However, water and water-related devices are often overlooked as potential sources of HAI outbreaks. This systematic review examines the role of water and water-related devices in the transmission of AMR bacteria responsible for HAIs, discussing common waterborne devices, pathogens, and surveillance strategies. AMR strains of previously described waterborne pathogens including Pseudomonas aeruginosa, Mycobacterium spp., and Legionella spp. were commonly isolated. However, methicillin-resistant Staphylococcus aureus and carbapenem-resistant Enterobacteriaceae that are not typically associated with water were also isolated. Biofilms were identified as a hot spot for the dissemination of genes responsible for survival functions. A limitation identified was a lack of consistency between environmental screening scope, isolation methodology, and antimicrobial resistance characterization. Broad universal environmental surveillance guidelines must be developed and adopted to monitor AMR pathogens, allowing prediction of future threats before waterborne infection outbreaks occur.

Legionella pneumophila is an opportunistic waterborne pathogen of public health concern. It is the causative agent of Legionnaires’ disease (LD) and Pontiac fever and is ubiquitous in manufactured water systems, where protozoan hosts and complex microbial communities provide protection from disinfection procedures. This review collates the literature describing interactions between L. pneumophila and protozoan hosts in hospital and municipal potable water distribution systems. The effectiveness of currently available water disinfection protocols to control L. pneumophila and its protozoan hosts is explored. The studies identified in this systematic literature review demonstrated the failure of common disinfection procedures to achieve long term elimination of L. pneumophila and protozoan hosts from potable water. It has been demonstrated that protozoan hosts facilitate the intracellular replication and packaging of viable L. pneumophila in infectious vesicles; whereas, cyst-forming protozoans provide protection from prolonged environmental stress. Disinfection procedures and protozoan hosts also facilitate biogenesis of viable but non-culturable (VBNC) L. pneumophila which have been shown to be highly resistant to many water disinfection protocols. In conclusion, a better understanding of L. pneumophila-protozoan interactions and the structure of complex microbial biofilms is required for the improved management of L. pneumophila and the prevention of LD.

Like many of us who had the great fortune to work with Bill Paul, my science life was immeasurably altered by my interactions with him. Although intimidating at first because of his stature in the immunology world, it was soon clear that he not only truly cared about the specific research we were doing together, but he wished to convey to his trainees an approach to science that was open, always questioning, and infinitely fun. His enthusiasm was infectious and after my training with him, despite stresses due to funding and publishing hurdles, I never regretted the path I took. My research took a sharp turn from the studies of adaptive immunity I had planned on pursuing after my fellowship with Bill to a life long quest to understand the wonders of the mast cell, a relatively rare innate immune cell. This came about because Bill’s curiosity and expectation of the unexpected allowed him to view, in retrospect, a rather mundane observation we made together involving a non-physiological transformed mast cell line as something that might be really interesting. I have never forgotten that lesson: Look at the data with an eye on the big picture. Sometimes the unexpected is more interesting than predicted results. His example in this regard was incredibly important when as an independent investigator a mistake in mouse sex determination led to unexpected and very confusing data. Yet, these data ultimately revealed a role for mast cells in male-specific protection in experimental autoimmune encephalomyelitis, the mouse model of multiple sclerosis. Bill’s influence in immunology is far-reaching and will continue to be felt as those of us who train our own students and post-doctoral fellows pass on his wisdom and approach to scientific research.

Multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis, are autoimmune CNS inflammatory diseases. As a result of a breakdown in the relatively impermeable blood–brain barrier (BBB) in affected individuals, myelin-specific CD4+ and CD8+ T cells gain entry into the immune privileged CNS and initiate myelin, oligodendrocyte, and nerve axon destruction. However, despite the absolute requirement for T cells, there is increasing evidence that innate immune cells also play critical amplifying roles in disease pathogenesis. By modulating the character and magnitude of the myelin-reactive T cell response and regulating BBB integrity, innate cells affect both disease initiation and progression. Two classes of innate cells, mast cells and innate lymphoid cells (ILCs), have been best studied in models of allergic and gastrointestinal inflammatory diseases. Yet, there is emerging evidence that these cell types also exert a profound influence in CNS inflammatory disease. Both cell types are residents within the meninges and can be activated early in disease to express a wide variety of disease-modifying cytokines and chemokines. In this review, we discuss how mast cells and ILCs can have either disease-promoting or -protecting effects on MS and other CNS inflammatory diseases and how sex hormones may influence this outcome. These observations suggest that targeting these cells and their unique mediators can be exploited therapeutically.

Adoption is sometimes considered paradoxical from an evolutionary perspective because the costs spent supporting an adopted child would be better spent on rearing one's own. Kin selection theory is commonly used to solve this paradox, because the adoption of closely related kin contributes to the inclusive fitness of the adoptive parent. In this paper, we perform a novel test of kin selection theory in the context of adoption by asking whether adopted daughters-in-law, who contribute directly (i.e. genealogically) to the perpetuation of their adoptive families' lineages, experience lower mortality than daughters adopted for other purposes in historical Taiwan. We show that both classes of adopted daughter suffer lower mortality than biological daughters, but that the protective effect of adoption is stronger among daughters who were not adopted with the intention of perpetuating the family lineage. We speculate as to the possible benefits of such a pattern and emphasize the need to move beyond typological definitions of adoption to understand the specific costs and benefits involved in different forms of caring for others' children.

In recent years, effective treatment of infections caused by Acinetobacter baumannii has become challenging due to the ability of the bacterium to acquire or up-regulate antimicrobial resistance determinants. Two component signal transduction systems are known to regulate expression of virulence factors including multidrug efflux pumps. Here, we investigated the role of the AdeRS two component signal transduction system in regulating the AdeAB efflux system, determined whether AdeA and/or AdeB can individually confer antimicrobial resistance, and explored the interplay between pentamidine resistance and growth conditions in A. baumannii ATCC 17978. Results identified that deletion of adeRS affected resistance towards chlorhexidine and 4',6-diamidino-2-phenylindole dihydrochloride, two previously defined AdeABC substrates, and also identified an 8-fold decrease in resistance to pentamidine. Examination of ΔadeA, ΔadeB and ΔadeAB cells augmented results seen for ΔadeRS and identified a set of dicationic AdeAB substrates. RNA-sequencing of ΔadeRS revealed transcription of 290 genes were ≥2-fold altered compared to the wildtype. Pentamidine shock significantly increased adeA expression in the wildtype, but decreased it in ΔadeRS, implying that AdeRS activates adeAB transcription in ATCC 17978. Investigation under multiple growth conditions, including the use of Biolog phenotypic microarrays, revealed resistance to pentamidine in ATCC 17978 and mutants could be altered by bioavailability of iron or utilization of different carbon sources. In conclusion, the results of this study provide evidence that AdeAB in ATCC 17978 can confer intrinsic resistance to a subset of dicationic compounds and in particular, resistance to pentamidine can be significantly altered depending on the growth conditions.

BACKGROUND:It is a wide-spread assumption about footbinding that footbound girls and women were more of an economic burden on their families than those never bound. It is often presumed that government policies and missionary campaigns ended footbinding. METHODS/ OBJECTIVES:We use regression and log-likelihood tests, with bootstrapping for confirmation, to analyze which of a series of ethnographically and historically hypothesized variables significantly correlate with footbinding. We also consider an indirect measure of government prohibitions. We analyze two large datasets based on oral surveys with elderly women of the last footbound generations from 12 inland Chinese provinces. CONCLUSIONS:Handicraft production, particularly commercial handicraft production, correlates with whether Chinese girls were subjected to footbinding before 1950. Girlhood knowledge of government prohibitions against footbinding, an indirect measure of awareness by the adults who decided whether to bind a girl's feet, did not correlate with whether women were ever footbound. Spinning cotton thread for commercial purposes (sale, wage, direct exchange) correlated with greater daily production, with great county-level variation in quantity produced. Moreover, Chinese commercial spinners labored more years before marriage than domestic spinners. IMPLICATIONS:Chinese daughters-whether footbound or not-made important economic contributions to rural households, thus suggesting a need to revise our understanding of China's gender and economic history. Further implications of our results are that research is warranted on the assumed efficacy of government prohibitions-in both rural and urban areas-and on the presumption that footbinding among elite Chinese women was unrelated to economic concerns, including handicraft production. The demonstrated economic correlates of footbinding in inland, rural China also suggest a need to reevaluate whether contemporary customs controlling and cloistering girls and women, such as female genital cutting in Africa and the threat of honor killings of girls and women in South Asia, might have economic correlates.