Trends in antibiotic prescribing in primary care out-of-hours doctors' services in Ireland.

Background: Infections are a common reason for patient consultation in out-of-hours (OOH) doctors' services. Surveillance of antibiotic prescribing in OOH settings is important to develop tailored antimicrobial stewardship (AMS) interventions. Objectives: To evaluate antibiotic prescribing patterns in OOH services in the Cork Kerry region, Ireland to inform future AMS interventions. Methods: A retrospective, observational cohort study was conducted of all oral antibiotic prescriptions in OOH doctors' consultations between 1 December 2019 and 31 December 2021 in the region. Data were gathered on age, gender, date and time of consultation, consultation method (in person, remote), antibiotic and its indication. Data were analysed using Microsoft Excel v.2018 and SPSS v.28. Results: Overall, 17% (69 017 of 406 812) of the OOH doctors' consultations resulted in an antibiotic prescription during the study period. This varied from 31% of OOH consultations in December 2019 to less than 2% of OOH consultations in April 2020. Of the antibiotics prescribed, 21% were for children under 6 years old. Respiratory tract infections (RTIs) were the most common indication for antibiotics (59%). Amoxicillin was the most commonly prescribed antibiotic (40% of all prescriptions). Red (reserved) antibiotics accounted for 19% of all prescriptions. During the COVID-19 pandemic period of the study, 66% of 49 421 of antibiotic prescriptions were issued from remote consultations. Conclusions: Low antibiotic prescribing levels during the early stages of the pandemic were not sustained. Antibiotic prescriptions from remote consultations were common. A key opportunity for AMS is addressing the volume of antibiotic prescribing for RTIs, particularly in children.

Quality assurance standards and their use in the preparation of parenteral systemic anticancer therapy products in healthcare establishments: a scoping review.

OBJECTIVES: The use of parenteral systemic anticancer therapy (SACT) has led to improved cancer survival. A quality assurance (QA) system of the aseptic compounding process is necessary to ensure safe and consistent production of parenteral SACT. This scoping review identifies international evidence and practice relating to QA standards in the preparation of parenteral SACT in healthcare establishments. METHODS: Standards relating to aseptic compounding in hospital pharmacies and literature exploring the aseptic compounding of parenteral SACT were included. Literature relating to the non-aseptic compounding of medicines and records specific to sterile manufacturing in industrial settings were excluded. A search of several electronic databases, trial registries, the grey literature and websites of key European hospital pharmacy groups and accreditation bodies was conducted on 16 March 2022. A narrative discussion was performed by country, and content analysis of articles was conducted. RESULTS: Thirty-seven records were included. Standards reviewed covered the work environment, the preparation process and the safety of the workers who are potentially exposed to hazardous chemicals. It was a common practice to include frequent audits to ensure adherence to standards. Some standards also recommended external inspections to allow for further learnings. Periodic reviews are encouraged to ensure standards maintain relevance. National standards of the countries reviewed were based on international standards, with minor adaptations for local conditions. CONCLUSIONS: The main limitation of this review is that it is limited to countries with a high human development index. The review shows that the use of an internationally recognised standard as a basis for national standards is best practice, and will allow for relevance into the future.

A novel erythromycin resistance plasmid from Bacillus sp. strain HS24, isolated from the marine sponge Haliclona simulans.

A better understanding of the origin and natural reservoirs of resistance determinants is fundamental to efficiently tackle antibiotic resistance. This paper reports the identification of a novel 5.8 kb erythromycin resistance plasmid, from Bacillus sp. HS24 isolated from the marine sponge Haliclona simulans. pBHS24B has a mosaic structure and carries the erythromycin resistance gene erm(T). This is the first report of an erythromycin resistance plasmid from a sponge associated bacteria and of the Erm(T) determinant in the genus Bacillus.

Sporulation during growth in a gut isolate of Bacillus subtilis.

Sporulation by Bacillus subtilis is a cell density-dependent response to nutrient deprivation. Central to the decision of entering sporulation is a phosphorelay, through which sensor kinases promote phosphorylation of Spo0A. The phosphorelay integrates both positive and negative signals, ensuring that sporulation, a time- and energy-consuming process that may bring an ecological cost, is only triggered should other adaptations fail. Here we report that a gastrointestinal isolate of B. subtilis sporulates with high efficiency during growth, bypassing the cell density, nutritional, and other signals that normally make sporulation a post-exponential-phase response. Sporulation during growth occurs because Spo0A is more active per cell and in a higher fraction of the population than in a laboratory strain. This in turn, is primarily caused by the absence from the gut strain of the genes rapE and rapK, coding for two aspartyl phosphatases that negatively modulate the flow of phosphoryl groups to Spo0A. We show, in line with recent results, that activation of Spo0A through the phosphorelay is the limiting step for sporulation initiation in the gut strain. Our results further suggest that the phosphorelay is tuned to favor sporulation during growth in gastrointestinal B. subtilis isolates, presumably as a form of survival and/or propagation in the gut environment.

Subtilomycin: a new lantibiotic from Bacillus subtilis strain MMA7 isolated from the marine sponge Haliclona simulans.

Bacteriocins are attracting increased attention as an alternative to classic antibiotics in the fight against infectious disease and multidrug resistant pathogens. Bacillus subtilis strain MMA7 isolated from the marine sponge Haliclona simulans displays a broad spectrum antimicrobial activity, which includes Gram-positive and Gram-negative pathogens, as well as several pathogenic Candida species. This activity is in part associated with a newly identified lantibiotic, herein named as subtilomycin. The proposed biosynthetic cluster is composed of six genes, including protein-coding genes for LanB-like dehydratase and LanC-like cyclase modification enzymes, characteristic of the class I lantibiotics. The subtilomycin biosynthetic cluster in B. subtilis strain MMA7 is found in place of the sporulation killing factor (skf) operon, reported in many B. subtilis isolates and involved in a bacterial cannibalistic behaviour intended to delay sporulation. The presence of the subtilomycin biosynthetic cluster appears to be widespread amongst B. subtilis strains isolated from different shallow and deep water marine sponges. Subtilomycin possesses several desirable industrial and pharmaceutical physicochemical properties, including activity over a wide pH range, thermal resistance and water solubility. Additionally, the production of the lantibiotic subtilomycin could be a desirable property should B. subtilis strain MMA7 be employed as a probiotic in aquaculture applications.

Pseudovibrio axinellae sp. nov., isolated from an Irish marine sponge.

A Gram-negative, motile, rod-shaped bacterial strain, designated Ad2(T), was isolated from a marine sponge, Axinella dissimilis, which was collected from a semi-enclosed marine lake in Ireland. Strain Ad2(T) grew optimally at 24 °C, at pH 7.0 and in the presence of 3 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Ad2(T) clustered with members of the genus Pseudovibrio, and showed 97.3-98.2 % sequence similarity to the type strains of recognized Pseudovibrio species. DNA-DNA relatedness values between strain Ad2(T) and the type strains of other Pseudovibrio species were <27 %. The DNA G+C content of strain Ad2(T) was 50.5 mol%. The major fatty acid was 18 : 1ω7c. Differences in phenotypic properties, together with phylogenetic and DNA-DNA hybridization analyses, indicated that strain Ad2(T) represented a novel species of the genus Pseudovibrio. The name Pseudovibrio axinellae sp. nov. is proposed, with Ad2(T) (= DSM 24994(T) = NCIMB 14761(T)) as the type strain.

Tetracycline resistance-encoding plasmid from Bacillus sp. strain #24, isolated from the marine sponge Haliclona simulans.

Knowledge of the nature of resistance determinants in natural habitats is fundamental to increasing our understanding of the development of antibiotic resistance in clinical settings. Here we provide the first report of a tetracycline resistance-encoding plasmid, pBHS24, from a marine sponge-associated bacterium, Bacillus sp. strain #24, isolated from Haliclona simulans.

Isolation and characterization of new thiamine-deregulated mutants of Bacillus subtilis.

In bacteria, thiamine pyrophosphate (TPP) is an essential cofactor that is synthesized de novo. Thiamine, however, is not an intermediate in the biosynthetic pathway but is salvaged from the environment and phosphorylated to TPP. We have isolated and characterized new mutants of Bacillus subtilis that deregulate thiamine biosynthesis and affect the export of thiamine products from the cell. Deletion of the ydiA gene, which shows significant similarity to the thiamine monophosphate kinase gene of Escherichia coli (thiL), did not generate the expected thiamine auxotroph but instead generated a thiamine bradytroph that grew to near-wild-type levels on minimal medium. From this DeltathiL deletion mutant, two additional ethyl methanesulfonate-induced mutants that derepressed the expression of a thiC-lacZ transcriptional reporter were isolated. One mutant, Tx1, contained a nonsense mutation within the B. subtilis yloS (thiN) gene that encodes a thiamine pyrophosphokinase, a result which confirmed that B. subtilis contains a single-step, yeast-like thiamine-to-TPP pathway in addition to the bacterial TPP de novo pathway. A second mutant, strain Tx26, was shown to contain two lesions. Genetic mapping and DNA sequencing indicated that the first mutation affected yuaJ, which encodes a thiamine permease. The second mutation was located within the ykoD cistron of the ykoFEDC operon, which putatively encodes the ATPase component of a unique thiamine-related ABC transporter. Genetic and microarray studies indicated that both the mutant yuaJ and ykoD genes were required for the derepression of thiamine-regulated genes. Moreover, the combination of the four mutations (the DeltathiL, thiN, yuaJ, and ykoD mutations) into a single strain significantly increased the production and excretion of thiamine products into the culture medium. These results are consistent with the proposed "riboswitch" mechanism of thiamine gene regulation (W. C. Winkler, A. Nahvi, and R. R. Breaker, Nature 419:952-956, 2002).

Screening for bacillus isolates in the broiler gastrointestinal tract.

Spores from a number of different Bacillus species are currently being used as human and animal probiotics, although their mechanisms of action remain poorly understood. Here we describe the isolation of 237 presumptive gut-associated Bacillus spp. isolates that were obtained by heat and ethanol treatment of fecal material from organically reared broilers followed by aerobic plating. Thirty-one representative isolates were characterized according to their morphological, physiological, and biochemical properties as well as partial 16S rRNA gene sequences and screening for the presence of plasmid DNA. The Bacillus species identified included B. subtilis, B. pumilus, B. licheniformis, B. clausii, B. megaterium, B. firmus, and species of the B. cereus group, whereas a number of our isolates could not be classified. Intrinsic properties of potential importance for survival in the gut that could be advantageous for spore-forming probiotics were further investigated for seven isolates belonging to five different species. All isolates sporulated efficiently in the laboratory, and the resulting spores were tolerant to simulated gastrointestinal tract conditions. They also exhibited antimicrobial activity against a broad spectrum of bacteria, including food spoilage and pathogenic organisms such as Bacillus spp., Clostridium perfringens, Staphylococcus aureus, and Listeria monocytogenes. Importantly, the isolates were susceptible to most of the antibiotics tested, arguing that they would not act as donors for resistance determinants if introduced in the form of probiotic preparations. Together, our results suggest that some of the sporeformers isolated in this study have the potential to persist in or transiently associate with the complex gut ecosystem.

Characterization of Bacillus probiotics available for human use.

Bacillus species (Bacillus cereus, Bacillus clausii, Bacillus pumilus) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties. Three B. cereus strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize. Spores of one B. cereus strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids. Spores of all strains were immunogenic when they were given orally to mice, but the B. pumilus strain was found to generate particularly high anti-spore immunoglobulin G titers. Spores of B. pumilus and of a laboratory strain of B. subtilis were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of B. pumilus and B. subtilis induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon. The B. pumilus strain and one B. cereus strain (B. cereus var. vietnami) were found to produce a bacteriocin-like activity against other Bacillus species. The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect. However, the three B. cereus strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use.

The Escherichia coli transcriptional regulator MarA directly represses transcription of purA and hdeA.

The Escherichia coli MarA protein mediates a response to multiple environmental stresses through the activation or repression in vivo of a large number of chromosomal genes. Transcriptional activation for a number of these genes has been shown to occur via direct interaction of MarA with a 20-bp degenerate asymmetric "marbox" sequence. It was not known whether repression by MarA was also direct. We found that purified MarA was sufficient in vitro to repress transcription of both purA and hdeA. Transcription and electrophoretic mobility shift experiments in vitro using mutant promoters suggested that the marbox involved in the repression overlapped the -35 promoter motif and was in the "backward" orientation. This organization contrasts with that of the class II promoters activated by MarA, in which the marbox also overlaps the -35 motif but is in the "forward" orientation. We conclude that MarA, a member of the AraC/XylS family, can act directly as a repressor or an activator, depending on the position and orientation of the marbox within a promoter.

Activation of the Escherichia coli nfnB gene by MarA through a highly divergent marbox in a class II promoter.

MarA is a global regulator that mediates resistance to multiple environmental hazards such as antibiotics, disinfectants and oxidative stress agents by modulating the expression of a large number of genes in the Escherichia coli chromosome. Two E. coli MarA homologues, SoxS and Rob also control, to different extents, genes in the mar/sox/rob regulon. The controlling element for these proteins is a 20 bp 'marbox' sequence in the promoter region of regulated genes. Using in vitro assays and mutagenesis of promoter fusions in whole cells, we identified the cis regulatory element involved in MarA upregulation of the oxygen-insensitive nitroreductase nfnB gene. MarA binds to a marbox that is highly divergent from the previously proposed consensus (eight differences out of 14 specified nucleotides). Although purified SoxS and Rob proteins, like MarA, activated nfnB transcription in vitro, only constitutive expression of chromosomal marA, but not of soxS and rob genes, affected nfnB expression in whole cells. Increased expression, but limited as compared with MarA, was only achieved by plasmid-mediated overexpression of SoxS and Rob. This study shows that MarA can regulate gene expression through a functional marbox that is considerably divergent from the current consensus sequence. The data suggest that MarA is preferred over SoxS and Rob in upregulating nfnB. The findings imply that other different but physiologically important marbox DNA-MarA interactions take place in the regulation of still uncharacterized members of the mar regulon.

The impact of antibiotic use on resistance development and persistence.

The intense use and misuse of antibiotics are undoubtedly the major forces associated with the high numbers of resistant pathogenic and commensal bacteria worldwide. Both the volume and the way antibiotics are applied contributes to the selection of resistant strains. Still, other social, ecological and genetic factors affect a direct relationship between use and frequency of resistance. Resistant bacteria, following their emergence and evolution in the presence of antibiotics, appear to acquire a 'life of their own'. They proliferate and maintain the resistance traits even in the absence of antibiotics, thus jeopardizing the reversal of bacterial resistance by simple reduction in antibiotic use. Reversing resistance requires restoration of the former susceptible flora in people and in the environment. Copyright 2000 Harcourt Publishers Ltd.