IntroductionFrancisella tularensis is the causative agent of tularemia, a severe infection that can be fatal if untreated. There is a need for improved therapeutic options to enhance the efficacy of existing antibiotic regimens for severe tularemia and to reduce the risk of disease relapse. Novel formulations of ciprofloxacin have demonstrated protection against inhalational tularemia in murine and marmoset models; however, a detailed immunological characterisation of treatment response is required. MethodsBlood samples were collected from marmosets infected with a lethal dose of inhalational tularemia and treated with either oral ciprofloxacin or inhaled ciprofloxacin (Apulmiq®). Immunological responses were assessed longitudinally and compared with immunological changes previously observed in recovering human cases of tularemia.ResultsInfected marmosets exhibited increased neutrophil levels, which, together with reduced HLA-DR expression, indicated severe infection. Rising circulating interferon-gamma (IFN γ) levels were followed by classical activation of macrophages as an initial attempt to control bacterial dissemination. Progressive disease was characterised by a marked depletion of neutrophils, excessive IFN γ production, and elevated levels of overactivated macrophages. In animals that responded successfully to treatment, these immunological parameters returned to baseline levels. Recovery was subsequently associated with increased T cell populations, particularly gamma delta T (γδ T) cells, and limited antibody production, suggesting the development of long-term immunity comparable to that observed in humans. DiscussionThe observed immunological changes in marmosets closely mirror those reported in human tularemia, supporting the relevance of this model for studying disease progression and recovery. The capacity to monitor these immune parameters demonstrates the utility of the marmoset model in assessing the efficacy of new and novel antibiotic treatments for tularemia.

Background/Objectives: Q fever, caused by Coxiella burnetii, is typically treated with doxycycline, but its efficacy is limited in chronic cases and may be poorly tolerated. Systemic ciprofloxacin shows limited activity for acute Q fever. However, inhaled liposomal formulations may provide therapeutic benefit. Methods: Two inhaled ciprofloxacin formulations (Lipoquin® and Apulmiq®) were evaluated in an A/J mouse model of Q fever and compared with intraperitoneal ciprofloxacin and oral doxycycline. Initially, pharmacokinetic studies were performed to determine an appropriate dosing regimen for the inhaled ciprofloxacin formulations. A separate cohort of mice were then infected with C. burnetii and treated once daily via nebulisation with Lipoquin or Apulmiq, initiated at 24, 48, or 72 h post-challenge. Clinical signs, weight change, splenomegaly, bacterial burden, and lung histopathology were evaluated. Results: Pharmacokinetic analysis confirmed sustained lung concentrations of inhaled ciprofloxacin, supporting once-daily dosing. Inhaled Lipoquin and Apulmiq significantly reduced clinical signs, weight loss, splenomegaly, and pulmonary bacterial burden compared to untreated controls and doxycycline-treated mice. Histopathology revealed decreased lung inflammation and lesion severity following inhalational dosing. Systemic ciprofloxacin slightly reduced splenic bacterial burden but was less effective in controlling pulmonary infection. Conclusions: Inhaled liposomal ciprofloxacin demonstrated superior protection and reduced respiratory manifestations of Q fever compared to doxycycline and systemic ciprofloxacin. These findings suggest inhaled formulations may represent a viable alternative for the treatment of Q fever pneumonia. Further studies are needed to evaluate clinical applicability and long-term outcomes.

The development of humane endpoints is critical for refining scientific studies involving animals. Body weight and clinical signs of disease data collected in four recent studies assessing medical countermeasures for utility against the disease melioidosis in mice were further analyzed. Here we used this information to ascertain whether a suitable alternative humane endpoint could be identified. A total of 66 possible alternative humane endpoints were explored, which varied the threshold values of the ‘percentage body weight loss post-challenge’ and ‘the clinical signs over time’ following cessation of treatment. The findings indicated a suitable alternative endpoint of a percentage weight loss threshold of 25

A range of technologies are being developed to modulate the human gut microbiome, aimed at resolving gut dysbiosis and restoring normal host function. Although limited, a subset of studies have begun to evaluate these technologies within healthy human populations. This could provide approaches to mitigate the impact of occupational stressors on military personnel to ensure their operational effectiveness and resilience is maintained, and could also extend to enhancing the physical or cognitive performance of an individual beyond their baseline potential. Research using in vivo models and healthy human populations suggest that cognition, mineral absorption, muscle resilience, endurance and structural integrity, and injury recovery are modified by the gut microbiome. However, the regulations that govern the use of these technologies are largely focused on their use in treating disease and promoting health, which could hinder such applications. Therefore, whilst the use of gut microbiome modulation could present opportunities to enhance resilience and performance in military personnel, more research in healthy human cohorts is needed, alongside the development of effective regulatory frameworks supporting wider applications.

Background: Inhalational antibiotics have been used effectively to treat chronic diseases such as Pseudomonas aeruginosa infections associated with cystic fibrosis. This approach may enhance treatment options for difficult-to-treat, acute pneumonic diseases. Liposomal encapsulated ciprofloxacin (Lipoquin and/or Apulmiq) has provided protection in murine models of plague, anthrax, Q fever and tularemia. Development of the ability to deliver these drugs to nonhuman primates (NHPs) would enable further extrapolation of the data observed in small animal models of infection to humans. Methods: In this study, the methodology was established to deliver Apulmiq to common marmosets (Callithrix jacchus). Marmosets were anaesthetised with a novel, reversible anaesthetic comprising fentanyl, medetomidine and midazolam (FMM). They were placed into plethysmography tubes with their heads in an exposure chamber. The LC Sprint jet nebuliser or Pari eFlow Rapid nebuliser were used to aerosolise Apulmiq into the exposure chamber. Animals were euthanised after dosing and the concentration of ciprofloxacin was assessed in the plasma and lungs of the animals. Results: Non-compartmental pharmacokinetic analysis determined that a 30 min exposure of drug was required to reach a human-equivalent target dose of 0.8 mg/kg body weight in the lungs. Conclusions: This approach can now be used to assess the efficacy of inhalational liposomal ciprofloxacin in NHP infection models.

The development of medical countermeasures against pathogens of biodefense concern remains critical to protecting military and public health. This review compares data detailing antibacterial activity and efficacy for a selection of antibiotics evaluated against potential bacterial biothreat pathogens. The human safety and tolerability of these formulations were also considered. This review includes finafloxacin, levofloxacin, delafloxacin, omadacycline, gepotidacin, tebipenem and sulopenem. The selection criteria of these antibiotics were 1) the availability of an oral formulation, 2) the regulatory status (licensed by a regulatory authority or in an advanced stage of development) and 3) the availability of publicly available information on the biodefence pathogens of concern. We hope to highlight approved or advanced clinical candidates that have significant and unique potential in the biodefense space which may be deployed to protect both the public and warfighter against these bacterial infections.

Francisella tularensis is a gram-negative, intracellular bacterium that causes the disease tularemia. Tularemia is prevalent in North America, Europe, and Asia and is typically treated with injected and orally administered antibiotics, including streptomycin, gentamicin, doxycycline, and ciprofloxacin, administered for 10 to 21 days. New therapeutic options are required to reduce the potential of a relapse of disease. Inhaled liposomal-encapsulated ciprofloxacin has demonstrated protection in a murine model of tularemia. The efficacy was further assessed in a nonhuman primate model of tularemia. Mixed-sex common marmosets were challenged with F. tularensis by the inhalational route, and the efficacy of ciprofloxacin delivered by either the inhalational (Apulmiq liposomal formulation) or oral route was compared. Antibiotics were initiated either at 24 h post-challenge (post-exposure prophylaxis) or at the onset of fever (treatment) and continued for 7 days. All control (untreated) animals succumbed to infection by 8 days post-challenge. All animals that received antibiotics, by either route, survived the duration of the study, with bacterial clearance in all but one animal that received inhalational ciprofloxacin. Antibiotic treatment also reduced the physiological and immunological responses observed when compared to animals that received no antibiotics. Histological changes in the lungs were less frequent, although mild, resolving lesions were present in animals treated with ciprofloxacin delivered at the onset of fever by either route.

Melioidosis is a disease that is difficult to treat due to the causative organism, Burkholderia pseudomallei being inherently antibiotic resistant and it having the ability to invade, survive, and replicate in an intracellular environment. Combination therapy approaches are routinely being evaluated in animal models with the aim of improving the level of protection and clearance of colonizing bacteria detected. In this study, a subunit vaccine layered with the antibiotic finafloxacin was evaluated in vivo against an inhalational infection with B. pseudomallei in Balb/c mice. Groups of mice vaccinated, infected, and euthanized at antibiotic initiation had a reduced bacterial load compared to those that had not been immunized. In addition, the subunit vaccine provided a synergistic effect when it was delivered with a CpG ODN and finafloxacin was initiated at 48 h post-challenge. Vaccination was also shown to improve the outcome, in a composite measure of survival and clearance. In summary, layering a subunit vaccine with the antibiotic finafloxacin is a promising therapeutic alternative for use in the treatment of B. pseudomallei infections.

The interactions between a virus and its host vary in space and time and are affected by the presence of molecules that alter the physiology of either the host or the virus. Determining the molecular mechanisms at the basis of these interactions is paramount for predicting the fate of bacterial and phage populations and for designing rational phage-antibiotic therapies. We study the interactions between stationary phase Burkholderia thailandensis and the phage ΦBp-AMP1. Although heterogeneous genetic resistance to phage rapidly emerges in B. thailandensis, the presence of phage enhances the efficacy of three major antibiotic classes, the quinolones, the beta-lactams and the tetracyclines, but antagonizes tetrahydrofolate synthesis inhibitors. We discovered that enhanced antibiotic efficacy is facilitated by reduced antibiotic efflux in the presence of phage. This new phage-antibiotic therapy allows for eradication of stationary phase bacteria, whilst requiring reduced antibiotic concentrations, which is crucial for treating infections in sites where it is difficult to achieve high antibiotic concentrations.

Antimicrobial resistance is a global issue, and the investigation of alternative therapies that are not traditional antibiotics are warranted. Novel bacterial type II topoisomerase inhibitors (NBTIs) have recently emerged as a novel class of antibiotics with reduced potential for cross-resistance to fluoroquinolones due to their novel mechanism of action. This study investigated the in vitro activity of a series of cyclohexyl–oxazolidinone bacterial topoisomerase inhibitors against type strains of Francisella tularensis and Burkholderia pseudomallei. Broth microdilution, time-kill, and cell infection assays were performed to determine activity against these biothreat pathogens. Two candidates were identified that demonstrated in vitro activity in multiple assays that in some instances was equivalent to ciprofloxacin and doxycycline. These data warrant the further evaluation of these novel NBTIs and future iterations in vitro and in vivo.

Burkholderia pseudomallei is the causative agent of the tropical disease, melioidosis. It is intrinsically resistant to many antimicrobials and treatment requires an onerous regimen of intravenous and orally administered drugs. Relapse of disease and high rates of mortality following treatment are common, demonstrating the need for new anti-Burkholderia agents. The cationic bola-amphiphile, 12,12 '-(dodecane-1,12-diyl) bis (9-amino-1,2,3,4-tetrahydroacridinium), referred to as 12-bis-THA, is a molecule with the potential to treat Burkholderia infections. 12-bis-THA spontaneously forms cationic nanoparticles that bind anionic phospholipids in the prokaryotic membrane and are readily internalized. In this study, we examine the antimicrobial activity of 12-bis-THA against strains of Burkholderia thailandensis. As B. pseudomallei produces a polysaccharide capsule we first examined if this extra barrier influenced the activity of 12-bis-THA which is known to act on the bacterial envelope. Therefore two strains of B. thailandensis were selected for further testing, strain E264 which does not produce a capsule and strain E555 which does produce a capsule that is chemically similar to that found in B. pseudomallei. In this study no difference in the minimum inhibitory concentration (MIC) was observed when capsulated (E555) and unencapsulated (E264) strains of B. thailandensis were compared, however time-kill analysis showed that the unencapsulated strain was more susceptible to 12-bis-THA. The presence of the capsule did not affect the membrane permeation of 12-bis-THA at MIC concentrations. Proteomic and metabolomic analyses showed that 12-bis-THA causes a shift in central metabolism away from glycolysis and glyoxylate cycle, and suppressed the production of the F-1 domain of ATP synthase. In summary, we provide insight into the molecular mechanisms underpinning the activity of 12-bis-THA against B. thailandensis and discuss its potential for further development.

Burkholderia pseudomallei , the causative agent of melioidosis, has two phases of infection. The acute phase occurs shortly after infection and is associated with bacterial sepsis, potentially leading to death, whilst the chronic phase occurs when infection persists for longer periods or is asymptomatic for months or years. BALB/c mice are more susceptible to melioidosis compared to C57BL/6 mice and are routinely models for the acute phase of infection. However, in some instances when medical countermeasures are being evaluated, mice continue to succumb to disease throughout the course of the experimental infection. Whilst B. pseudomallei is not known to be transmitted from mouse-to-mouse, we hypothesized that mice that have recovered from infection after medical countermeasure intervention may become reinfected from chronically infected mice. We tested this hypothesis by cohousing naïve mice with mice exposed to B. pseudomallei by the inhalational or intraperitoneal routes in either static or ventilated caging. Mice that were exposed to aerosolized B. pseudomallei transmitted the bacterium to approximately 4% of their naïve cagemates, whereas mice that were infected by the intraperitoneal route transmitted to approximately 8% of their naïve cagemates. Whilst the exact route of transmission remains to be determined, the results of this study showed that low levels of mouse-to-mouse transmission of B. pseudomallei are possible. We conclude that although the chance of reinfection is low amongst mice housed in the same cage, this possible scenario should be considered when interpreting data from the BALB/c mouse model of melioidosis in lengthy studies.

Burkholderia pseudomallei, the causative agent of the disease melioidosis, has been isolated from the environment in 45 countries. The treatment of melioidosis is complex, requiring lengthy antibiotic regimens, which can result in the relapse of the disease following treatment cessation. It is important that novel therapies to treat infections with B. pseudomallei be assessed in appropriate animal models, and discussions regarding the different protocols used between laboratories are critical. A 'deep dive' was held in October 2020 focusing on the use of the BALB/c mouse model and the inhalational route of infection to evaluate new antibiotic therapies.

Previous work indicated that the incidence of travellers' diarrhoea (TD) is higher in soldiers of British origin, when compared to soldiers of Nepalese descent (Gurkhas). We hypothesise that the composition of the gut microbiota may be a contributing factor in the risk of developing TD in soldiers of British origin. This study aimed to characterise the gut microbial composition of Gurkha and non-Gurkha soldiers of the British Army. Recruitment of 38 soldiers (n = 22 Gurkhas, n = 16 non-Gurkhas) and subsequent stool collection, enabled shotgun metagenomic sequencing-based analysis of the gut microbiota. The microbiota of Gurkhas had significantly (P < 0.05) lower diversity, for both Shannon and Simpson diversity indices, using species level markers than the gut microbiota of non-Gurkha soldiers. Non-metric Multidimensional Scaling (NMDS) of the Bray-Curtis distance matrix revealed a significant difference in the composition of the gut microbiota between Gurkhas and non-Gurkha soldiers, at both the species level (P = 0.0178) and the genus level (P = 0.0483). We found three genera and eight species that were significantly enriched in the non-Gurkha group and one genus (Haemophilus) and one species (Haemophilus parainfluenzae) which were enriched in the Gurkha group. The difference in the microbiota composition between Gurkha soldiers and soldiers of British origin may contribute to higher colonization resistance against diarrhoeal pathogens in the former group. Our findings may enable further studies into interventions that modulate the gut microbiota of soldiers to prevent TD during deployment.

The interactions between bacteria and bacteriophage have important roles in the global ecosystem; in turn changes in environmental parameters affect the interactions between bacteria and phage. However, there is a lack of knowledge on whether clonal bacterial populations harbour different phenotypes that respond to phage in distinct ways and whether the abundance of such phenotypes within bacterial populations is affected by variations in environmental parameters. Here we study the impact of variations in nutrient availability, bacterial growth rate and phage abundance on the interactions between the phage T4 and individual Escherichia coli cells confined in spatial refuges. Surprisingly, we found that fast growing bacteria survive together with all of their clonal kin cells, whereas slow growing bacteria survive in isolation. We also discovered that the number of bacteria that survive in isolation decreases at increasing phage doses possibly due to lysis inhibition in the presence of secondary adsorptions. We further show that these changes in the phenotypic composition of the E. coli population have important consequences on the bacterial and phage population dynamics and should therefore be considered when investigating bacteria-phage interactions in ecological, health or food production settings in structured environments.

This study determined the in vitro activity of finafloxacin against panels of bacterial strains, representative of those associated with infection in cystic fibrosis patients and predominately isolated from clinical cases of respiratory disease. Many of these isolates were resistant to various antimicrobials evaluated including the aminoglycosides, cephalosporins, carbapenems and fluoroquinolones. Broth microdilution assays were performed at neutral and acidic pH, to determine antimicrobial activity. Finafloxacin demonstrated superior activity at reduced pH for all of the bacterial species investigated, highlighting the requirement to determine the activity of antimicrobials in host-relevant conditions.

ObjectivesTo conduct public engagement work to establish the views of key stakeholders on the feasibility, acceptability, key purposes, and data structure of the first national linked longitudinal research database for fetal alcohol spectrum disorder (FASD) in the UK. MethodsWe conducted stakeholder mapping and identified contacts through collaborator networks and online searches. We consulted with stakeholders using a range of formats, including: online focus groups (one for adults with FASD [and their supporters] N=5; one for guardians of people with FASD N=7), 1:1/small-team video calls and email communication with clinicians, data providers, policy-makers, data-governance experts, third-sector representatives and researchers [N=~85]), and one hybrid workshop (N=16 [N=10 in-person, 6 online attendees; 15 clinical and one third-sector]). Consultations included discussions on data sharing, harmonisation, perceived benefits, and challenges of a national linked database for FASD. We analysed consultation transcripts and notes using thematic analysis. ResultsOur tailored, multi-method approach resulted in high levels of engagement with diverse stakeholders. Overall, stakeholders expressed strong support for a national linked database for FASD. For people living with FASD and third-sector representatives, the main perceived benefit was the potential for increased understanding, awareness, and support for FASD. Clinicians reported that a national database could provide new insights into FASD profiles, supporting more efficient diagnosis. Researchers highlighted potential for increased knowledge of FASD epidemiology and impacts. Policy-makers noted its clear alignment with contemporary FASD and data transformation policies. Common concerns were around privacy and data-sharing, particularly the tension between public good and disclosure risks if sample numbers were low. Clinicians expressed the importance of retaining space for clinical judgement and flexibility alongside the potential insights gained from quantitative analysis of data. ConclusionMulti-method and multidisciplinary stakeholder engagement demonstrated the feasibility and acceptability of establishing the first UK national linked database for FASD. Perceived benefits and concerns varied by stakeholder group, demonstrating that flexible, diverse, embedded stakeholder collaboration will be essential as we seek to establish this database.

Teaching staff report poorer mental health and wellbeing than the general working population. Intervention to address this issue is imperative, as poor wellbeing is associated with burnout, presenteeism, and adverse student mental health outcomes. The Wellbeing in Secondary Education (WISE) intervention is a secondary school-based programme aimed at improving the mental health and wellbeing of teachers and students. There are three components: awareness-raising for staff; a peer support service delivered by staff trained in Mental Health First Aid (MHFA); and Schools and Colleges Mental Health First Aid (MHFA) training for teachers. A cluster randomised controlled trial with integrated process and economic evaluation was conducted with 25 secondary schools in the UK (2016-2018). The intervention was largely ineffective in improving teacher mental health and wellbeing. This paper reports process evaluation data on acceptability to help understand this outcome. It adopts a complex systems perspective, exploring how acceptability is a dynamic and contextually contingent concept. Data sources were as follows: interviews with funders (n = 3); interviews with MHFA trainers (n = 6); focus groups with peer supporters (n = 8); interviews with headteachers (n = 12); and focus groups with teachers trained in Schools and Colleges MHFA (n = 7). Results indicated that WISE intervention components were largely acceptable. Initially, the school system was responsive, as it had reached a 'tipping point' and was prepared to address teacher mental health. However, as the intervention interacted with the complexities of the school context, acceptability became more ambiguous. The intervention was seen to be largely inadequate in addressing the structural determinants of teacher mental health and wellbeing (e.g. complex student and staff needs, workload, and system culture). Future teacher mental health interventions need to focus on coupling skills training and support with whole school elements that tackle the systemic drivers of the problem.

The efficacy of finafloxacin as a component of a layered defense treatment regimen was determined in vitro and in vivo against an infection with Burkholderia pseudomallei. Doxycycline was down-selected from a panel of antibiotics evaluated in vitro and used in combination with finafloxacin in a Balb/c mouse model of inhalational melioidosis. When treatment was initiated at 24 h post-infection with B. pseudomallei, there were no differences in the level of protection offered by finafloxacin or doxycycline (as monotherapies) when compared to the combination therapy. There was evidence for improved bacterial control in the groups treated with finafloxacin (as monotherapies or in combination with doxycycline) when compared to mice treated with doxycycline. Survival comparisons of finafloxacin and doxycycline (as monotherapies) or in combination initiated at 36 h post-infection indicated that finafloxacin was superior to doxycycline. Doxycycline was also unable to control the levels of bacteria within tissues to the extent that doxycycline and finafloxacin used in combination or finafloxacin (as a sole therapy) could. In summary, finafloxacin is a promising therapy for use in the event of exposure to B. pseudomallei.

Bacteria have developed unique mechanisms to adapt to environmental stresses and challenges of the immune system. Here, we report that Burkholderia pseudomallei, the causative agent of melioidosis, and its laboratory surrogate, Burkholderia thailandensis, utilize distinct mechanisms for surviving starvation at different incubation temperatures. At 21°C, Burkholderia are present as short rods which can rapidly reactivate and form colonies on solid media. At 4°C, Burkholderia convert into coccoid forms that cannot be cultured on solid agar but can be resuscitated in liquid media supplemented with supernatant obtained from logarithmic phase cultures of B. thailandensis, or catalase and Tween 80, thus displaying characteristics of differentially culturable bacteria (DCB). These DCB have low intensity fluorescence when stained with SYTO 9, have an intact cell membrane (propidium iodide negative), and contain 16S rRNA at levels comparable with growing cells. We also present evidence that lytic transglycosylases, a family of peptidoglycan-remodeling enzymes, are involved in the generation of coccoid forms and their resuscitation to actively growing cells. A B. pseudomallei ΔltgGCFD mutant with four ltg genes deleted did not produce coccoid forms at 4°C and could not be resuscitated in the liquid media evaluated. Our findings provide insights into the adaptation of Burkholderia to nutrient limitation and the generation of differentially culturable bacteria. IMPORTANCE Bacterial pathogens exhibit physiologically distinct forms that enable their survival in an infected host, the environment and following exposure to antimicrobial agents. B. pseudomallei causes the disease melioidosis, which has a high mortality rate and is difficult to treat with antibiotics. The bacterium is endemic to several countries and detected in high abundance in the environment. Here, we report that during starvation at low temperature, B. pseudomallei produces coccoid forms that cannot grow in standard media and which, therefore, can be challenging to detect using common tools. We provide evidence that the formation of these cocci is mediated by cell wall-specialized enzymes and lytic transglycosylases, and that resuscitation of these forms occurs following the addition of catalase and Tween 80. Our findings have important implications for the disease control and detection of B. pseudomallei, an agent of both public health and defense interest.