Spatial distribution of trace metals and associated transport proteins during bacterial infection.

Innate immune systems alter the concentrations of trace elements in host niches in response to invading pathogens during infection. This work reports the interplay between d-block metal ions and their associated biomolecules using hyphenated elemental techniques to spatially quantify both elemental distributions and the abundance of specific transport proteins. Here, lung tissues were collected for analyses from naïve and Streptococcus pneumoniae-infected mice fed on a zinc-restricted or zinc-supplemented diet. Spatiotemporal distributions of manganese (55Mn), iron (56Fe), copper (63Cu), and zinc (66Zn) were determined by quantitative laser ablation-inductively coupled plasma-mass spectrometry. The murine transport proteins ZIP8 and ZIP14, which are associated with zinc transport, were also imaged by incorporation of immunohistochemistry techniques into the analytical workflow. Collectively, this work demonstrates the potential of a single instrumental platform suitable for multiplex analyses of tissues and labelled antibodies to investigate complex elemental interactions at the host-pathogen interface. Further, these methods have the potential for broad application to investigations of biological pathways where concomitant measurement of elements and biomolecules is crucial to understand the basis of disease and aid in development of new therapeutic approaches.

Zinc acquisition and its contribution to Klebsiella pneumoniae virulence.

Klebsiella pneumoniae is a World Health Organization priority pathogen and a significant clinical concern for infections of the respiratory and urinary tracts due to widespread and increasing resistance to antimicrobials. In the absence of a vaccine, there is an urgent need to identify novel targets for therapeutic development. Bacterial pathogens, including K. pneumoniae, require the d-block metal ion zinc as an essential micronutrient, which serves as a cofactor for ~6% of the proteome. During infection, zinc acquisition necessitates the use of high affinity uptake systems to overcome niche-specific zinc limitation and host-mediated nutritional immunity. Here, we report the identification of ZnuCBA and ZniCBA, two ATP-binding cassette permeases that are highly conserved in Klebsiella species and contribute to K. pneumoniae AJ218 zinc homeostasis, and the high-resolution structure of the zinc-recruiting solute-binding protein ZniA. The Znu and Zni permeases appear functionally redundant with abrogation of both systems required to reduce K. pneumoniae zinc accumulation. Disruption of both systems also exerted pleiotropic effects on the homeostasis of other d-block elements. Zinc limitation perturbed K. pneumoniae cell morphology and compromised resistance to stressors, such as salt and oxidative stress. The mutant strain lacking both systems showed significantly impaired virulence in acute lung infection models, highlighting the necessity of zinc acquisition in the virulence and pathogenicity of K. pneumoniae.

Host-Mediated Copper Stress Is Not Protective against Streptococcus pneumoniae D39 Infection.

Metal ions are required by all organisms for the chemical processes that support life. However, in excess they can also exert toxicity within biological systems. During infection, bacterial pathogens such as Streptococcus pneumoniae are exposed to host-imposed metal intoxication, where the toxic properties of metals, such as copper, are exploited to aid in microbial clearance. However, previous studies investigating the antimicrobial efficacy of copper in vivo have reported variable findings. Here, we use a highly copper-sensitive strain of S. pneumoniae, lacking both copper efflux and intracellular copper buffering by glutathione, to investigate how copper stress is managed and where it is encountered during infection. We show that this strain exhibits highly dysregulated copper homeostasis, leading to the attenuation of growth and hyperaccumulation of copper in vitro. In a murine infection model, whole-tissue copper quantitation and elemental bioimaging of the murine lung revealed that infection with S. pneumoniae resulted in increased copper abundance in specific tissues, with the formation of spatially discrete copper hot spots throughout the lung. While the increased copper was able to reduce the viability of the highly copper-sensitive strain in a pneumonia model, copper levels in professional phagocytes and in a bacteremic model were insufficient to prosecute bacterial clearance. Collectively, this study reveals that host copper is redistributed to sites of infection and can impact bacterial viability in a hypersusceptible strain. However, in wild-type S. pneumoniae, the concerted actions of the copper homeostatic mechanisms are sufficient to facilitate continued viability and virulence of the pathogen. IMPORTANCE Streptococcus pneumoniae (the pneumococcus) is one of the world's foremost bacterial pathogens. Treatment of both localized and systemic pneumococcal infection is becoming complicated by increasing rates of multidrug resistance globally. Copper is a potent antimicrobial agent used by the mammalian immune system in the defense against bacterial pathogens. However, unlike other bacterial species, this copper stress is unable to prosecute pneumococcal clearance. This study determines how the mammalian host inflicts copper stress on S. pneumoniae and the bacterial copper tolerance mechanisms that contribute to maintenance of viability and virulence in vitro and in vivo. This work has provided insight into the chemical biology of the host-pneumococcal interaction and identified a potential avenue for novel antimicrobial development.

Dysregulation of Streptococcus pneumoniae zinc homeostasis breaks ampicillin resistance in a pneumonia infection model.

Streptococcus pneumoniae is the primary cause of community-acquired bacterial pneumonia with rates of penicillin and multidrug-resistance exceeding 80% and 40%, respectively. The innate immune response generates a variety of antimicrobial agents to control infection, including zinc stress. Here, we characterize the impact of zinc intoxication on S. pneumoniae, observing disruptions in central carbon metabolism, lipid biogenesis, and peptidoglycan biosynthesis. Characterization of the pivotal peptidoglycan biosynthetic enzyme GlmU indicates a sensitivity to zinc inhibition. Disruption of the sole zinc efflux pathway, czcD, renders S. pneumoniae highly susceptible to ß-lactam antibiotics. To dysregulate zinc homeostasis in the wild-type strain, we investigated the safe-for-human-use ionophore 5,7-dichloro-2-[(dimethylamino)methyl]quinolin-8-ol (PBT2). PBT2 rendered wild-type S. pneumoniae strains sensitive to a range of antibiotics. Using an invasive ampicillin-resistant strain, we demonstrate in a murine pneumonia infection model the efficacy of PBT2 + ampicillin treatment. These findings present a therapeutic modality to break antibiotic resistance in multidrug-resistant S. pneumoniae.

The influence of pain, kinesiophobia and psychological comorbidities on the accuracy of rating of perceived exertion in UK military spinal rehabilitation.

INTRODUCTION: Chronic low back pain (CLBP) is a leading cause of disability in the UK Military. Pain and psychological comorbidities have been reported to influence the rating of perceived exertion (RPE). Exercise rehabilitation can be monitored using RPE; however, the accuracy of RPE in inpatient CLBP rehabilitation is unknown. METHODS: A prospective cohort correlation study of 40 UK Military inpatients with CLBP was completed. Disability (ODI), kinesiophobia (TSK), anxiety (GAD-7) and depression (PHQ-9) were subjectively reported at the beginning and end of a 3 week intervention. Pain (VAS) and HR were recorded in the first aerobic exercise (AE) session (T1) and the final aerobic exercise session (T2). RPE was reported for each AE session. RESULTS: At T1, a positive correlation was observed between RPE accuracy (-7.2±20.9), and pre-exercise pain (2.7 mm ±1.6 mm) (p>0.001) and ODI (31.0±16.9) (p>0.05), and a negative relationship between RPE accuracy and average HR (135 bpm ±22 bpm) (p>0.001) was observed. At T2, there was no significant correlation between RPE accuracy (-4.4±22.6) and pre-exercise pain (2.8 mm ±1.6 mm) or ODI (34.0±16.5) (p>0.05). The strong negative relationship between RPE accuracy and average HR (137 bpm ±20 bpm) remained at T2. Improved RPE accuracy over the 3-week rehabilitation programme was correlated to the change in average HR (r=-0.314, p<0.05). CONCLUSIONS: Comorbidities may negatively affect RPE accuracy in CLBP, but the magnitude of the influence reduces over intensive rehabilitation.

Dietary zinc and the control of Streptococcus pneumoniae infection.

Human zinc deficiency increases susceptibility to bacterial infection. Although zinc supplementation therapies can reduce the impact of disease, the molecular basis for protection remains unclear. Streptococcus pneumoniae is a major cause of bacterial pneumonia, which is prevalent in regions of zinc deficiency. We report that dietary zinc levels dictate the outcome of S. pneumoniae infection in a murine model. Dietary zinc restriction impacts murine tissue zinc levels with distribution post-infection altered, and S. pneumoniae virulence and infection enhanced. Although the activation and infiltration of murine phagocytic cells was not affected by zinc restriction, their efficacy of bacterial control was compromised. S. pneumoniae was shown to be highly sensitive to zinc intoxication, with this process impaired in zinc restricted mice and isolated phagocytic cells. Collectively, these data show how dietary zinc deficiency increases sensitivity to S. pneumoniae infection while revealing a role for zinc as a component of host antimicrobial defences.

Cephamycins inhibit pathogen sporulation and effectively treat recurrent Clostridioides difficile infection.

Spore-forming bacteria encompass a diverse range of genera and species, including important human and animal pathogens, and food contaminants. Clostridioides difficile is one such bacterium and is a global health threat because it is the leading cause of antibiotic-associated diarrhoea in hospitals. A crucial mediator of C. difficile disease initiation, dissemination and re-infection is the formation of spores that are resistant to current therapeutics, which do not target sporulation. Here, we show that cephamycin antibiotics inhibit C. difficile sporulation by targeting spore-specific penicillin-binding proteins. Using a mouse disease model, we show that combined treatment with the current standard-of-care antibiotic, vancomycin, and a cephamycin prevents disease recurrence. Cephamycins were found to have broad applicability as an anti-sporulation strategy, as they inhibited sporulation in other spore-forming pathogens, including the food contaminant Bacillus cereus. This study could directly and immediately affect treatment of C. difficile infection and advance drug development to control other important spore-forming bacteria that are problematic in the food industry (B. cereus), are potential bioterrorism agents (Bacillus anthracis) and cause other animal and human infections.

Bovine antibodies targeting primary and recurrent Clostridium difficile disease are a potent antibiotic alternative.

The increased incidence of antibiotic resistant 'superbugs' has amplified the use of broad spectrum antibiotics worldwide. An unintended consequence of antimicrobial treatment is disruption of the gastrointestinal microbiota, resulting in susceptibility to opportunistic pathogens, such as Clostridium difficile. Paradoxically, treatment of C. difficile infections (CDI) also involves antibiotic use, leaving patients susceptible to re-infection. This serious health threat has led to an urgent call for the development of new therapeutics to reduce or replace the use of antibiotics to treat bacterial infections. To address this need, we have developed colostrum-derived antibodies for the prevention and treatment of CDI. Pregnant cows were immunised to generate hyperimmune bovine colostrum (HBC) containing antibodies that target essential C. difficile virulence components, specifically, spores, vegetative cells and toxin B (TcdB). Mouse infection and relapse models were used to compare the capacity of HBC to prevent or treat primary CDI as well as prevent recurrence. Administration of TcdB-specific colostrum alone, or in combination with spore or vegetative cell-targeted colostrum, prevents and treats C. difficile disease in mice and reduces disease recurrence by 67%. C. difficile-specific colostrum should be re-considered as an immunotherapeutic for the prevention or treatment of primary or recurrent CDI.

Effects of relative age on physical and physiological performance characteristics in youth soccer.

AIM: In youth sport, birth-date positioning of performers has significant implications for future success. This phenomenon is particularly evident in soccer and is identified as the "Relative Age Effect" (RAE). To date, limited work has been conducted into the RAE from a laboratory setting. METHODS: Subjects completed a modified cycling intermittent sprint protocol (mCISP) of 15, 6 s sprints against a resistance of 7.5%·body mass, interspersed with 120 s active recovery. Nineteen (male) athletes (mean±SD) age 14.2±0.7 y; height 164±7.9 cm and body mass 54±8.7 kg participated and were separated into six-month groups, Early-Born (EB) or Late-Born (LB) based on birth-month. Ethical approval was granted by the University Ethics Committee. RESULTS: Statistically significant differences (P<0.05) were found between EB and LB peak power outputs (PPO) and mean power outputs (MPO) (absolute and relative to body mass). EB subjects had significantly higher inter-sprint MPOs and PPOs for 15 and 13 sprints respectively. Borderline significance was observed for height and fat free mass (P=0.11). CONCLUSION: The present study suggests EB individuals are often more physically and physiologically mature than LB counterparts.

A pilot randomised controlled trial of medical versus nurse clerking for minor surgery.

BACKGROUND: Nurse led clerking is currently practiced in a growing number of UK centres, but there is a paucity of evidence to underpin the safety of this innovation. AIM: To assess the safety of nurse led clerking in paediatric day case and minor surgery. METHODS: Children aged 3 months to 15 years were randomly assigned to clerking by either a nurse or a senior house officer (SHO) (resident). All children were then independently reassessed by a specialist registrar anaesthetist to provide a "gold standard" against which practitioner performance could be judged. RESULTS: In 60 children studied, nurses identified a significantly greater proportion of the detectable abnormalities present in the sample (p = 0.16). This difference is attributable to nurses' greater accuracy in history taking (p = 0.04); no conclusions regarding the comparability of nurses' and SHOs' skills in physical examination can be derived from the current study. CONCLUSION: Evidence attests to the likelihood of nursing having superior skills in history taking to SHOs. Exploration of nursing safety in undertaking physical examination, however, requires the conduct of a large scale equivalence study. Only then can conclusions be drawn as to whether nurse led physical assessment offers children a standard of care equivalent to that which they currently receive from SHOs.

Evaluation of a spring-loaded syringe driver for the subcutaneous administration of narcotics.

A simple spring-loaded syringe driver was tested for the subcutaneous administration of narcotic analgesics and antiemetics. With concentrations of 2 to 10 mg/mL of hydromorphone and 10 to 50 mg/mL of morphine, the infusion rate during preclinical testing was 1.01 +/- 0.1 mL/hr (range 0.70-1.2 mL/hr). The rate of infusion was not modified by the concentration of narcotic in solution. Clinical trials were performed with morphine in 17 patients, and with hydromorphone in 11 patients. The duration of the infusion was 21 +/- 11 days. The most frequent reason for discontinuation was death (22 cases). The average duration of the site of infusion was 6.3 +/- 4 days. When used subcutaneously, the rate of infusion of the device was 1 +/- 0.15 mL/hr (range 0.70-1.30 mL/hr). Patients and nurses were satisfied with the simplicity and safety of the device. Cost analysis shows that this device is significantly less expensive than currently available portable infusion devices. We conclude that the Medifuse Pump is an inexpensive, safe and effective device for the subcutaneous infusion of narcotics and antiemetics.

Structural studies on bio-active compounds. Part 9. 2,4-Diamino-6-azidoquinazoline hydrochloride.

C8H7N7.HCl has a formula weight Mr237.7, and crystallizes in space group P21/n with a = 6.498(1), b = 10.333(8), c = 15.406(5) A, beta = 91.72(2) degrees. The final R factor was 0.049 for 1010 unique observed reflections. The azido substituent is almost linear, coplanar with the heterocycle, and parallel to the C(7)-C(6) ring bond. The heterocycle is protonated at N(1) and extensively hydrogen bonded. The 6-azido compound is at least a 100-fold better inhibitor of Escherichia coli dihydrofolate reductase than the analogue lacking the azido group, according to I50 values. Superimposing the quinazoline moiety upon the pteridine ring of the methotrexate-dihydrofolate reductase (E. coli) complex reveals two orientations of the N3 group in which it can fit the enzyme, making van der Waals contacts. Ab initio molecular orbital calculations suggest that one of these conformations, with torsion angle phi = C(5)-C(6)-N(61)-N(62) = -163.8 degrees, is low in energy.

Major disaster planning.

In 1983 a coach crash brought a hospital's major disaster plan into operation. The surgical aspects of the plan were assessed to see how well they matched up to three major aims: saving life, relieving pain and distress, and completing primary treatment of open wounds within eight hours of the accident. The last goal was not met for most of the 21 victims, mainly children with multiple deep dirty abrasions and extensive tissue loss. Having determined that none of the victims were in immediate danger the surgeons reassessed the priorities--in several cases disturbing dressings for a third or fourth time. The total time spent in theatre (in five theatres) was 37 hours, as opposed to the original estimated 10-15 hours. The experience gained in this accident suggests that a disaster plan should indicate the number of patients a single hospital can admit and that a senior surgeon should act as a coordinator and get surgeons working as soon as patients arrive, keeping two theatres reserved for lifesaving surgery. In this way primary treatment of wounds may be completed within eight hours of injury and the risk of infection reduced.

Effect of nutritional status on the lung surfactant system: food deprivation and caloric restriction.

It has been reported that food deprivation may result in decreased synthesis of lung surfactant phospholipids while the ratio of saturated and unsaturated fatty acids of phosphatidylcholine is maintained. To further investigate this effect, rats were deprived of diet for 24, 48, 72, or 96 hr or fed a nutritionally complete but calorie-deficient diet for 48, 96, 144, and 192 hr. The surfactant was separated into extracellular and intracellular fractions, with the remaining lung tissue pooled as a nonsurfactant fraction. The total phospholipids, phosphatidylcholine, phosphatidylglycerol, and disaturated phosphatidylcholine of each surfactant fraction and the remaining lung tissue were analyzed. The data suggest that on a DNA basis, food and caloric deprivation results temporarily in an altered quantity of pulmonary phospholipids. By 96 hr of fasting or 192 hr of calorie-deficient diet, the pool sizes were replenished and no longer significantly different from the control state. In addition, the distribution of phosphatidylglycerol and phosphatidylcholine was modified in extracellular and intracellular surfactant fractions and in the nonsurfactant tissue fraction. Although the percentage of total phospholipids found as phosphatidylcholine was decreased, the relationship between saturated and unsaturated phosphatidylcholine was preserved in the extracellular surfactant fraction. These results demonstrate that during food and caloric deprivation, the lung quickly adapts to maintain the quality and quantity of surfactant on the alveolar surface.

Issues confronting the new health professionals.

New health practitioners (NHPs) is a generic term referring to mid-level health workers such as physician's assistants and nurse practitioners who perform tasks traditionally within the purview of physicians. In the little over ten years since the first program to train physician assistants was initiated at Duke University, 6,500 physician's assistants (PAs) have completed formal training programs. Similarly, programs to train nurses for extended roles have prepared more than 8,500 nurse practitioners over the same decade. This paper considers the comparative achievements of these two major new health professions during the last decade and identifies eight crucial issues which may influence new health professionals in the ten years which lie ahead: (1) How many NHPs are enough? (2) What impact are NHPs making on distribution? (3) Do we know what clinical difference NHPs make? (4) Are NHPs bringing about cost control in health care? (5) What is the status of the controversy between organized medicine and nursing with regard to NHPs? (6) Are there differences between nurse practitioners and physician's assistants? (7) How are NHPs certified? and (8) Finally, can a better name than "new health practitioners" be found?