Improved Antibacterial Activity of 1,3,4-Oxadiazole-Based Compounds That Restrict Staphylococcus aureus Growth Independent of LtaS Function.

The lipoteichoic acid (LTA) biosynthesis pathway has emerged as a promising antimicrobial therapeutic target. Previous studies identified the 1,3,4 oxadiazole compound 1771 as an LTA inhibitor with activity against Gram-positive pathogens. We have succeeded in making six 1771 derivatives and, through subsequent hit validation, identified the incorporation of a pentafluorosulfanyl substituent as central in enhancing activity. Our newly described derivative, compound 13, showed a 16- to 32-fold increase in activity compared to 1771 when tested against a cohort of multidrug-resistant Staphylococcus aureus strains while simultaneously exhibiting an improved toxicity profile against mammalian cells. Molecular techniques were employed in which the assumed target, lipoteichoic acid synthase (LtaS), was both deleted and overexpressed. Neither deletion nor overexpression of LtaS altered 1771 or compound 13 susceptibility; however, overexpression of LtaS increased the MIC of Congo red, a previously identified LtaS inhibitor. These data were further supported by comparing the docking poses of 1771 and derivatives in the LtaS active site, which indicated the possibility of an additional target(s). Finally, we show that both 1771 and compound 13 have activity that is independent of LtaS, extending to cover Gram-negative species if the outer membrane is first permeabilized, challenging the classification that these compounds are strict LtaS inhibitors.

Triclosan-resistant small-colony variants of Staphylococcus aureus produce less capsule, less phenol-soluble modulins, and are attenuated in a Galleria mellonella model of infection.

In recent work we identified genes that confer the slow-growing and antibiotic-resistant small-colony variant (SCV) form of Staphylococcus aureus, as associated with the amount of capsule the bacteria produce. In this study we isolated a triclosan-resistant SCV (tr-SCV) and demonstrated that it produces significantly less capsule, an effect that appears to be mediated at the transcriptional stage. As with other SCVs, we found that the tr-SCV produces less toxins, and when compared to both a capsule and an Agr mutant we found the tr-SCV to be significantly attenuated in an insect model of infection.

Wall Teichoic Acids Facilitate the Release of Toxins from the Surface of Staphylococcus aureus.

A major feature of the pathogenicity of Staphylococcus aureus is its ability to secrete cytolytic toxins. This process involves the translocation of the toxins from the cytoplasm through the bacterial membrane and the cell wall to the external environment. The process of their movement through the membrane is relatively well defined, involving both general and toxin-specific secretory systems. Movement of the toxins through the cell wall was considered to involve the passive diffusion of the proteins through the porous cell wall structures; however, recent work suggests that this is more complex, and here we demonstrate a role for the wall teichoic acids (WTA) in this process. Utilizing a genome-wide association approach, we identified a polymorphism in the locus encoding the WTA biosynthetic machinery as associated with the cytolytic activity of the bacteria. We verified this association using an isogenic mutant set and found that WTA are required for the release of several cytolytic toxins from the bacterial cells. We show that this effect is mediated by a change in the electrostatic charge across the cell envelope that results from the loss of WTA. As a major target for the development of novel therapeutics, it is important that we fully understand the entire process of cytolytic toxin production and release. These findings open up a new aspect to the process of toxin release by a major human pathogen while also demonstrating that clinical isolates can utilize WTA production to vary their cytotoxicity, thereby altering their pathogenic capabilities. IMPORTANCE The production and release of cytolytic toxins is a critical aspect for the pathogenicity of many bacterial pathogens. In this study, we demonstrate a role for wall teichoic acids, molecules that are anchored to the peptidoglycan of the bacterial cell wall, in the release of toxins from S. aureus cells into the extracellular environment. Our findings suggest that this effect is mediated by a gradient of electrostatic charge which the presence of the negatively charged WTA molecules create across the cell envelope. This work brings an entirely new aspect to our understanding of the cytotoxicity of S. aureus and demonstrates a further means by which this major human pathogen can adapt its pathogenic capabilities.

CidA and LrgA: a "Hole" Lot More than Programmed Cell Death.

What do programmed cell death (PCD) and carbohydrate metabolism by-product transport have in common? Intriguingly, both processes involve the cidABC and lrgAB operons in the major human pathogen Staphylococcus aureus. Previously, CidA and LrgA have been studied in the context of programmed cell death, but a second function in overflow metabolism is increasingly evident. New work from J. L. Endres, S. S. Chaudhari, X. Zhang, J. Prahlad, et al. (mBio 13:e02827-21, 2022, https://doi.org/10.1128/mBio.02827-21) combining a lysis cassette, mutagenesis, and classic microbiology demonstrates that CidA and LrgA function as holins to support endolysin-induced lysis. But that's not all-the lrgAB operon also facilitates pyruvate uptake during microaerobic and anaerobic growth. This commentary highlights the main findings from this work and places them in context of the literature to date. Finally, as these proteins are highly conserved and carry out disparate functions of great importance, it is tempting to speculate future work will elucidate the link between S. aureus lysis and pyruvate metabolism.

Targeted control of pneumolysin production by a mobile genetic element in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major human pathogen that can cause severe invasive diseases such as pneumonia, septicaemia and meningitis. Young children are at a particularly high risk, with an estimated 3-4 million cases of severe disease and between 300 000 and 500 000 deaths attributable to pneumococcal disease each year. The haemolytic toxin pneumolysin (Ply) is a primary virulence factor for this bacterium, yet despite its key role in pathogenesis, immune evasion and transmission, the regulation of Ply production is not well defined. Using a genome-wide association approach, we identified a large number of potential affectors of Ply activity, including a gene acquired horizontally on the antibiotic resistance-conferring Integrative and Conjugative Element (ICE) ICESp23FST81. This gene encodes a novel modular protein, ZomB, which has an N-terminal UvrD-like helicase domain followed by two Cas4-like domains with potent ATP-dependent nuclease activity. We found the regulatory effect of ZomB to be specific for the ply operon, potentially mediated by its high affinity for the BOX repeats encoded therein. Using a murine model of pneumococcal colonization, we further demonstrate that a ZomB mutant strain colonizes both the upper respiratory tract and lungs at higher levels when compared to the wild-type strain. While the antibiotic resistance-conferring aspects of ICESp23FST81 are often credited with contributing to the success of the S. pneumoniae lineages that acquire it, its ability to control the expression of a major virulence factor implicated in bacterial transmission is also likely to have played an important role.

The MpsB protein contributes to both the toxicity and immune evasion capacity of Staphylococcus aureus.

Understanding the role specific bacterial factors play in the development of severe disease in humans is critical if new approaches to tackle such infections are to be developed. In this study we focus on genes we have found to be associated with patient outcome following bacteraemia caused by the major human pathogen Staphylococcus aureus. By examining the contribution these genes make to the ability of the bacteria to survive exposure to the antibacterial factors found in serum, we identify three novel serum resistance-associated genes, mdeA, mpsB and yycH. Detailed analysis of an MpsB mutant supports its previous association with the slow growing small colony variant (SCV) phenotype of S. aureus, and we demonstrate that the effect this mutation has on membrane potential prevents the activation of the Agr quorum sensing system, and as a consequence the mutant bacteria do not produce cytolytic toxins. Given the importance of both toxin production and immune evasion for the ability of S. aureus to cause disease, we believe that these findings explain the role of the mpsB gene as a mortality-associated locus during human disease.

A Small Membrane Stabilizing Protein Critical to the Pathogenicity of Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen, and the emergence of antibiotic-resistant strains is making all types of S. aureus infections more challenging to treat. With a pressing need to develop alternative control strategies to use alongside or in place of conventional antibiotics, one approach is the targeting of established virulence factors. However, attempts at this have had little success to date, suggesting that we need to better understand how this pathogen causes disease if effective targets are to be identified. To address this, using a functional genomics approach, we have identified a small membrane-bound protein that we have called MspA. Inactivation of this protein results in the loss of the ability of S. aureus to secrete cytolytic toxins, protect itself from several aspects of the human innate immune system, and control its iron homeostasis. These changes appear to be mediated through a change in the stability of the bacterial membrane as a consequence of iron toxicity. These pleiotropic effects on the ability of the pathogen to interact with its host result in significant impairment in the ability of S. aureus to cause infection in both a subcutaneous and sepsis model of infection. Given the scale of the effect the inactivation of MspA causes, it represents a unique and promising target for the development of a novel therapeutic approach.

An evaluation of nurses' experiences of mentoring pre-registration students.

Nurse education in the UK has undergone a radical change over the past 30 years. The integration of nursing students within practice has evolved from an apprenticeship style to bespoke mentoring support. To act as mentors, registered nurses must have met stage 2 outcomes of the Nursing and Midwifery Council (NMC) Standards to Support Learning and Assessment in Practice, which clearly stipulate that mentors should have a reduced clinical commitment when supporting students, with one hour per week being protected, in addition to the 40% of time through direct or indirect supervision with their mentor/sign off mentor when facilitating a student on their final 12-week experience. However, this does not seem to be the case in reality. A qualitative study comprising six semi-structured interviews was undertaken across one health and social care trust. Data were analysed using Braun and Clarke's thematic analysis. A number of themes and subthemes were identified: engagement (barriers versus strategies), support (inclusivity versus exclusivity), and lack of recognition (strategic versus organisational). Due to the expected changes of supervising and assessing nursing students in practice, it is imperative that an innovative, collaborative and engaged approach is facilitated from all key stakeholders to ensure the sustainability of supporting and assessing students by registered nurses and the safeguarding of the public within clinical practice.

Neutrophil activation by Candida glabrata but not Candida albicans promotes fungal uptake by monocytes.

Candida albicans and Candida glabrata account for the majority of candidiasis cases worldwide. Although both species are in the same genus, they differ in key virulence attributes. Within this work, live cell imaging was used to examine the dynamics of neutrophil activation after confrontation with either C. albicans or C. glabrata. Analyses revealed higher phagocytosis rates of C. albicans than C. glabrata that resulted in stronger PMN (polymorphonuclear cells) activation by C. albicans. Furthermore, we observed differences in the secretion of chemokines, indicating chemotactic differences in PMN signalling towards recruitment of further immune cells upon confrontation with Candida spp. Supernatants from co-incubations of neutrophils with C. glabrata primarily attracted monocytes and increased the phagocytosis of C. glabrata by monocytes. In contrast, PMN activation by C. albicans resulted in recruitment of more neutrophils. Two complex infection models confirmed distinct targeting of immune cell populations by the two Candida spp.: In a human whole blood infection model, C. glabrata was more effectively taken up by monocytes than C. albicans and histopathological analyses of murine model infections confirmed primarily monocytic infiltrates in C. glabrata kidney infection in contrast to PMN-dominated infiltrates in C. albicans infection. Taken together, our data demonstrate that the human opportunistic fungi C. albicans and C. glabrata are differentially recognized by neutrophils and one outcome of this differential recognition is the preferential uptake of C. glabrata by monocytes.

Host response to Candida albicans bloodstream infection and sepsis.

Candida albicans is a major cause of bloodstream infection which may present as sepsis and septic shock - major causes of morbidity and mortality world-wide. After invasion of the pathogen, innate mechanisms govern the early response. Here, we outline the models used to study these mechanisms and summarize our current understanding of innate immune responses during Candida bloodstream infection. This includes protective immunity as well as harmful responses resulting in Candida induced sepsis. Neutrophilic granulocytes are considered principal effector cells conferring protection and recognize C. albicans mainly via complement receptor 3. They possess a range of effector mechanisms, contributing to elimination of the pathogen. Neutrophil activation is closely linked to complement and modulated by activated mononuclear cells. A thorough understanding of these mechanisms will help in creating an individualized approach to patients suffering from systemic candidiasis and aid in optimizing clinical management.

Defining the transcriptomic landscape of Candida glabrata by RNA-Seq.

Candida glabrata is the second most common pathogenic Candida species and has emerged as a leading cause of nosocomial fungal infections. Its reduced susceptibility to antifungal drugs and its close relationship to Saccharomyces cerevisiae make it an interesting research focus. Although its genome sequence was published in 2004, little is known about its transcriptional dynamics. Here, we provide a detailed RNA-Seq-based analysis of the transcriptomic landscape of C. glabrata in nutrient-rich media, as well as under nitrosative stress and during pH shift. Using RNA-Seq data together with state-of-the-art gene prediction tools, we refined the annotation of the C. glabrata genome and predicted 49 novel protein-coding genes. Of these novel genes, 14 have homologs in S. cerevisiae and six are shared with other Candida species. We experimentally validated four novel protein-coding genes of which two are differentially regulated during pH shift and interaction with human neutrophils, indicating a potential role in host-pathogen interaction. Furthermore, we identified 58 novel non-protein-coding genes, 38 new introns and condition-specific alternative splicing. Finally, our data suggest different patterns of adaptation to pH shift and nitrosative stress in C. glabrata, Candida albicans and S. cerevisiae and thus further underline a distinct evolution of virulence in yeast.

'With a bit of tweaking we could be great'. An exploratory study of the perceptions of students on working with older people in a preregistration BSc (Hons) Nursing course.

BACKGROUND: With ageing demographics, it is important that nurse education curriculum can prepare students to work with older people. AIMS AND OBJECTIVES: To explore students' perceptions of working with older people and the extent to which their preregistration curriculum is preparing them for this role. DESIGN: A qualitative research design, incorporating focus groups in data collection. METHODS: Four focus groups were held in January 2011, involving a total of 32 students undertaking a preregistration BSc (Hons) nursing degree course. RESULTS: An overt focus in the preregistration curriculum on acute and critical care and perceived deficits in care of older people content left some students feeling underprepared to work with older people and to challenge ritualistic practice. Clinical placement experience and mentor support appeared to be influencing students' decisions about whether they would consider working with older people in the future. CONCLUSION: Education providers should ensure that students are adequately prepared to work with older people and that students are supported when they observe poor practice. A finding that observation of ritualistic practice could prompt some students to consider working with older people, warrants further research. Implications for practice. Nurse educators should evaluate the content and delivery of their preregistration courses to ensure that the prerequisite knowledge, skills and attitudes required to work with older people are accorded appropriate value and attention.

Perceptions of older people on disaster response and preparedness.

Most disasters occur in developing countries but in the last decade due to the increasing threat of floods, air disasters and terrorist threat, disaster response and preparedness is a growing global concern. Due to an ageing population across the world, older people now constitute a significant proportion of those at risk from disasters. This paper reports on a qualitative study carried out in Sri Lanka and in the United States where a group of older people were asked about aspects of disaster response and preparedness. The group from Sri Lanka (n=9) who had direct experience of the 2004 Indian Ocean Tsunami were asked how they perceived international aid relief and a group of white Caucasians from East Coast USA (n=8) were asked about disaster preparedness. Findings indicate that both groups had similar issues albeit that they were looking at different phases of the disaster cycle and from different cultural perspectives. Both groups identified issues related to, protecting the rights of the older person and preventing loss of independence in responding and preparing for a disaster, mistrust of government and access to resources and all expressed strong feelings of self-responsibility.

Addressing the imbalance: empowering older people in disaster response and preparedness.

This paper explores the role of the nurse in empowering older people at all stages in the disaster cycle. Evidence points to the need to increase the level of consultation and inclusivity of older people on this topic. Caution is called for in relation to classifying older people as a vulnerable group in disasters without first recognising that older people are probably the richest resource within any culture when it comes to emergency planning and\or coping with disasters. Practical advice on how nurses can best assist older people is presented.