Outcomes of a National, Cross-Sector Antimicrobial Stewardship Training Initiative for Pharmacists in England.

(1) Background: Pharmacists play a pivotal role in tackling Antimicrobial resistance through antimicrobial stewardship (AMS) and are well placed to lead behaviour change interventions across the healthcare system; (2) Methods: A cross-sector AMS training initiative for pharmacists was implemented across England, with three cohorts between 2019-2021. Each cohort took part in an introductory workshop, followed- by a workplace-based quality improvement project supported by peer-assisted learning sessions. Completion of training was determined by an end of training assessment after three to four months. Outcome data and learner survey results were collated, anonymised, and analysed by the training provider. (3) Results: In total, 118 pharmacists participated in the introductory workshop, 70% of these subsequently undertook an improvement project, and 48% engaged workplace stakeholders in the process. Interventions were designed by 57% of learners and 18% completed a at least one Plan-Do-Study-Act cycle. Approximately a quarter of learners met the requirements for a Certificate of Completion. Knowledge quiz scores were obtained from 115 learners pre-training and 28 learners post-training. Paired t-tests conducted for 28 learners showed a statistically significant improvement in mean score from 67.7% to 81.1% (p < 0.0001). Sixty-two learner survey responses were received during the training and 21 follow-up survey responses 6 to 12 months post training. Of the 21 responses to the follow-up survey, ongoing quality improvement work and improvement outcomes were reported by nine and six learners, respectively. (4) Conclusions: The delivery of workplace-based training at scale can be challenging, however this study demonstrates that coupling learning with workplace implementation and peer support can promote behaviour change in learners. Further study into the impact of providing pharmacists across sectors and geographies with access to this type of training will help inform ongoing workforce development interventions.

Impact of the Mk VI SkinSuit on skin microbiota of terrestrial volunteers and an International Space Station-bound astronaut.

Microgravity induces physiological deconditioning due to the absence of gravity loading, resulting in bone mineral density loss, atrophy of lower limb skeletal and postural muscles, and lengthening of the spine. SkinSuit is a lightweight compression suit designed to provide head-to-foot (axial) loading to counteract spinal elongation during spaceflight. As synthetic garments may impact negatively on the skin microbiome, we used 16S ribosomal RNA (rRNA) gene amplicon procedures to define bacterial skin communities at sebaceous and moist body sites of five healthy male volunteers undergoing SkinSuit evaluation. Each volunteer displayed a diverse, distinct bacterial population at each skin site. Short (8 h) periods of dry hyper-buoyancy flotation wearing either gym kit or SkinSuit elicited changes in the composition of the skin microbiota at the genus level but had little or no impact on community structure at the phylum level or the richness and diversity of the bacterial population. We also determined the composition of the skin microbiota of an astronaut during pre-flight training, during an 8-day visit to the International Space Station involving two 6-7 h periods of SkinSuit wear, and for 1 month after return. Changes in composition of bacterial skin communities at five body sites were strongly linked to changes in geographical location. A distinct ISS bacterial microbiota signature was found which reversed to a pre-flight profile on return. No changes in microbiome complexity or diversity were noted, with little evidence for colonisation by potentially pathogenic bacteria; we conclude that short periods of SkinSuit wear induce changes to the composition of the skin microbiota but these are unlikely to compromise the healthy skin microbiome.

Epicatechin gallate, a naturally occurring polyphenol, alters the course of infection with ß-lactam-resistant Staphylococcus aureus in the zebrafish embryo.

(-)-epicatechin gallate (ECg) substantially modifies the properties of Staphylococcus aureus and reversibly abrogates ß-lactam resistance in methicillin/oxacillin resistant (MRSA) isolates. We have determined the capacity of ECg to alter the course of infection in zebrafish embryos challenged with epidemic clinical isolate EMRSA-16. At 30 h post fertilization (hpf), embryos were infected by injection of 1-5 × 10(3) colony forming units (CFU) of EMRSA-16 into the circulation valley or yolk sac. Infection by yolk sac injection was lethal with a challenge dose above 3 × 10(3) CFU, with no survivors at 70 hpf. In contrast, survival at 70 hpf after injection into the circulation was 83 and 44% following challenge with 3 × 10(3) and 1-5 × 10(3) CFU, respectively. No significant increases in survival were noted when infected embryos were maintained in medium containing 12.5-100 µg/mL ECg with or without 4 or 16 µg/mL oxacillin. However, when EMRSA-16 was grown in medium containing 12.5 µg/mL ECg and the bacteria used to infect embryos by either the circulation valley or yolk sac, there were significant increases in embryo survival in both the presence and absence of oxacillin. ECg-modified and unmodified, GFP-transformed EMRSA-16 bacteria were visualized within phagocytic cells in the circulation and yolk sac; pre-treatment with ECg also significantly increased induction of the respiratory burst and suppressed increases in IL-1ß expression typical of infection with untreated EMRSA-16. We conclude that exposure to ECg prior to infection reduces the lethality of EMRSA-16, renders cells more susceptible to elimination by immune processes and compromises their capacity to establish an inflammatory response in comparison to non-exposed bacteria.

Impact of the ß-Lactam Resistance Modifier (-)-Epicatechin Gallate on the Non-Random Distribution of Phospholipids across the Cytoplasmic Membrane of Staphylococcus aureus.

The polyphenol (-)-epicatechin gallate (ECg) inserts into the cytoplasmic membrane (CM) of methicillin-resistant Staphylococcus aureus (MRSA) and reversibly abrogates resistance to ß-lactam antibiotics. ECg elicits an increase in MRSA cell size and induces thickened cell walls. As ECg partially delocalizes penicillin-binding protein PBP2 from the septal division site, reduces PBP2 and PBP2a complexation and induces CM remodelling, we examined the impact of ECg membrane intercalation on phospholipid distribution across the CM and determined if ECg affects the equatorial, orthogonal mode of division. The major phospholipids of the staphylococcal CM, lysylphosphatidylglycerol (LPG), phosphatidylglycerol (PG), and cardiolipin (CL), were distributed in highly asymmetric fashion; 95%-97% of LPG was associated with the inner leaflet whereas PG (~90%) and CL (~80%) were found predominantly in the outer leaflet. ECg elicited small, significant changes in LPG distribution. Atomic force microscopy established that ECg-exposed cells divided in similar fashion to control bacteria, with a thickened band of encircling peptidoglycan representing the most recent plane of cell division, less distinct ribs indicative of previous sites of orthogonal division and concentric rings and "knobbles" representing stages of peptidoglycan remodelling during the cell cycle. Preservation of staphylococcal membrane lipid asymmetry and mode of division in sequential orthogonal planes appear key features of ECg-induced stress.

Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a.

Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes ß-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function.

Staphylococcal phenotypes induced by naturally occurring and synthetic membrane-interactive polyphenolic ß-lactam resistance modifiers.

Galloyl catechins, in particular (-)-epicatechin gallate (ECg), have the capacity to abrogate ß-lactam resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA); they also prevent biofilm formation, reduce the secretion of a large proportion of the exoproteome and induce profound changes to cell morphology. Current evidence suggests that these reversible phenotypic traits result from their intercalation into the bacterial cytoplasmic membrane. We have endeavoured to potentiate the capacity of ECg to modify the MRSA phenotype by stepwise removal of hydroxyl groups from the B-ring pharmacophore and the A:C fused ring system of the naturally occurring molecule. ECg binds rapidly to the membrane, inducing up-regulation of genes responsible for protection against cell wall stress and maintenance of membrane integrity and function. Studies with artificial membranes modelled on the lipid composition of the staphylococcal bilayer indicated that ECg adopts a position deep within the lipid palisade, eliciting major alterations in the thermotropic behaviour of the bilayer. The non-galloylated homolog (-)-epicatechin enhanced ECg-mediated effects by facilitating entry of ECg molecules into the membrane. ECg analogs with unnatural B-ring hydroxylation patterns induced higher levels of gene expression and more profound changes to MRSA membrane fluidity than ECg but adopted a more superficial location within the bilayer. ECg possessed a high affinity for the positively charged staphylococcal membrane and induced changes to the biophysical properties of the bilayer that are likely to account for its capacity to disperse the cell wall biosynthetic machinery responsible for ß-lactam resistance. The ability to enhance these properties by chemical modification of ECg raises the possibility that more potent analogs could be developed for clinical evaluation.

Transcriptome analysis of responses to rhodomyrtone in methicillin-resistant Staphylococcus aureus.

Rhodomyrtone, purified from Rhodomyrtus tomentosa (Aiton) Hassk, exhibits a high degree of potency against methicillin-resistant Staphylococcus aureus (MRSA). We recently demonstrated that exposure of MRSA to a subinhibitory concentration (0.174 µg/ml) of rhodomyrtone resulted in the alteration of expression of several functional classes of bacterial proteins. To provide further insight into the antibacterial mode of action of this compound, we determined the impact of exposure to rhodomyrtone on the gene transcriptional profile of MRSA using microarray analysis. Exposure of MRSA to subinhibitory concentrations (0.5MIC; 0.5 µg/ml) of rhodomyrtone revealed significant modulation of gene expression, with induction of 64 genes and repression of 35 genes. Prominent changes in response to exposure to rhodomyrtone involved genes encoding proteins essential to metabolic pathways and processes such as amino acid metabolism, membrane function, ATP-binding cassette (ABC) transportation and lipoprotein and nucleotide metabolism. Genes involved in the synthesis of the aspartate family of amino acids, in particular proteins encoded by the dap operon were prominent. The diaminopimelate (DAP) biosynthetic pathway is the precursor of lysine synthesis and is essential for peptidoglycan biosynthesis. However, phenotypic analysis of the peptidoglycan amino acid content of rhodomyrtone-treated MRSA did not differ significantly from that extracted from control cells. Genes involved in the biosynthesis of amino acids and peptidoglycan, and a high affinity ATP-driven K ((+)) transport system, were investigated by quantitative reverse transcription-PCR (qRT-PCR) using EMRSA-16 1, 4, or 18 h after exposure to rhodomyrtone and in general the data concurred with that obtained by microarray, highlighting the relevance of the DAP biosynthetic pathway to the mode of action of rhodomyrtone.

Antistaphylococcal activity of DNA-interactive pyrrolobenzodiazepine (PBD) dimers and PBD-biaryl conjugates.

OBJECTIVES: Pyrrolobenzodiazepine (PBD) dimers, tethered through inert propyldioxy or pentyldioxy linkers, possess potent bactericidal activity against a range of Gram-positive bacteria by virtue of their capacity to cross-link duplex DNA in sequence-selective fashion. Here we attempt to improve the antibacterial activity and cytotoxicity profile of PBD-containing conjugates by extension of dimer linkers and replacement of one PBD unit with phenyl-substituted or benzo-fused heterocycles that facilitate non-covalent interactions with duplex DNA. METHODS: DNase I footprinting was used to identify high-affinity DNA binding sites. A staphylococcal gene microarray was used to assess epidemic methicillin-resistant Staphylococcus aureus 16 phenotypes induced by PBD conjugates. Molecular dynamics simulations were employed to investigate the accommodation of compounds within the DNA helix. RESULTS: Increasing the length of the linker in PBD dimers led to a progressive reduction in antibacterial activity, but not in their cytotoxic capacity. Complex patterns of DNA binding were noted for extended PBD dimers. Modelling of DNA strand cross-linking by PBD dimers indicated distortion of the helix. A majority (26 of 43) of PBD-biaryl conjugates possessed potent antibacterial activity with little or no helical distortion and a more favourable cytotoxicity profile. Bactericidal activity of PBD-biaryl conjugates was determined by inability to excise covalently bound drug molecules from bacterial duplex DNA. CONCLUSIONS: PBD-biaryl conjugates have a superior antibacterial profile compared with PBD dimers such as ELB-21. We have identified six PBD-biaryl conjugates as potential drug development candidates.

The minor groove-binding agent ELB-21 forms multiple interstrand and intrastrand covalent cross-links with duplex DNA and displays potent bactericidal activity against methicillin-resistant Staphylococcus aureus.

OBJECTIVES: The antistaphylococcal pyrrolobenzodiazepine dimer ELB-21 forms multiple adducts with duplex DNA through covalent interactions with appropriately spaced guanine residues; it is now known to form interstrand and intrastrand adducts with oligonucleotide sequences of variable length. We determined the DNA sequence preferences of ELB-21 in relation to its capacity to exert a bactericidal effect by damaging DNA. METHODS: Formation of adducts by ELB-21 and 12- to 14-mer DNA duplexes was investigated using ion-pair reversed phase liquid chromatography and mass spectrometry. Drug-induced changes in gene expression were measured in prophage-free Staphylococcus aureus RN4220 by microarray analysis. RESULTS: ELB-21 preferentially formed intrastrand adducts with guanines separated by three nucleotide base pairs. Interstrand and intrastrand adducts were formed with duplexes both longer and shorter than the preferred target sequences. ELB-21 elicited rapid bactericidal effects against prophage-carrying and prophage-free S. aureus strains; cell lysis occurred following activation and release of resident prophages. Killing appeared to be due to irreparable damage to bacterial DNA and susceptibility to ELB-21 was governed by the capacity of staphylococci to repair DNA lesions through induction of the SOS DNA damage response mediated by the RecA-LexA pathway. CONCLUSIONS: The data support the contention that ELB-21 arrests DNA replication, eliciting formation of ssDNA-RecA filaments that inactivate LexA, the SOS repressor, and phage repressors such as Cl, resulting in activation of the DNA damage response and de-repression of resident prophages. Above the MIC threshold, DNA repair is ineffective.