High-throughput screening of small-molecules libraries identified antibacterials against clinically relevant multidrug-resistant A. baumannii and K. pneumoniae.

BACKGROUND: The current pipeline for new antibiotics fails to fully address the significant threat posed by drug-resistant Gram-negative bacteria that have been identified by the World Health Organization (WHO) as a global health priority. New antibacterials acting through novel mechanisms of action are urgently needed. We aimed to identify new chemical entities (NCEs) with activity against Klebsiella pneumoniae and Acinetobacter baumannii that could be developed into a new treatment for drug-resistant infections. METHODS: We developed a high-throughput phenotypic screen and selection cascade for generation of hit compounds active against multidrug-resistant (MDR) strains of K. pneumoniae and A. baumannii. We screened compound libraries selected from the proprietary collections of three pharmaceutical companies that had exited antibacterial drug discovery but continued to accumulate new compounds to their collection. Compounds from two out of three libraries were selected using "eNTRy rules" criteria associated with increased likelihood of intracellular accumulation in Escherichia coli. FINDINGS: We identified 72 compounds with confirmed activity against K. pneumoniae and/or drug-resistant A. baumannii. Two new chemical series with activity against XDR A. baumannii were identified meeting our criteria of potency (EC50 ≤50 µM) and absence of cytotoxicity (HepG2 CC50 ≥100 µM and red blood cell lysis HC50 ≥100 µM). The activity of close analogues of the two chemical series was also determined against A. baumannii clinical isolates. INTERPRETATION: This work provides proof of principle for the screening strategy developed to identify NCEs with antibacterial activity against multidrug-resistant critical priority pathogens such as K. pneumoniae and A. baumannii. The screening and hit selection cascade established here provide an excellent foundation for further screening of new compound libraries to identify high quality starting points for new antibacterial lead generation projects. FUNDING: BMBF and GARDP.

Differences in the gut microbiota between Gurkhas and soldiers of British origin.

Previous work indicated that the incidence of travellers' diarrhoea (TD) is higher in soldiers of British origin, when compared to soldiers of Nepalese descent (Gurkhas). We hypothesise that the composition of the gut microbiota may be a contributing factor in the risk of developing TD in soldiers of British origin. This study aimed to characterise the gut microbial composition of Gurkha and non-Gurkha soldiers of the British Army. Recruitment of 38 soldiers (n = 22 Gurkhas, n = 16 non-Gurkhas) and subsequent stool collection, enabled shotgun metagenomic sequencing-based analysis of the gut microbiota. The microbiota of Gurkhas had significantly (P < 0.05) lower diversity, for both Shannon and Simpson diversity indices, using species level markers than the gut microbiota of non-Gurkha soldiers. Non-metric Multidimensional Scaling (NMDS) of the Bray-Curtis distance matrix revealed a significant difference in the composition of the gut microbiota between Gurkhas and non-Gurkha soldiers, at both the species level (P = 0.0178) and the genus level (P = 0.0483). We found three genera and eight species that were significantly enriched in the non-Gurkha group and one genus (Haemophilus) and one species (Haemophilus parainfluenzae) which were enriched in the Gurkha group. The difference in the microbiota composition between Gurkha soldiers and soldiers of British origin may contribute to higher colonization resistance against diarrhoeal pathogens in the former group. Our findings may enable further studies into interventions that modulate the gut microbiota of soldiers to prevent TD during deployment.

Parallel loss of type VI secretion systems in two multi-drug-resistant Escherichia coli lineages.

The repeated emergence of multi-drug-resistant (MDR) Escherichia coli clones is a threat to public health globally. In recent work, drug-resistant E. coli were shown to be capable of displacing commensal E. coli in the human gut. Given the rapid colonization observed in travel studies, it is possible that the presence of a type VI secretion system (T6SS) may be responsible for the rapid competitive advantage of drug-resistant E. coli clones. We employed large-scale genomic approaches to investigate this hypothesis. First, we searched for T6SS genes across a curated dataset of over 20 000 genomes representing the full phylogenetic diversity of E. coli. This revealed large, non-phylogenetic variation in the presence of T6SS genes. No association was found between T6SS gene carriage and MDR lineages. However, multiple clades containing MDR clones have lost essential structural T6SS genes. We characterized the T6SS loci of ST410 and ST131 and identified specific recombination and insertion events responsible for the parallel loss of essential T6SS genes in two MDR clones.

From bench to bedside: 64Cu/177Lu 1C1m-Fc anti TEM-1: mice-to-human dosimetry extrapolations for future theranostic applications.

The development of diagnostic and therapeutic radiopharmaceuticals is an hot topic in nuclear medicine. Several radiolabeled antibodies are under development necessitating both biokinetic and dosimetry extrapolations for effective human translation. The validation of different animal-to-human dosimetry extrapolation methods still is an open issue. This study reports the mice-to-human dosimetry extrapolation of 64Cu/177Lu 1C1m-Fc anti-TEM-1 for theranostic application in soft-tissue sarcomas. We adopt four methods; direct mice-to-human extrapolation (M1); dosimetry extrapolation considering a relative mass scaling factor (M2), application of a metabolic scaling factor (M3) and combination of M2 and M3 (M4). Predicted in-human dosimetry for the [64Cu]Cu-1C1m-Fc resulted in an effective dose of 0.05 mSv/MBq. Absorbed dose (AD) extrapolation for the [177Lu]Lu-1C1m-Fc indicated that the AD of 2 Gy and 4 Gy to the red-marrow and total-body can be reached with 5-10 GBq and 25-30 GBq of therapeutic activity administration respectively depending on applied dosimetry method. Dosimetry extrapolation methods provided significantly different absorbed doses in organs. Dosimetry properties for the [64Cu]Cu-1C1m-Fc are suitable for a diagnostic in-human use. The therapeutic application of [177Lu]Lu-1C1m-Fc presents challenges and would benefit from further assessments in animals' models such as dogs before moving into the clinic.

The highly diverse plasmid population found in Escherichia coli colonizing travellers to Laos and its role in antimicrobial resistance gene carriage.

Increased colonization by antimicrobial-resistant organisms is closely associated with international travel. This study investigated the diversity of mobile genetic elements involved with antimicrobial resistance (AMR) gene carriage in extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli that colonized travellers to Laos. Long-read sequencing was used to reconstruct complete plasmid sequences from 48 isolates obtained from the daily stool samples of 23 travellers over a 3 week period. This method revealed a collection of 105 distinct plasmids, 38.1 % (n=40) of which carried AMR genes. The plasmids in this population were diverse, mostly unreported and included 38 replicon types, with F-type plasmids (n=23) the most prevalent amongst those carrying AMR genes. Fine-scale analysis of all plasmids identified numerous AMR gene contexts and emphasized the importance of IS elements, specifically members of the IS6/IS26 family, in the evolution of complex multidrug resistance regions. We found a concerning convergence of ESBL and colistin resistance determinants, with three plasmids from two different F-type lineages carrying bla CTX-M and mcr genes. The extensive diversity seen here highlights the worrying probability that stable new vehicles for AMR will evolve in E. coli populations that can disseminate internationally through travel networks.

Phosphodiesterase Type 5 Inhibitors and Oral Nitrates in Male Patients with Ischemic Heart Disease.

PURPOSE OF REVIEW: This review sought to define the mechanism of the drug-drug interaction between phosphodiesterase-type-5 (PDE-5) inhibitors and organic nitrates as well as the clinical impact and recommended management across different clinical scenarios. RECENT FINDINGS: This drug-drug interaction results in hemodynamically significant hypotension during episodic PDE-5 use and acute nitrate administration mainly during cardiovascular emergencies with multiple studies describing the expected impact. Chronic co-administration of long-acting nitrates and PDE-5 inhibitors has been observed in practice in a small percentage of patients despite the labeled contraindication without noted adverse effects. Acute nitrate therapy should be avoided in the context of episodic PDE-5 exposure, likely identified through systematic processes. Few data exist defining risk with lower-intensity daily PDE-5 administration. Chronic co-administration is not recommended but may be navigated with careful risk-benefit determination. Future directions also aim to identify potential areas where nitrate synergy may achieve clinical benefit.

RND pumps across the genus Acinetobacter: AdeIJK is the universal efflux pump.

Acinetobacter are generally soil-dwelling organisms that can also cause serious human infections. A. baumannii is one of the most common causative agents of Acinetobacter infections and is often multidrug resistant. However, an additional 25 species within the genus have also been associated with infection. A. baumannii encodes six resistance nodulation division (RND) efflux pumps, the most clinically relevant class of efflux pumps for antibiotic export, but the distribution and types of RND efflux pumps across the genus is currently unknown. Sixty-four species making up the genus Acinetobacter were searched for RND systems within their genomes. We also developed a novel method using conserved RND residues to predict the total number of RND proteins including currently undescribed RND pump proteins. The total number of RND proteins differed both within a species and across the genus. Species associated with infection tended to encode more pumps. AdeIJK/AdeXYZ was found in all searched species of Acinetobacter, and through genomic, structural and phenotypic work we show that these genes are actually homologues of the same system. This interpretation is further supported by structural analysis of the potential drug-binding determinants of the associated RND-transporters, which reveal their close similarity to each other, and distinctiveness from other RND-pumps in Acinetobacter, such as AdeB. Therefore, we conclude that AdeIJK is the fundamental RND system for species in the genus Acinetobacter. AdeIJK can export a broad range of antibiotics and provides crucial functions within the cell, for example lipid modulation of the cell membrane, and therefore it is likely that all Acinetobacter require AdeIJK for survival and homeostasis. In contrast, additional RND systems, such as AdeABC and AdeFGH, were only found in a subset of Acinetobacter that are associated with infection. By understanding the roles and mechanisms of RND efflux systems in Acinetobacter, treatments for infections can avoid efflux-mediated resistance and improve patient outcomes.

Evolutionary Responses to Acquiring a Multidrug Resistance Plasmid Are Dominated by Metabolic Functions across Diverse Escherichia coli Lineages.

Multidrug resistance (MDR) plasmids drive the spread of antibiotic resistance between bacterial lineages. The immediate impact of MDR plasmid acquisition on fitness and cellular processes varies among bacterial lineages, but how the evolutionary processes enabling the genomic integration of MDR plasmids vary is less well understood, particularly in clinical pathogens. Using diverse Escherichia coli lineages experimentally evolved for ~700 generations, we show that the evolutionary response to gaining the MDR plasmid pLL35 was dominated by chromosomal mutations affecting metabolic and regulatory functions, with both strain-specific and shared mutational targets. The expression of several of these functions, such as anaerobic metabolism, is known to be altered upon acquisition of pLL35. Interactions with resident mobile genetic elements, notably several IS-elements, potentiated parallel mutations, including insertions upstream of hns that were associated with its upregulation and the downregulation of the plasmid-encoded extended-spectrum beta-lactamase gene. Plasmid parallel mutations targeted conjugation-related genes, whose expression was also commonly downregulated in evolved clones. Beyond their role in horizontal gene transfer, plasmids can be an important selective force shaping the evolution of bacterial chromosomes and core cellular functions. IMPORTANCE Plasmids drive the spread of antimicrobial resistance genes between bacterial genomes. However, the evolutionary processes allowing plasmids to be assimilated by diverse bacterial genomes are poorly understood, especially in clinical pathogens. Using experimental evolution with diverse E. coli lineages and a clinical multidrug resistance plasmid, we show that although plasmids drove unique evolutionary paths per lineage, there was a surprising degree of convergence in the functions targeted by mutations across lineages, dominated by metabolic functions. Remarkably, these same metabolic functions show higher evolutionary rates in MDR-lineages in nature and in some cases, like anaerobic metabolism, their expression is directly manipulated by the plasmid. Interactions with other mobile elements resident in the genomes accelerated adaptation by disrupting genes and regulatory sequences that they inserted into. Beyond their role in horizontal gene transfer, plasmids are an important selective force driving the evolution of bacterial genomes and core cellular functions.

Management of Iron Deficiency in Heart Failure: A Review of Evidence.

ABSTRACT: Iron deficiency is common in patients with heart failure and has been associated with worse outcomes, including increases in mortality, disease progression, and hospitalizations. As such, several studies have evaluated the role of iron supplementation in mitigating these risks. Evidence for the role of intravenous iron in improving exercise capacity, quality of life, and hospitalizations is promising, although the benefits of oral iron remain less clear. This review will evaluate the literature surrounding iron supplementation in heart failure and provide practical recommendations for its management.

Whole-genome sequencing enhances existing pathogen and antimicrobial-resistance surveillance schemes within a neonatal unit.

In some neonatal units, the screening of isolates for antimicrobial-resistant organisms is a matter of routine, with theoretical benefits including the prevention or early detection of outbreaks. This study sought to use whole-genome sequencing (WGS) retrospectively to characterize the genomic epidemiology of Gram-negative organisms obtained from a screening programme in a 32-bed unit providing intensive, high-dependency and special care at City Hospital, Birmingham, UK, identifying occult transmission events and clinically important antimicrobial-resistance (AMR) genes. WGS was performed for 155 isolates collected from rectal and umbilical screening swabs over a 2 month period from 44 individual neonates. Genomic epidemiological analysis showed possible transmission events involving Escherichia coli, Enterobacter cloacae, Klebsiella oxytoca and Klebsiella pneumoniae not detected by routine screening, with eight putative clusters involving different individuals identified. Within phylogenetic clusters, the relatedness of organisms - as determined by the abundance of SNPs - varied widely, indicating that a variety of transmission routes may be implicated. While clinically important AMR genes were not present in the putative transmission clusters, our observation of suspected interspecies horizontal transfer of blaCTX-M-15 within individuals highlights the potential for their spread between organisms as well as individuals in this environment, with implications for surveillance. Our data show that WGS may reveal occult Gram-negative transmission events, demonstrating the potential of sequencing-based surveillance systems for nosocomial pathogens. Challenges remain in understanding how to utilize WGS surveillance to maximum effect in real-world settings.

SARS-CoV-2 Testing in the Community: Testing Positive Samples with the TaqMan SARS-CoV-2 Mutation Panel To Find Variants in Real Time.

Genome sequencing is a powerful tool for identifying SARS-CoV-2 variant lineages; however, there can be limitations due to sequence dropout when used to identify specific key mutations. Recently, ThermoFisher Scientific has developed genotyping assays to help bridge the gap between testing capacity and sequencing capability to generate real-time genotyping results based on specific variants. Over a 6-week period during the months of April and May 2021, we set out to assess the ThermoFisher TaqMan mutation panel genotyping assay, initially for three mutations of concern and then for an additional two mutations of concern, against SARS-CoV-2-positive clinical samples and the corresponding COVID-19 Genomics UK Consortium (COG-UK) sequencing data. We demonstrate that genotyping is a powerful in-depth technique for identifying specific mutations, is an excellent complement to genome sequencing, and has real clinical health value potential, allowing laboratories to report and take action on variants of concern much more quickly.

Association of colchicine use for acute gout with clinical outcomes in acute decompensated heart failure.

BACKGROUND: Gout is a common comorbidity in heart failure (HF) patients and is frequently associated with acute exacerbations during treatment for decompensated HF. Although colchicine is often used to manage acute gout in HF patients, its impact on clinical outcomes when used during acute decompensated HF is unknown. METHODS: This was a single center, retrospective study of hospitalized patients treated for an acute HF exacerbation with and without acute gout flare between March 2011 and December 2020. We assessed clinical outcomes in patients treated with colchicine for a gout flare compared to those who did not experience a gout flare or receive colchicine. The primary outcome was in-hospital all-cause mortality. RESULTS: Among 1047 patient encounters for acute HF during the study period, there were 237 encounters (22.7%) where the patient also received colchicine for acute gout during admission. In-hospital all-cause mortality was significantly reduced in the colchicine group compared with the control group (2.1% vs. 6.5%, p = .009). The colchicine group had increased length of stay (9.93 vs. 7.96 days, p < .001) but no significant difference in 30-day readmissions (21.5% vs. 19.5%, p = .495). In a Cox proportional hazards model adjusted for age, inpatient colchicine use was associated with improved survival to discharge (hazards ratio [HR] 0.163, 95% confidence interval [CI] 0.051-0.525, p = .002) and a reduced rate of in-hospital CV mortality (HR 0.184, 95% CI 0.044-0.770, p = .021). CONCLUSION: Among patients with a HF exacerbation, treatment with colchicine for a gout flare was associated with significantly lower in-hospital mortality compared with those not treated for acute gout.

Amantadine-induced bilateral corneal edema in a pediatric patient.

Amantadine was originally developed as an antiviral agent for influenza A. However, it also has off-label uses for Parkinson disease, multiple sclerosis, and in the management of extrapyramidal symptoms. The mechanism of action in these conditions has yet to be elucidated. Ocular side effects from systemic amantadine are rare but have been described in three previous reports of amantadine-associated corneal edema in the pediatric population. We present an additional case of amantadine-associated transient visual impairment in a patient, which was associated with significant regression and worsening of his underlying neurodevelopmental status.

A cell-based phenotypic library selection and screening approach for the de novo discovery of novel functional chimeric antigen receptors.

Anti-tumor therapies that seek to exploit and redirect the cytotoxic killing and effector potential of autologous or syngeneic T cells have shown extraordinary promise and efficacy in certain clinical settings. Such cells, when engineered to express synthetic chimeric antigen receptors (CARs) acquire novel targeting and activation properties which are governed and orchestrated by, typically, antibody fragments specific for a tumor antigen of interest. However, it is becoming increasingly apparent that not all antibodies are equal in this regard, with a growing appreciation that 'optimal' CAR performance requires a consideration of multiple structural and contextual parameters. Thus, antibodies raised by classical approaches and intended for other applications often perform poorly or not at all when repurposed as CARs. With this in mind, we have explored the potential of an in vitro phenotypic CAR library discovery approach that tightly associates antibody-driven bridging of tumor and effector T cells with an informative and functionally relevant CAR activation reporter signal. Critically, we demonstrate the utility of this enrichment methodology for 'real world' de novo discovery by isolating several novel anti-mesothelin CAR-active scFv candidates.

Arm race among closely-related carbapenem-resistant Klebsiella pneumoniae clones.

Multiple carbapenem-resistant Klebsiella pneumoniae (CRKP) clones typically co-exist in hospital wards, but often certain clones will dominate. The factors driving this dominance are largely unclear. This study began from a genomic epidemiology analysis and followed by multiple approaches to identify the potential mechanisms driving the successful spread of a dominant clone. 638 patients in a 50-bed ICU were screened. 171 (26.8%) and 21 had CRKP from swabs and clinical specimens, respectively. Many (39.8% of those with ≥7-day ICU stay) acquired CRKP. After removing 18 unable to recover, 174 CRKP isolates were genome sequenced and belonged to six sequence types, with ST11 being the most prevalent (n = 154, 88.5%) and most (n = 169, 97.1%) carrying blaKPC-2. The 154 ST11 isolates belonged to 7 clones, with one (clone 1, KL64 capsular type) being dominant (n = 130, 84.4%). Clone 1 and the second-most common clone (clone 2, KL64, n = 15, 9.7%) emerged simultaneously, which was also detected by genome-based dating. Clone 1 exhibited decreased biofilm formation, shorter environment survival, and attenuated virulence. In murine gut, clone 1 outcompeted clone 2. Transcriptomic analysis showed significant upregulation of the ethanolamine operon in clone 1 when competing with clone 2. Clone 1 exhibited increased utilization of ethanolamine as a nitrogen source. This highlights that reduced virulence and enhanced ability to utilize ethanolamine may promote the success of nosocomial multidrug-resistant clones.

Copper-64-Labeled 1C1m-Fc, a New Tool for TEM-1 PET Imaging and Prediction of Lutetium-177-Labeled 1C1m-Fc Therapy Efficacy and Safety.

1C1m-Fc, a promising anti-TEM-1 DOTA conjugate, was labeled with 64Cu to target cancer cells for PET imaging and predicting the efficacy and safety of a previously studied [177Lu]Lu-1C1m-Fc companion therapy. DOTA-conjugated 1C1m-Fc was characterized by mass spectrometry, thin layer chromatography and immunoreactivity assessment. PET/CT and biodistribution studies were performed in human neuroblastoma xenografted mice. Absorbed doses were assessed from biodistribution results and extrapolated to 177Lu based on the [64Cu]Cu-1C1m-Fc data. The immunoreactivity was ≥ 70% after 48 h of incubation in serum, and the specificity of [64Cu]Cu-1C1m-Fc for the target was validated. High-resolution PET/CT images were obtained, with the best tumor-to-organ ratios reached at 24 or 48 h and correlated with results of the biodistribution study. Healthy organs receiving the highest doses were the liver, the kidneys and the uterus. [64Cu]Cu-1C1m-Fc could be of interest to give an indication of 177Lu dosimetry for parenchymal organs. In the uterus and the tumor, characterized by specific TEM-1 expression, the 177Lu-extrapolated absorbed doses are overestimated because of the lack of later measurement time points. Nevertheless, 1C1m-Fc radiolabeled with 64Cu for imaging would appear as an interesting radionuclide companion for therapeutic application with [177Lu]Lu-1C1m-Fc.

Optimizing anticoagulation for patients receiving Impella support.

Anticoagulation of patients treated with the Impella percutaneous mechanical circulatory support (MCS) devices is complex and lacks consistency across centers, potentially increasing the risk of complications. In order to optimize safety and efficacy, an expert committee synthesized all available evidence evaluating anticoagulation for patients receiving Impella support in order to provide consensus recommendations for the management of anticoagulation with these devices. The evidence synthesis led to the creation of 42 recommendations to improve anticoagulation management related to the use of the Impella devices. Recommendations address purge solution management, intravenous anticoagulation, monitoring, evaluation and management of heparin-induced thrombocytopenia (HIT), and management during combination MCS support. The use of a heparinized, dextrose-containing purge solution is critical for optimal device function, and a bicarbonate-based purge solution may be an alternative in certain situations. Likewise, intravenous (ie, systemic) anticoagulation with heparin is often necessary, although evidence supporting the optimal assay and target range for monitoring the level of anticoagulation is generally lacking. Patients treated with an Impella MCS device may develop HIT, which is more difficult to evaluate and treat in this setting. Lastly, the use of Impella with extracorporeal membrane oxygenation or for biventricular support creates additional anticoagulation challenges.

Soluble trivalent engagers redirect cytolytic T cell activity toward tumor endothelial marker 1.

Tumor endothelial marker 1 (TEM1) is an emerging cancer target with a unique dual expression profile. First, TEM1 is expressed in the stroma and neo-vasculature of many human carcinomas but is largely absent from healthy adult tissues. Second, TEM1 is expressed by tumor cells of mesenchymal origin, notably sarcoma. Here, we present two fully human anti-TEM1 single-chain variable fragment (scFv) reagents, namely, 1C1m and 7G22, that recognize distinct regions of the extracellular domain and possess substantially different affinities. In contrast to other, well-described anti-TEM1 binders, these fragments confer cytolytic activity when expressed as 2nd generation chimeric antigen receptors (CARs). Moreover, both molecules selectively redirect human T cell effector functions toward TEM1+ tumor cells when incorporated into experimental soluble bispecific trivalent engagers that we term TriloBiTEs (tBs). Furthermore, systemic delivery of 1C1m-tB prevents the establishment of Ewing sarcoma tumors in a xenograft model. Our observations confirm TEM1 as a promising target for cancer immunotherapy and illustrate the prospective translational potential of certain scFv-based reagents.

Validation testing to determine the sensitivity of lateral flow testing for asymptomatic SARS-CoV-2 detection in low prevalence settings: Testing frequency and public health messaging is key.

Lateral flow devices (LFDs) are quickly being implemented for use in large-scale population surveillance programs for SARS-CoV-2 infection in the United Kingdom. These programs have been piloted in city-wide screening in the city of Liverpool and are now being rolled out to support care home visits and the return home of University students for the Christmas break. Here, we present data on the performance of LFDs to test almost 8,000 students at the University of Birmingham between December 2 and December 9, 2020. The performance is validated against almost 800 samples using PCR performed in the University Pillar 2 testing lab and theoretically validated on thousands of Pillar 2 PCR testing results performed on low-prevalence care home testing samples. Our data show that LFDs do not detect infections presenting with PCR Ct values over 29 to 30 as determined using the Thermo Fisher TaqPath asssay. This may be of particular importance in detecting individuals that are either at the early, or late stages of infection, and reinforces the need for frequent, recurrent testing.