OXA- and GES-type beta-lactamases predominate in extensively drug-resistant Acinetobacter baumannii isolates from a Turkish University Hospital

This study was supported by grants from Recep Tayyip Erdogan University (BAP-2012.106.01.11 and BAP-2011.102.03.3). AYP was supported by the Australian National Health and Medical Research Council (APP1047916 and APP1010114).We determined the antibiotic susceptibility and genetic mechanisms of resistance in clinical strains of Acinetobacter baumannii from Istanbul, Turkey. A total of 101 clinical strains were collected between November 2011 and July 2012. Antimicrobial susceptibility was performed using the Vitek 2 Compact system and E-test. Multiplex PCR was used for detecting bla(OXA-51-like), bla(OXA-23-like), bla(OXA-40-like) and bla(OXA-58-like) genes. ISAba1, bla(IMP-like), bla(VIM-like), bla(GES), bla(VEB), bla(PER-2), aac-3-Ia and aac-6'-Ib and NDM-1 genes were detected by PCR and sequencing. By multiplex PCR, all strains were positive for bla(OXA-51), 79 strains carried bla(OXA-23) and one strain carried bla(OXA-40). bla(OXA-51) and bla(OXA-23) were found together in 79 strains. ISAba1 element was detected in 81 strains, and in all cases it was found upstream of bla(OXA-51). GES-type carbapenemases were found in 24 strains (GES-11 in 16 strains and GES-22 in 8 strains) while bla(PER-2), bla(VEB-1), bla(NDM-1), bla(IMP)- and bla(VIM)-type carbapenemases were not observed. Aminoglycoside modifying enzyme (aac-3-Ia and aac-6'Ib) genes were detected in 13 and 15 strains, respectively. Ninety-seven (96%) A. baumannii strains were defined as MDR and of these, 98% were extensively drug resistant (sensitive only to colistin). Colistin remains the only active compound against all clinical strains. As seen in other regions, OXA-type carbapenemases, with or without an upstream ISAba1, predominate but GES-type carbapenemases also appear to have a significant presence. REP-PCR analysis was performed for molecular typing and all strains were collected into 12 different groups. To our knowledge, this is the first report of GES-11 and OXA-40 in A. baumannii from Turkey

The benefits of steroids versus steroids plus antivirals for treatment of Bell’s palsy: a meta-analysis

Objective To determine whether steroids plus antivirals provide a better degree of facial muscle recovery in patients with Bell’s palsy than steroids alone

Closing the gap in surveillance and audit of invasive mold diseases for antifungal stewardship using machine learning

Clinical audit of invasive mold disease (IMD) in hematology patients is inefficient due to the difficulties of case finding. This results in antifungal stewardship (AFS) programs preferentially reporting drug cost and consumption rather than measures that actually reflect quality of care. We used machine learning-based natural language processing (NLP) to non-selectively screen chest tomography (CT) reports for pulmonary IMD, verified by clinical review against international definitions and benchmarked against key AFS measures. NLP screened 3014 reports from 1 September 2008 to 31 December 2017, generating 784 positives that after review, identified 205 IMD episodes (44% probable-proven) in 185 patients from 50,303 admissions. Breakthrough-probable/proven-IMD on antifungal prophylaxis accounted for 60% of episodes with serum monitoring of voriconazole or posaconazole in the 2 weeks prior performed in only 53% and 69% of episodes, respectively. Fiberoptic bronchoscopy within 2 days of CT scan occurred in only 54% of episodes. The average turnaround of send-away bronchoalveolar galactomannan of 12 days (range 7-22) was associated with high empiric liposomal amphotericin consumption. A random audit of 10% negative reports revealed two clinically significant misses (0.9%, 2/223). This is the first successful use of applied machine learning for institutional IMD surveillance across an entire hematology population describing process and outcome measures relevant to AFS. Compared to current methods of clinical audit, semi-automated surveillance using NLP is more efficient and inclusive by avoiding restrictions based on any underlying hematologic condition, and has the added advantage of being potentially scalable

Chemoenzymatic surface decoration of Nisin-shelled nanoemulsions: novel targeted drug-nanocarriers for cancer applications

Nisin, a peptide used as a natural food preservative, is employed in this work for the development of a novel nanocarrier system. Stable and uniform nisin-shelled nanoemulsions (NSNE) with a diameter of 100 ± 20 nm were successfully prepared using 20 kHz flow-through ultrasonication technique. The NSNE showed limited toxicity, high bactericidal activity and high drug loading capacity (EE 65 % w/w). In addition, the nisin shell was exploited for the site-specific attachment of a recombinantly produced cancer targeting ligand (αHER2LPETG IgG). Employing a unique two phases (bio-click) approach which involved both Sortase A mediated Azide Bioconjugation (SMAB) and Strain Promoted Azide Alkyne Cycloaddition (SPAAC) reactions, targeted NSNE (NSNEDOX-αHER2 IgG) were successfully assembled and loaded with the chemotherapeutic drug Doxorubicin (DOX). Finally, NSNEDOX-αHER2 IgG showed cancer-specific binding and augmented cytotoxicity to HER2 expressing tumour cells

Species identification within Acinetobacter calcoaceticus-baumannii complex using MALDI-TOF MS

Acinetobacter baumannii, one of the more clinically relevant species in the Acinetobacter genus is well known to be multi-drug resistant and associated with bacteremia, urinary tract infection, pneumonia, wound infection and meningitis. However, it cannot be differentiated from closely related species such as Acinetobacter calcoaceticus, Acinetobacter pittii and Acinetobacter nosocomialis by most phenotypic tests and can only be differentiated by specific, time consuming genotypic tests with very limited use in clinical microbiological laboratories. As a result, these species are grouped into the A. calcoaceticus-. A. baumannii (Acb) complex. Herein we investigated the mass spectra of 73 Acinetobacter spp., representing ten different species, using an AB SCIEX 5800 MALDI-TOF MS to differentiate members of the Acinetobacter genus, including the species of the Acb complex. RpoB gene sequencing, 16S rRNA sequencing, and gyrB multiplex PCR were also evaluated as orthogonal methods to identify the organisms used in this study. We found that whilst 16S rRNA and rpoB gene sequencing could not differentiate A. pittii or A. calcoaceticus, they can be differentiated using gyrB multiplex PCR and MALDI-TOF MS. All ten Acinetobacter species investigated could be differentiated by their MALDI-TOF mass spectra

Evolution of carbapenem resistance in Acinetobacter baumannii during a prolonged infection.

Acinetobacter baumannii is a common causative agent of hospital-acquired infections and a leading cause of infection in burns patients. Carbapenem-resistant A. baumannii is considered a major public-health threat and has been identified by the World Health Organization as the top priority organism requiring new antimicrobials. The most common mechanism for carbapenem resistance in A. baumannii is via horizontal acquisition of carbapenemase genes. In this study, we sampled 20 A. baumannii isolates from a patient with extensive burns, and characterized the evolution of carbapenem resistance over a 45 day period via Illumina and Oxford Nanopore sequencing. All isolates were multidrug resistant, carrying two genomic islands that harboured several antibiotic-resistance genes. Most isolates were genetically identical and represented a single founder genotype. We identified three novel non-synonymous substitutions associated with meropenem resistance: F136L and G288S in AdeB (part of the AdeABC efflux pump) associated with an increase in meropenem MIC to ≥8 µg ml-1; and A515V in FtsI (PBP3, a penicillin-binding protein) associated with a further increase in MIC to 32 µg ml-1. Structural modelling of AdeB and FtsI showed that these mutations affected their drug-binding sites and revealed mechanisms for meropenem resistance. Notably, one of the adeB mutations arose prior to meropenem therapy but following ciprofloxacin therapy, suggesting exposure to one drug whose resistance is mediated by the efflux pump can induce collateral resistance to other drugs to which the bacterium has not yet been exposed

Antibiotic resistance and host immune evasion in Staphylococcus aureus mediated by a metabolic adaptation

Staphylococcus aureus is a notorious human bacterial pathogen with considerable capacity to develop antibiotic resistance. We have observed that human infections caused by highly drug-resistant S. aureus are more prolonged, complicated, and difficult to eradicate. Here we describe a metabolic adaptation strategy used by clinical S. aureus strains that leads to resistance to the last-line antibiotic, daptomycin, and simultaneously affects host innate immunity. This response was characterized by a change in anionic membrane phospholipid composition induced by point mutations in the phospholipid biosynthesis gene, cls2, encoding cardiolipin synthase. Single cls2 point mutations were sufficient for daptomycin resistance, antibiotic treatment failure, and persistent infection. These phenotypes were mediated by enhanced cardiolipin biosynthesis, leading to increased bacterial membrane cardiolipin and reduced phosphatidylglycerol. The changes in membrane phospholipid profile led to modifications in membrane structure that impaired daptomycin penetration and membrane disruption. The cls2 point mutations also allowed S. aureus to evade neutrophil chemotaxis, mediated by the reduction in bacterial membrane phosphatidylglycerol, a previously undescribed bacterial-driven chemoattractant. Together, these data illustrate a metabolic strategy used by S. aureus to circumvent antibiotic and immune attack and provide crucial insights into membrane-based therapeutic targeting of this troublesome pathogen

Multicentre stepped-wedge cluster randomised controlled trial of an antimicrobial stewardship programme in residential aged care: protocol for the START trial.

INTRODUCTION: Antimicrobial resistance is a growing global health threat, driven by increasing inappropriate use of antimicrobials. High prevalence of unnecessary use of antimicrobials in residential aged care facilities (RACFs) has driven demand for the development and implementation of antimicrobial stewardship (AMS) programmes. The Stepped-wedge Trial to increase antibiotic Appropriateness in Residential aged care facilities and model Transmission of antimicrobial resistance (START) will implement and evaluate the impact of a nurse-led AMS programme on antimicrobial use in 12 RACFs. METHODS AND ANALYSIS: The START trial will implement and evaluate a nurse-led AMS programme via a stepped-wedge cluster randomised controlled trial design in 12 RACFs over 16 months. The AMS programme will incorporate education, aged care-specific treatment guidelines, documentation forms, and audit and feedback strategies that will target aged care staff, general practitioners, pharmacists, and residents and their families. The intervention will primarily focus on urinary tract infections, lower respiratory tract infections, and skin and soft tissue infections. RACFs will transition from control to intervention phases in random order, two at a time, every 2 months, with a 2-month transition, wash-in period. The primary outcome is the cumulative proportion of residents within each facility prescribed an antibiotic during each month and total days of antibiotic use per 1000 occupied bed days. Secondary outcomes include the number of courses of systemic antimicrobial therapy, antimicrobial appropriateness, antimicrobial resistant organisms, Clostridioides difficile infection, change in antimicrobial susceptibility profiles, hospitalisations and all-cause mortality. Analyses will be conducted according to the intention-to-treat principle. ETHICS AND DISSEMINATION: Ethics approval has been granted by the Alfred Hospital Human Research Ethics Committee (HREC/18/Alfred/591). Research findings will be disseminated through peer-reviewed publications, conferences and summarised reports provided to participating RACFs. TRIAL REGISTRATION NUMBER: NCT03941509

BonA from Acinetobacter baumannii Forms a Divisome-Localized Decamer That Supports Outer Envelope Function

Acinetobacter baumannii is a high-risk pathogen due to the rapid global spread of multidrug-resistant lineages. Its phylogenetic divergence from other ESKAPE pathogens means that determinants of its antimicrobial resistance can be difficult to extrapolate from other widely studied bacteria. A recent study showed that A. baumannii upregulates production of an outer membrane lipoprotein, which we designate BonA, in response to challenge with polymyxins. Here, we show that BonA has limited sequence similarity and distinct structural features compared to lipoproteins from other bacterial species. Analyses through X-ray crystallography, small-angle X-ray scattering, electron microscopy, and multiangle light scattering demonstrate that BonA has a dual BON (Bacterial OsmY and Nodulation) domain architecture and forms a decamer via an unusual oligomerization mechanism. This analysis also indicates this decamer is transient, suggesting dynamic oligomerization plays a role in BonA function. Antisera recognizing BonA shows it is an outer membrane protein localized to the divisome. Loss of BonA modulates the density of the outer membrane, consistent with a change in its structure or link to the peptidoglycan, and prevents motility in a clinical strain (ATCC 17978). Consistent with these findings, the dimensions of the BonA decamer are sufficient to permeate the peptidoglycan layer, with the potential to form a membrane-spanning complex during cell division. IMPORTANCE The pathogen Acinetobacter baumannii is considered an urgent threat to human health. A. baumannii is highly resistant to treatment with antibiotics, in part due to its protective cell envelope. This bacterium is only distantly related to other bacterial pathogens, so its cell envelope has distinct properties and contains components distinct from those of other bacteria that support its function. Here, we report the discovery of BonA, a protein that supports A. baumannii outer envelope function and is required for cell motility. We determine the atomic structure of BonA and show that it forms part of the cell division machinery and functions by forming a complex, features that mirror those of distantly related homologs from other bacteria. By improving our understanding of the A. baumannii cell envelope this work will assist in treating this pathogen

Whole Genome Characterization of the Mechanisms of Daptomycin Resistance in Clinical and Laboratory Derived Isolates of Staphylococcus aureus

Background: Daptomycin remains one of our last-line anti-staphylococcal agents. This study aims to characterize the genetic evolution to daptomycin resistance in S. aureus. Methods: Whole genome sequencing was performed on a unique collection of isogenic, clinical (21 strains) and laboratory (12 strains) derived strains that had been exposed to daptomycin and developed daptomycin-nonsusceptibility. Electron microscopy (EM) and lipid membrane studies were performed on selected isolates. Results: On average, six coding region mutations were observed across the genome in the clinical daptomycin exposed strains, whereas only two mutations on average were seen in the laboratory exposed pairs. All daptomycin-nonsusceptible strains had a mutation in a phospholipid biosynthesis gene. This included mutations in the previously described mprF gene, but also in other phospholipid biosynthesis genes, including cardiolipin synthase (cls2) and CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase (pgsA). EM and lipid membrane composition analyses on two clinical pairs showed that the daptomycin-nonsusceptible strains had a thicker cell wall and an increase in membrane lysyl-phosphatidylglycerol. Conclusion: Point mutations in genes coding for membrane phospholipids are associated with the development of reduced susceptibility to daptomycin in S. aureus. Mutations in cls2 and pgsA appear to be new genetic mechanisms affecting daptomycin susceptibility in S. aureus