Improving paediatric antimicrobial stewardship in hospital-based settings: why, where and how?

BACKGROUND: Antimicrobial resistance (AMR) is being recognized as a priority by healthcare organizations across the world. However, many children are managed on IV antimicrobials in hospital with very little consideration of antimicrobial stewardship issues. OBJECTIVES: A nurse-led paediatric ambulatory outpatient parenteral antimicrobial therapy (OPAT) service, managing children with common infections being ambulated on short courses of IV antimicrobials, was introduced within Southampton Children's Hospital in January 2018. We evaluated the impact of this service in terms of the quality of antimicrobial prescribing and timing of ambulation in children presenting with common infections. METHODS: All cases managed within the service were reviewed in two separate 2 month time periods: prior to introduction of the service (September-October 2016) and then prospectively after its introduction (September-October 2018). RESULTS: A total of 96% of IV antibiotic management decisions at 48 h were deemed appropriate in 2018, compared with 75% in 2016. A total of 64% of patients were ambulated on IV antibiotics at some point during their treatment course in 2018, compared with 19% in 2016. However, a significant proportion of antimicrobial decisions made at the point of presentation to hospital remained suboptimal in 2018. CONCLUSIONS: Children are commonly managed with IV antibiotics in hospital. We demonstrate marked improvements in appropriate antimicrobial use through the introduction of a nurse-led ambulatory OPAT service. In addition, such a service can promote a greater proportion of children being ambulated from hospital, freeing up valuable inpatient beds and potentially delivering cost savings that can be used to fund such services.

Ricin antitoxins based on lyotropic mesophases containing galactose amphiphiles.

Lyotropic mesophases of the inverse hexagonal or cubic type are nanostructured materials that result from the self-assembly of amphiphilic surfactant molecules in water. The extremely large area of the surfactant-water interface inherent within these structures makes them attractive media for sorbent or encapsulant systems. Here, we report on the development of a new class of polyvalent materials that are based on the incorporation of bioactive ligands within lyotropic mesophases. In particular, we have studied the potential for these materials to behave as polyvalent antitoxins by incorporating synthetic galactose amphiphiles, which mimic the natural cell surface ligand for the protein toxin ricin. The study demonstrates that cubic morphology lyotropic mesophases containing galactose amphiphiles exhibit high specificity ricin uptake, with favorably high dissociation constants and high capacities. We suggest that lyotropic mesophase polyvalent ligands are thus promising materials for the incorporation of a broad range of cell surface recognition moieties and hence may have wide applicability as materials capable of partaking in biological recognition processes.

Monovalent and polyvalent carbohydrate inhibitors of ricin binding to a model of the cell-surface receptor.

A selection of galactose and lactose analogues was evaluated for their potency in inhibiting the binding of ricin to immobilised asialofetuin, which is a model of the cell-surface receptor for ricin. The aim was to identify compounds that could be used as antagonists of ricin toxicity in vivo, and as more selective, and therefore safer, antitoxins. Although one of these analogues had been identified by molecular modelling in a previous study as a potentially potent inhibitor, it and the other carbohydrates studied were less effective than galactose and lactose themselves (I(50) = 1.39 and 0.74 mM, respectively). In an attempt to increase the potency of carbohydrate-based inhibitors, galactose was coupled to the surface of dendrimers. No synergistic interactions were observed from this multivalent approach. Encouraging results, however, were obtained with a self-assembled lyotropic mesophase gel containing novel synthetic galactose-based surfactants, which was able to sequester ricin from aqueous solution in a 2-phase system.

An investigation of the degradation of the plant toxin, ricin, by sodium hypochlorite.

The toxin, ricin (0.4 microg/microl), was exposed to a range of sodium hypochlorite concentrations. SDS PAGE showed that hypochlorite caused the ricin to smear and decrease in mobility and, ultimately, caused a loss of silver staining. Cytotoxicity assays using dye uptake by Hep2 cells showed that treatment with 3 mM hypochlorite inactivated the ricin. Western blotting and ELISAs showed that binding by polyclonal antibodies raised against native ricin, or partially degraded ricin, diminished as hypochlorite degradation of the ricin increased.

Characterization of the Asialofetuin microtitre plate-binding assay for evaluating inhibitors of ricin lectin activity.

Optimum conditions for the binding of ricin to the glycoprotein asialofetuin immobilized on microtitre plates were investigated for the purpose of evaluating inhibitors of ricin B-chain lectin activity. Such inhibitors are of potential value in the use of immunotoxins based on ricin. This assay was first reported in 1986, but has not been characterized fully. Maximum binding of asialofetuin to the plate was observed at a concentration of ca. 4 microg ml(-1). Binding increased with time of incubation (1-24 h), pH (7.4-9.9) and temperature (2-37 degrees C). The pH effects were more marked at lower temperatures. Saturable binding of ricin to immobilized asialofetuin was observed, and at least 80% of maximum binding was observed by 10 min of incubation time. The binding was found to be very tight, such that an appreciable proportion of ricin added to the wells was bound at low concentrations, and binding was only partially reversible by addition of free galactose. Consequently, only estimates of the ricin-asialofetuin and ricin-galactose dissociation constants could be determined: 1.9 nM and 83 microM, respectively. Binding of ricin A- and B-chains was found to be 47% (at a 200-fold higher concentration) and 26% (at a twofold higher concentration) of that of the whole ricin molecule, respectively. The assay permits qualitative comparison of inhibitors of ricin B-chain lectin activity.

Isolation and molecular analysis of colonising and non-colonising strains of Campylobacter jejuni and Campylobacter coli following experimental infection of young chickens.

Fourteen-day-old chickens were inoculated with selected Campylobacter coli and C. jejuni strains. C. jejuni strains were of two subgroups based on a polymorphism detected using a DNA probe and represented the profiles typical for the majority of strains of either chicken or human origin. All C. coli strains previously isolated from humans colonised chickens, whereas from 4/7 C. jejuni strains of human origin, failed to colonise. Of 12 Campylobacter strains of chicken origin, 10 established a persistent colonisation in the chickens, and 2 strains colonised poorly or not at all. Four strains that failed to colonise chickens were each inoculated into groups of five birds. Three strains again did not colonise any of the chickens and the fourth strain colonised four out of the five chickens, but was poorly excreted. When infected chickens were placed in the same enclosure to facilitate interchange of strains, C. jejuni strain 331 was found to be dominant and colonised all 12 chickens by 21 days, displacing all other strains. C. jejuni strain 331, was then inoculated into groups of five birds with previously established colonisation by C. jejuni and C. coli strains. Strain 331 was able to replace the C. jejuni strain in all five birds but established co-colonisation with C. coli strain. Naturally occurring co-colonisation by two C. jejuni strains was detected in one chicken out of 200 tested. There was no obvious correlation between the type of DNA polymorphism in strains of chicken origin and their ability to colonise chickens.

Characterization of immunorelated peptides to porcidin P1.

Porcidin P1, an antimicrobial peptide purified from the granules of porcine polymorphonuclear neutrophils (PMN) using ultrafiltration and reverse phase high performance liquid chromatography (RP-HPLC), was covalently conjugated to BSA and used to generate monospecific polyclonal ascites. Antibodies raised against porcidin P1 were covalently coupled to an Affi-gel Hz affinity column and used for immunoaffinity chromatography of peptides from porcine PMN cell extract. Eleven immunorelated peptides were eluted from the column from neutrophil cell extracts and purified to homogeneity by HPLC. The molecular weights of the immunorelated peptides were determined by mass spectral analysis and ranged in size from 1.91 to 10.65 kDa. Of the 11 immunorelated peptides which were bound to the affinity column, only six peptides were recognized by the anti-porcidin antibodies after HPLC purification. Three immunoreactive peptides displayed potent antibacterial activity towards Staphylococcus aureus and Escherichia coli, reducing viability by as much as 99.9% (> 3 log reduction in CFU) when 5 mu g/mL of each purified peptide was used. The polyclonal monospecific antibodies also reacted with proteins from ovine and human PMN, illustrating possible structural relationships between small antibacterial peptides from the different species.

Antibacterial proteins from porcine polymorphonuclear neutrophils.

Antibacterial peptides were purified from porcine neutrophil granules collected from healthy pigs. Granule proteins, extracted with 0.2 mol/L sodium acetate were subjected to ion-exchange chromatography and five peaks (designated A to E) were detected. Individual porcine neutrophil granule proteins were shown to inhibit the growth of target organisms Escherichia coli and Staphylococcus aureus. The antimicrobial activity was shown to be concentration and time dependent. Peak D showed strong antimicrobial activity against S. aureus and peak C (with a greater number of eluted proteins) was shown to be active against both S. aureus and E. coli. One of the peptides was purified further by reverse-phase HPLC from peak fraction C. The MW of this peptide was approximately 5500 Da as determined by SDS-PAGE and mass spectral analysis and was active against both E. coli and S. aureus in vitro sustaining a > 90% decrease, respectively, in CFU after a 2 h exposure with 50 micrograms of this peptide. Amino acid analysis showed the peptide was rich in aspartate/aspartic acid, glutamine/glutamic acid, proline, arginine and threonine. The antimicrobial activity of this peptide and other novel proteins in porcine neutrophilic granules demonstrates the probable role of these proteins and peptides in host defence of porcine neutrophils against bacterial infection.

Campylobacter hyoilei sp. nov., associated with porcine proliferative enteritis.

Campylobacter hyoilei sp. nov. is the name proposed for an organism formerly described as strain RMIT 32AT (T = type strain) and a group of similar bacteria isolated from intestinal lesions of pigs with proliferative enteritis. The phenotypic characteristics of these organisms indicated that they are closely related to each other and are not strains of other Campylobacter spp. commonly isolated from pigs. The results of probing of ClaI-, EcoRV-, or BglII-cleaved genomic DNAs from C. hyoilei strains with a radiolabeled DNA probe that distinguishes between Campylobacter jejuni and Campylobacter coli indicated that C. hyoilei and C. coli are closely related. However, the 16S rRNA sequence of the reference strain of C. hyoilei, RMIT 32AT, was four bases different from the 16S rRNA sequence of C. jejuni CCUG 11284T and five bases different from the 16S rRNA sequence of C. jejuni subsp. doylei CCUG 24567T, suggesting that C. hyoilei is more closely related to C. jejuni than to C. coli. Hybridization between DNA from C. hyoilei type strain RMIT 32A and DNAs from selected type and reference strains of other Campylobacter species and subspecies, including C. jejuni, C. jejuni subsp. doylei, C. coli, Campylobacter mucosalis, and Campylobacter hyointestinalis, as well as the other C. hyoilei strains (the RMIT 32AT-like isolates), revealed that high levels of DNA hybridization (> 70%) occurred only between the reference strain and other strains of C. hyoilei.

Identification of a novel group of Serpulina hyodysenteriae isolates by using a lipopolysaccharide-specific monoclonal antibody.

A monoclonal antibody to Serpulina hyodysenteriae 8930 was produced and was used to probe pronase-treated cell lysates of S. hyodysenteriae isolates in immunblots. The results showed that the monoclonal antibody was specific for only five closely related S. hyodysenteriae isolates: 8930, 5380, 70A, RMIT 88, and RMIT 97.

Experimental reproduction of porcine proliferative enteritis.

A campylobacter-like bacterium, isolated from the terminal ileum of a pig with porcine proliferative enteritis (PPE), was used to initiate PPE in healthy crossbred pigs. Post-mortem examinations of pigs infected orally with the bacterium revealed thickening of the terminal ileum, haemorrhage within the ileum, degeneration of ileal villi and large quantities of bile and mucous in the ileal contents. Histopathological examination revealed that the terminal ileum, ileo-caecal valve and caecum from infected pigs had partial or total loss of mucous membrane, hyperplasia of the crypt and glandular epithelial cells, campylobacter-like organisms in and around the Peyers' patches and lymphoid aggregates in the mucosa. The causative organism was re-isolated from the intestinal specimens of infected animals post-mortem. In control animals, kept under the same conditions, no evidence of abnormality was observed in the terminal ileum, ileo-caecal valve or caecum.

Production of a monoclonal antibody that recognizes the lipopolysaccharide of a Campylobacter-like organism.

A monoclonal antibody was produced to a Campylobacter-like organism (RMIT 32A) which was isolated from the terminal ileum of a pig with proliferative enteritis. Isotyping of the antibody revealed that it was an IgG2a with kappa light chains. Immunoblots using the antibody against proteinase-K-treated whole cell lysates of RMIT 32A, a selection of Campylobacter species and other enteric bacteria showed that the antibody was specific for RMIT 32A and was directed against the lipopolysaccharide. This antibody can be used for the specific detection of RMIT 32A.

Humoral responses and salmonellosis protection in chickens given a vitamin-dependent Salmonella typhimurium mutant.

Humoral responses in chickens inoculated with an aromatic vitamin dependent (Aro-) Salmonella typhimurium mutant (STM) were studied to ascertain the efficacy of the organism as a vaccine for salmonellosis and possibly as a delivery system for antigens from enteric pathogens of chickens. Serum antibody responses in chickens that were given oral or subcutaneous inoculations of the bacterium followed the classic order of antibody production, with IgM being detected first, followed by IgG and IgA. Antibody responses in the gut of orally inoculated chickens were restricted to IgG and IgA. Weight gain measured in chickens given high doses of STM (up to 5 x 10(9)) orally, revealed that the bacterium did not adversely affect the chickens; in fact, inoculated chickens had significantly higher body weights than controls at the same age. Salmonellosis protection of chickens by oral vaccination with STM was examined in a vaccination/challenge experiment. The experiment revealed that oral vaccination reduced excretion of a virulent S. typhimurium used as the challenge organism.