Impact of a sustained, collaborative antimicrobial stewardship programme in spinal cord injury patients.

Background: In patients with spinal cord injuries (SCIs), infections continue to be a leading cause of morbidity, mortality and hospital admission. Objectives: This study evaluated the long-term impact of a weekly, multidisciplinary Spinal/Antimicrobial Stewardship (AMS) meeting for acute-care SCI inpatients, on antimicrobial prescribing over 3 years. Methods: A retrospective, longitudinal, pre-post comparison of antimicrobial prescribing was conducted at our tertiary hospital in Melbourne. Antimicrobial prescribing was audited in 6 month blocks pre- (25 April 2017 to 24 October 2017), immediately post- (27 March 2018 to 25 September 2018) and 3 years post-implementation (2 March 2021 to 31 August 2021). Antimicrobial orders for patients admitted under the spinal unit at the meeting time were included. Results: The number of SCI patients prescribed an antimicrobial at the time of the weekly meeting decreased by 40% at 3 years post-implementation [incidence rate ratio (IRR) 0.63; 95% CI 0.51-0.79; P ≤ 0.001]. The overall number of antimicrobial orders decreased by over 22% at 3 years post-implementation (IRR 0.78; 95% CI 0.61-1.00; P = 0.052). A shorter antimicrobial order duration in the 3 year post-implementation period was observed (-28%; 95% CI -39% to -15%; P ≤ 0.001). This was most noticeable in IV orders at 3 years (-36%; 95% CI -51% to -16%; P = 0.001), and was also observed for oral orders at 3 years (-25%; 95% CI -38% to -10%; P = 0.003). Antimicrobial course duration (days) decreased for multiple indications: skin and soft tissue infections (-43%; 95% CI -67% to -1%; P = 0.045), pulmonary infections (-45%; 95% CI -67% to -9%; P = 0.022) and urinary infections (-31%; 95% CI -47% to -9%; P = 0.009). Ninety-day mortality rates were not impacted. Conclusions: This study showed that consistent, collaborative meetings between the Spinal and AMS teams can reduce antimicrobial exposure for acute-care SCI patients without adversely impacting 90 day mortality.

Evaluation of a pharmacist-led penicillin allergy de-labelling ward round: a novel antimicrobial stewardship intervention.

BACKGROUND: Antibiotic allergy labels (AALs), reported by up to 25% of hospitalized patients, are a significant barrier to appropriate prescribing and a focus of antimicrobial stewardship (AMS) programmes. METHODS: A prospective audit of a pharmacist-led AMS penicillin allergy de-labelling ward round at Austin Health (Melbourne, Australia) was evaluated. Eligible inpatients with a documented penicillin allergy receiving an antibiotic were identified via an electronic medical report and then reviewed by a pharmacist-led AMS team. The audit outcomes evaluated were: (i) AMS post-prescription review recommendations; (ii) direct de-labelling; (iii) inpatient oral rechallenge referral; (iv) skin prick testing/intradermal testing referral; and (v) outpatient antibiotic allergy clinic assessment. RESULTS: Across a 5 month period, 106 patients were identified from a real-time electronic prescribing antibiotic allergy report. The highest rate of penicillin allergy de-labelling was demonstrated in patients who were referred for an inpatient oral rechallenge with 95.2% (n = 21) successfully having their penicillin AAL removed. From the 22 patients with Type A reactions, 63.6% had their penicillin AAL removed. We demonstrated a significant decrease in the prescribing of restricted antibiotics (defined as third- or fourth-generation cephalosporins, fluoroquinolones, glycopeptides, carbapenems, piperacillin/tazobactam, lincosamides, linezolid or daptomycin) in patients reviewed (pre 42.5% versus post 17.9%, P = 0.0002). CONCLUSIONS: A pharmacist-led AMS penicillin allergy de-labelling ward round reduced penicillin AALs and the prescribing of restricted antibiotics. This model could be implemented at other hospitals with existing AMS programmes.

Expression of active spinach glycolate oxidase in Aspergillus nidulans.

The biocatalytic production of glyoxylic acid from glycolic acid requires two enzymes: glycolate oxidase, which catalyzes the oxidation of glycolic acid by oxygen to produce glyoxylic acid and hydrogen peroxide, and catalase, which decomposes the byproduct hydrogen peroxide. As an alternative to isolation from the leaf peroxisomes of spinach, glycolate oxidase has now been cloned and expressed in transformants of Aspergillus nidulans T580 at levels ranging from 1.7 to 36 IU/g dry wt. cells. The glycolate oxidase of transformant strain T17 comprises ca. 1.9% of total cell protein and is expressed at near 100% activity.

Expression of heterologous proteins in Aspergillus.

Filamentous fungi, in particular those of the genus Aspergillus have been well exploited for their ability to produce high levels of extracellular proteins in an inexpensive manner. Since many human proteins with the potential to be used therapeutically are secreted and require post-translational modification for biological activity, eukaryotic expression-secretion systems have been targeted for development. Recent developments in DNA-mediated transformation systems have allowed the utilization of Aspergillus as a host for the production of recombinant proteins. Several features such as well-characterized genetics and the availability of many mutants make Aspergillus nidulans the organism of choice for development of expression secretion systems. Recombinant strains contain integrated expression cassettes often in multiple copy, which are mitotically stable. In this review, we discuss the recent progress made in the use of Aspergillus as expression secretion hosts for the production of proteins of therapeutic significance.

An efficient cell-free translation system from Aspergillus nidulans and in vitro translocation of prepro-alpha-factor across Aspergillus microsomes.

We describe the preparation of an in vitro translation system from heat shock-treated Aspergillus nidulans, capable of supporting efficient and faithful synthesis of proteins from natural and in vitro transcribed eukaryotic messages. In vitro synthesized prepro-alpha-factor was translocated across Aspergillus nidulans microsomal membranes in either the homologous A. nidulans or a yeast cell-free system. The translocated prepro-alpha-factor was protected from digestion by protease and glycosylated to higher MW forms.

Binding of a 30-kDa protein to the pyruvate kinase gene of Neurospora crassa.

Extracts of a wild-type strain of Neurospora crassa, electrophoresed on SDS-polyacrylamide gels and electroblotted onto nitrocellulose sheets, were hybridized to an end-labelled pyruvate kinase (PK) gene fragment containing the 5' noncoding sequence and a large part of the coding region. A 30-kDa protein was found to bind strongly to the PK gene DNA, while binding weakly to plasmid pUC12 DNA and to total N. crassa DNA. Probing of blots with individual restriction fragments derived from the PK gene showed that the protein binding occurred primarily to the 5' noncoding region. Nonspecific DNA from pUC12, PK gene DNA from the recombinant plasmid pNP460 (pUC12 containing a 1.8-kilobase EcoRI insert of the PK gene DNA), along with a 0.7-kilobase EcoRI-AccI restriction fragment containing the 5' flanking region, were used in filter-binding experiments to analyze the kinetics of binding. Formation of protein-DNA complexes was demonstrated by monitoring the electrophoretic mobility of this fragment on nondenaturing gels.

Preparation of a cell-free translation system from a wild-type strain of Neurospora crassa.

We describe the preparation of an in vitro translation system from a wild-type strain of Neurospora crassa. The system is capable of supporting efficient and faithful translation of native and in vitro transcribed eukaryotic messages. The translation products have minimal background and can be clearly analyzed by SDS-polyacrylamide gel electrophoresis. The method of preparation of the lysate is simple, fast and reproducible. The procedure should be readily applicable to other filamentous fungi.

Some aspects of the regulation of pyruvate kinase levels in Neurospora crassa.

Pyruvate kinase levels were monitored in Neurospora crassa mycelium (grown on different carbon sources for varying time intervals) by immunoprecipitation using polyclonal antibodies raised against a purified enzyme preparation. Pyruvate kinase specific mRNA was demonstrated by hybridization of Northern and dot blots of total RNA with a N. crassa pyruvate kinase gene fragment. Two pyruvate kinase specific mRNA species were detected in mycelia of all ages examined. An age-dependent and carbon source dependent variation in the pyruvate kinase protein and mRNA levels was encountered: both registered an increase for up to about 20 h and a subsequent decline; growth on acetate and sucrose resulted in significantly higher yields of both, relative to that on medium containing ethanol and alanine. Stress caused by heat shock depressed the pyruvate kinase mRNA levels.

A study of the messenger RNA encoding pyruvate kinase of Neurospora crassa.

In Neurospora crassa, there is a single pyruvate kinase (PK) consisting of four identical subunits of approximately 60k daltons. Northern and dot blot hybridization studies, using most of the yeast pyruvate kinase gene as a probe, suggest the presence of two distinct mRNA species for pyruvate kinase, separable on the basis of the length of their polyadenylated tails, by oligo(dT)cellulose chromatography. These messages are present in polysomes, immuno-precipitated by anti-PK antibodies, indicating probable translation in vivo. Fractions containing both messages were translated in vitro in the heterologous systems as well as in a homologous N. crassa lysate, the newly-synthesized PK being detected by immunoadsorption. Protection studies using S1-nuclease suggest no major structural differences in the 5'-untranslated and most of the coding regions of the two messages.

Preparation of a cell-free translation system from a wild-type strain of Neurospora crassa and translation of pyruvate kinase messenger RNA.

A cell-free in vitro translation system exhibiting high activity has been developed from wild-type Neurospora crassa mycelium. The isolation is simple and fast, and the homogenization does not appear to affect the activity of mycelial proteases and nucleases. This system is capable of supporting efficient translation of exogenously added homologous RNA as demonstrated by the experiments with PK-specific mRNA. In addition, it translates heterologous RNA efficiently, shown by the translation of globin mRNA. We did not examine the Neurospora lysate for post-translational modification activity. The procedure used for the preparation of Neurospora cell-free extracts should be readily applicable to the other filamentous fungi.