bluuebunny/crossref_metadata_2025 huggingface dataset:
Improving shared decision-making for prosthetic care: A qualitative needs assessment of prosthetists and new lower-limb prosthesis users
Authors: Anderson Chelsey B. , Fatone Stefania , Mañago Mark M. , Swink Laura A. , Hager Emily R. , Kittelson Andrew J. , Christiansen Cory L. , Magnusson Dawn M.
Published: May 2022

Background:

Prosthesis design is complex and multiple appropriate options exist for any individual with lower-limb amputation. However, there is insufficient evidence for guiding decision-making. Shared decision-making (SDM) offers an opportunity to incorporate patient-specific values and preferences where evidence is lacking for prosthesis design decisions. To develop resources to facilitate SDM, and consistent with the International Patient Decision Aid Standards, it is necessary to identify the decisional needs of prosthetists and prosthesis users for prosthesis design decisions.

Objectives:

To assess the needs of prosthetists and new prosthesis users for SDM about the first prosthesis design.

Study design:

Qualitative descriptive design.

Methods:

Six focus groups were conducted with 38 prosthetists. Individual semistructured interviews were conducted with 17 new prosthesis users. Transcripts were analyzed using directed content analysis, with codes defined a priori using existing frameworks for SDM: the Three Talk Model for SDM and the Ottawa Decision Support Framework.

Results:

Four main themes were identified among prosthetists and prosthesis users: acknowledging complexity in communication, clarifying values, recognizing the role of experience to inform preferences, and understanding the prosthetic journey.

Conclusions:

Resources that support SDM for the first prosthesis design should consider methods for identifying individual communication needs, support with clarifying values, and resources such as experience for achieving informed preferences, within the context of the overall course of rehabilitation and recovery following lower-limb amputation. The themes identified in this work can inform SDM to promote collaborative discussion between prosthetists and new prosthesis users when making prosthesis design decisions.
68Ga-DOTA-FAPI-04 PET/CT Imaging of Tracheal Mucoepidermoid Carcinoma
Authors: Peng Dengsai , Chen Qian , Cao Jianpeng , Huang Yilin , Chen Yue
Published: May 2022

A 23-year-old woman presented with a dry cough and dyspnea. Contrast-enhanced CT revealed an intratracheal space-occupying lesion with continuous homogeneous enhancement and airway stenosis. The patient then underwent 68Ga-DOTA-FAPI-04 PET/CT, which showed increased uptake of FAPI-04 by the lesion. Postoperative pathology confirmed the lesion as tracheal mucoepidermoid carcinoma. This case reports a rare site of mucoepidermoid carcinoma and highlights the potential utility of 68Ga-DOTA-FAPI-04 PET/CT for the diagnosis of tracheal mucoepidermoid carcinoma.
Unexpected Detection of Skeletal Muscle Renal Cell Carcinoma Metastasis With 99mTc-EDDA/HYNIC-Tyr3-Octreotide (Tektrotyd) Scan
Authors: Fuoco Valentina , Barisella Marta , Lorenzoni Alice , Verzoni Elena , Maccauro Marco
Published: May 2022

Somatostatin receptor scintigraphy with 99mTc-Tektrotyd is widely used for the investigation of neuroendocrine tumors. Overexpression of somatostatin receptors has been shown in different tumor types including lymphomas, breast carcinoma, and renal cell carcinoma (RCC). Isolated case reports have shown that RCC metastases can be identified using somatostatin receptor imaging such as Octreoscan scintigraphy and 68Ga-DOTATATE PET/CT. We report the case of a 70-year-old man with a history of surgically removed RCC who referred to 99mTc-Tektrotyd scintigraphy for the evaluation of a pancreatic tail lesion. The scan revealed intense tracer uptake in a left splenius cervicis muscle lesion that on biopsy was consistent with metastatic RCC.
Hypermetabolic Esophagitis Dessicans Superficialis Mimicking Esophageal Cancer on FDG PET/CT
Authors: Mamaliga Tatiana , Liu Yiyan
Published: May 2022

An 83-year-old man with history of multiple myeloma obtained an FDG PET/CT for surveillance. Although there was no evidence of metabolically active myeloma, an incidental highly FDG-avid focus was noted at the gastroesophageal junction. Findings were interpreted as malignancy. The patient underwent esophagogastroduodenoscopy, which showed nonerythematous desquamation of the distal esophagus consistent with esophagitis dessicans superficialis (EDS) without any evidence of malignancy. There is paucity of literature regarding EDS and its image findings. This case illustrates an instance of focal mass-like uptake on FDG PET/CT, which represents an endoscopy- and pathology-confirmed EDS and mimics esophageal cancer.
Liver Outcome in Renal Transplant Recipients Who Acquired Hepatitis C Infection From an Infected Graft: Study Based on Liver Biopsy Findings
Authors: Kothadia Jiten P. , Bhalla Anshul , Molnar Miklos Z. , Mohan Rahul , Balaraman Vasanthi , Talwar Manish , Helmick Ryan , Eymard Corey , Clark Ian , Jain Richa , Faust Thomas W. , Vanatta Jason M. , Eason James D. , Nair Satheesh P.
Published: May 2022

Background.

Long-term liver outcome in hepatitis C virus (HCV)-negative kidney recipients who acquired HCV infection from viremic donors is of intense interest in the transplant community. We evaluated the incidence of fibrosis in liver biopsy specimens of recipients who were transplanted with HCV-infected grafts.

Methods.

Patients were evaluated in the hepatology clinic, and 29 patients agreed to undergo liver biopsy. The liver histology was scored by the meta-analysis of histological data in viral hepatitis scoring system and was assessed by hepatopathologists. The fibrosis score was compared between patients who initiated direct-acting antiviral (DAA) within 6 wk (n = 6) and after 6 wk (n = 29).

Results.

Eighty-nine aviremic patients were transplanted with HCV-infected grafts between March 2018 and October 2019. All patients developed HCV infection and were treated with DAA treatment after kidney transplantation (median, 70 d; interquartile range, 55–85 d). All patients (n = 89) achieved sustained virologic response with DAA. The median follow-up time from kidney transplant to liver biopsy was 28 mo (interquartile range, 26–30 mo). Twenty-five patients (86%) had F0, and 4 patients (14%) had F1 fibrosis. No patient had advanced fibrosis (F3–F4). Grade 1 inflammation was present in 6 (21%) patients, whereas 26 (90%) patients had iron accumulation in the hepatocytes and reticuloendothelial cells. There was no difference in the fibrosis score between patients who received treatment within 6 wk versus after 6 wk (P = 0.55).

Conclusions.

Kidney transplantation of HCV-infected graft to HCV-negative recipients is safe and has no long-term liver-related complications with successful eradication of HCV. In our cohort, delayed treatment did not affect sustained virologic response or liver histology.
Increasing the Cognitive Screening Efficiency of Global Phase III Trials in Early Alzheimer Disease
Authors: Doherty Thomas , Gee Michelle , Maruff Paul , Smith Robert , Murphy Jennifer , Marsh Julie , Koschalka Luke , Martinez Mairelys , Irizarry Michael , Albala Bruce
Published: May 2022

Purpose:

A Cognitive Task Force (CTF) was established for the MissionAD program with the aim of reducing the screen failure (SF) rate to ∼30% and thereby reduce unnecessary subject burden, site burden, and excess trial costs.

Methods/Subjects:

The MissionAD program consisted of 2 global phase 3 studies evaluating the BACE inhibitor elenbecestat in subjects with early Alzheimer disease. The CTF monitored and engaged with MissionAD clinical sites to provide support through collegial discussions to maximize the efficiency of the preconsent recruitment phase.

Results:

The CTF significantly improved cognitive screening efficiency in the MissionAD program, with a 24% decline in cognitive SF rate for the sites that the CTF contacted. The study-wide 11.5% reduction in cognitive SF rates were likely further driven by wider country-level initiatives in which CTF members held CTF-specific Investigator meetings with the recruitment staff, speaking to all sites on a country level regardless of their recruitment performance.

Conclusions:

The establishment of a CTF to support efficient cognitive screening is highly recommended for future Alzheimer disease studies. Additional benefits included improved site relationships, increased engagement in MissionAD and access to a group of cognitive experts for consulting, with a focus on achieving more efficient trial recruitment.
Detection of Extended Spectrum Beta-lactamase Gene (CTX-M) among Representative Multidrug-Resistant Gram Negative Bacterial Isolates from Patients with Urinary Tract Infection in Ekiti State, Nigeria
Authors: Ajayi Ayodele Oluwaseun , Osanyinlusi Samuel Ayodeji , Ojerinde Amos , Ogeneh Bryan
Published: May 2022

Urinary tract infection is huge public health burden and the emergence of extended spectrum beta lactamase producing bacterial pathogens increases the burden of infectious diseases in Nigeria. This study determined the current prevalence of cephalosporin resistance among Gram-negative bacteria isolated from patients with urinary tract infections between February 2018 and June 2018. Non-repetitive Gram–negative bacteria were recovered from 106 individuals with urinary tract infections who reported at two tertiary healthcare centers in Ekiti-State, Nigeria. A total of 106 bacterial isolates were obtained which included: Klebsiella pneumoniae 34 (29.1%), Klebsiella oxytoca 17 (16.0%), Proteus vulgaris 10 (9.4%), E. coli 24 (22.6%), Proteus mirabilis 18 (16.9%) and Pseudomonas aeruginosa 3 (2.8%). Sixty five of these organisms showed resistance to ceftazidime while 76 organisms showed resistance to ceftriaxone. Forty representative organisms were selected and tested for presence of extended spectrum beta-lactamase (ESBL) genes using primers specific for different ESBL genes. A total of eight (20.0%) organisms carried the blaCTX-M gene and other variants of the ESBL genes were not detected. The organisms carrying the blaCTX-M gene included E. coli 3 (37.5%), K. pneumoniae 1(12.5%), P. mirabilis 1(12.5%),) and K. oxytoca 3(37.5%). The high prevalence of cephalosporin resistant Gram-negative bacteria among patients with UTI is a serious threat to public health and efforts must be intensified to regulate the clinical use of the cephalosporins.
Phenotypic whole-cell screening identifies a protective carbohydrate epitope on Klebsiella pneumoniae
Authors: Berry Sophia , Caceres Carolina , Rust Steven , Minter Ralph , Grant Andrew
Published: May 2022

The increasing occurrence of recalcitrant multi-drug resistant (MDR) Klebsiella pneumoniae infections coupled with a diminishing pipeline of new antibioticswarrants the investigation of alternative antimicrobial therapies. We employed a target-agnostic phage display approach using live K. pneumoniaebacteria with the aim of isolating therapeutic monoclonal antibodies (mAbs) targetingconserved epitopes among clinically relevant strains. mAb targets were explored using ELISA and biolayer interferometry, and a high-throughput opsonophagocytic killing assay was developed to determine functional activity. Fluorescence-activated cell sorting was used to screen a global panel of clinical isolates, and high-content imaging further explored binding and functional activity. One mAb was tested in vivousing a lethal murine model of pneumonia. mAbs binding to carbohydrate epitopes were isolated in phage display selections enriched on wild-type and capsule-deficient strains. mAbs binding O1 lipopolysaccharide (LPS) and cross-binding O1/O2 LPS were identified. mAbs were shown to promote opsonophagocytic killing by human monocyte-derived macrophages, and clearance of macrophage-associated bacteria. One mAb, named B39, protected mice against MDR O1 and O2 strains when dosed therapeutically in a murine pneumonia model. Binding to a panel of O1 and O2 clinical isolates suggests B39 binds to both d-galactan-I and d-galactan-II of the LPS. With the rise of antimicrobial resistance among enteric pathogens, the discovery of a novel therapeutic mAb targeting the most prevalent K. pneumoniaeserotypes demonstrates a significant advancement in the field, and showcases the potential of alternative antimicrobial therapies for the treatment of MDR infections.
Use of long read sequencing to characterise the genomic architecture of mobile genetic elements encoding blaCTX-M-15 in Escherichia coli causing travellers’ diarrhoea
Authors: Bird Matthew , Nair Satheesh , Greig David , Dallman Timothy , Gharbia Saheer , Jenkins Claire
Published: May 2022

Increasing levels of antimicrobial resistance (AMR) have been documented in Escherichia colicausing travellers’ diarrhoea, particularly to the third-generation cephalosporins. Diarrhoeagenic E. coli (DEC) can act as a reservoir for the exchange of AMR genes between bacteria residing in the human gut, enabling them to survive and flourish through the selective pressures of antibiotic treatments.

Using Oxford Nanopore Technology (ONT), we sequenced eight different sequence types (ST) belonging to five different pathotypes of extended-beta-lactamase-producing DEC harbouring blaCTX-M-15from four patients recently returned to the UK from Pakistan. The aim of the study was to determine whether blaCTX-M-15 was chromosome or plasmid-encoded to better understand the mechanisms of onward transmission of AMR determinants.

In Patient A, blaCTX-M-15was plasmid-encoded in both DEC isolates (ST504/ST3032), whereas in Patient B blaCTX-M-15was located on the chromosome in both DEC isolates (ST227/1283). Patients C and D both had one isolate where blaCTX-M-15 was located on the plasmid and one chromosomally encoded (ST443/182 and ST38/99, respectively). The two plasmids associated with Patient A were different although one exhibited high similarity to the plasmid from Patient C. In the four isolates where blaCTX-M-15 was chromosomally encoded, the site of insertion and the characteristics of the inserted plasmid segment differed.

Analysis of long-read sequencing data enables us to characterise the genomic architecture of mobile genetic elements encoding AMR determinants. These data may contribute to a better understanding of persistence and onward transmission of AMR determinants in MDR E. coli causing gastrointestinal and extra-intestinal infections.
Systematic Reconstruction of the Complete Two-Component Sensorial Network in Staphylococcus aureus
Authors: Haag Andreas , Rapun-Araiz Beatriz , Cesare Virginia De , Dorado-Morales Pedro , Penadés José , Lasa Iñigo
Published: May 2022

In bacteria, adaptation to changes in the environment is mainly controlled through two-component signal transduction systems (TCSs). Most bacteria contain dozens of TCSs, each of them responsible for sensing a different range of signals and controlling the expression of a repertoire of target genes (regulon). Frequently, TCS control key physiological changes required for pathogenesis and/or antimicrobial resistance. Over the years, identification of the regulon controlled by each individual TCS in different bacteria has been a recurrent question. However, limitations associated with the classical approaches used have left our knowledge far from complete. In this report, using a pioneering approach in which a strain devoid of the complete nonessential TCS network was systematically complemented with the constitutively active form of each response regulator, we have reconstituted the regulon of each TCS of S. aureus in the absence of interference between members of the family. Transcriptome sequencing (RNA-Seq) and proteomics allowed us to determine the size, complexity, and insulation of each regulon and to identify the genes regulated exclusively by one or many TCSs. This gain-of-function strategy provides the first description of the complete TCS regulon in a living cell, which we expect will be useful to understand the pathobiology of this important pathogen.
Comparison of genome-derived and phenotypic antimicrobial resistance profiles of Shigella species isolated from patients with symptoms of gastrointestinal disease in England, 2015-2020
Authors: Poh Ching-Ying , Gentle Amy , Jenkins Claire
Published: May 2022

Increasing antimicrobial resistance (AMR) in Shigellaspecies is a global public health problem. We compared genotypic and phenotypic methods for the detection of AMR in Shigellaspecies to evaluate the use of genome data for surveillance and monitoring of emerging AMR.

Whole genome sequencing (WGS) data from 388 isolates of all four Shigella species were analysed for the presence/absence of specific AMR determinants and selected accordingly. Phenotypic antimicrobial susceptibility testing was performed using in-agar dilution on all viable and pure isolates (n=358). The genotypic and phenotypic AMR profiles were then compared.

There were 335 (93.6%) isolates resistant to at least one antimicrobial and 222 (62%) isolates were multi-drug resistant, of which the majority (77%) were associated with foreign travel. Out of a possible 2864 isolate/antimicrobial class combinations, we identified 119 unexpected results, giving an overall concordance of 96.8% between the two methods. There were 54 samples that had an AMR determinant expected to confer resistance that were phenotypically susceptible, of which 31/54 (57.4%) were associated with tetracycline resistance and trimethoprim-sulfamethoxazole resistance. There were 65 that were phenotypically resistant to a specific antimicrobial class, but no AMR determinant was detected, of which 32/65 (49.2%) were associated with isolates harbouring a single gyrA mutation and exhibiting an unexpectedly high minimum inhibitory concentration (MIC) to ciprofloxacin.

Although comparisons between both methods showed good correlation between the genotypic and phenotypic AMR profiles, phenotypic monitoring is required to identify novel AMR mechanisms and to update reference database used for WGS analysis.
Bioinformatic exploration of trimethylamine N-oxide metabolism in human gut bacteria
Authors: Dawson Samuel , Hoyles Lesley
Published: May 2022

Trimethylamine N-oxide (TMAO) is a microbial metabolite that has been shown to have protective effects on the blood–brain barrier, while elevated serum levels of TMAO and its precursors have been linked to cardiometabolic diseases in Western populations. Previous work examined the prevalence of TorA to determine which groups of bacteria were responsible for the metabolism of TMAO in the human gut. This study examined 6 TMAO metabolism pathways to provide a more in-depth analysis of bacterial TMAO metabolism. These results were then filtered for hits with >90% coverage and >70% identity.

Results showed that Tor proteins were largely limited to members of the Enterobacteriaceae, mostly appearing in Escherichia coli and Citrobacter spp. >1% of 9898 Klebsiella spp. genomes examined encode any Tor proteins, despite previous work highlighting Klebsiella spp. as one of the prevalent genera encoding TorA. Dms proteins were much more prevalent than TorA in other genera of bacteria, along with MsrP and BisC. 118 of the HGRGs were found to encode for at least 1 TMAO metabolism protein.

Overall, this work highlights the need for more comprehensive methods to be used to examine large genomic and metagenomic datasets and the need for in vitro work to be done alongside in silico analyses to improve functional annotations and our understanding of the roles of gut bacteria.
Recycling-derived Phosphorus Fertilizers as a Sustainable Alternative to Triple Superphosphate Fertilizers
Authors: Deinert Lea , Ikoyi Israel , Schmalenberger Achim
Published: May 2022

Microbial activity is adamant for the nutrient cycling in soil. Generally, mineral phosphorus (P) fertilizer is applied to soil to improve plant growth, however, significant amounts are immobilized quickly. Mineral fertilizer can cause soil degradation, affecting the microbial community. Alternative, recycling-derived fertilizers (RDFs) need to be evaluated as suitable replacement for finite mineral P fertilizer. The impact of four RDFs (two ashes, two struvites) on the soil microbiome in comparison with a P-free control and triple superphosphate (TSP) as mineral fertilizer was investigated in a pot trial and a subsequent microcosm trial (subset of samples). For both experiments, perennial ryegrass was cultivated for 54 days. The pot trial was conducted at P fertilization rates of 20 and 60 kg P ha-1 in quadruplicates. After the pot harvest, the bulk soil was stored until the microcosm trial was conducted, using the control, TSP and the two ashes at 60 kg P ha-1 in six replicates. Struvites displayed highest P bioavailability at high P application rates in the pot trial, yielding higher biomass on average. Furthermore, P solubilization from tri-calcium phosphate was enhanced in the RDFs treatments, while the TSP treatments were negatively affected. For the microcosm trial, most probable number (MPN) analysis showed that phytate-utilizing bacterial abundance was significantly increased in one of the ashes. Non-metric multidimensional scaling (NMDS) analysis of phoD illumina sequencing data showed significant separation between all treatments of the microcosm trial. Understanding the impact of RDF application on the soil P cycle is vital to sustainable agriculture.
Phenotypic diversity of a clonal Ralstonia solanacearum pathogen lineage is explained by accessory genome variation
Authors: Farnham Evie , Stoycheva Martina , Elphinstone John , Jeffares Daniel , Friman Ville
Published: May 2022

Ralstonia solanacearumis aplant pathogenic gram-negative bacterium capable of infecting several economically important crops such as potato and tomato. It can also persist in environmental reservoirs including soils, rivers and in asymptomatic wild hosts, causing disease outbreaks during pathogen spillover events when crossing agroecological interface. In the UK, R. solanacearum outbreaks originate from Solanum dulcamarawild hosts (woody nightshade) and river networks. To what extent selection in these natural environments drive R. solanacearumsurvival and life history evolution including virulence is unknown. To study this, we focused on a largely clonal R. solanacearum lineage inhabiting river networks across the UK consisting of a collection of 182 isolates spanning 30 years since the first outbreak in 1992. We first characterised strains phenotypically regarding 32 traits including resource catabolism, virulence and abiotic stress tolerance and then used microbial GWAS techniques to identify links between phenotypic traits and the presence of specific accessory genes. We found that isolates can be clustered into three phenotypic groups, which differed clearly regarding their resource specialism and stress tolerance. No effect of isolation location was found. However, isolates became more variable phenotypically along with time. While only few SNPs were found to vary among all isolates, the presence and absence of certain accessory genes, such asS-layer family protein,could be associated with phenotypic variation in terms ofsurvival in stressful environments. Together, our findings suggest that a low number of accessory genes can cause high phenotypic variability within highly clonal bacterial lineage.
Identifying long-term colonization factors of Vibrio cholerae O1 El Tor in the zebrafish natural host model
Authors: Walton Madison G. , Severin Geoffrey B. , Rhoades Christopher R. , Waters Christopher M. , Withey Jeffrey H.
Published: May 2022

The human disease cholera, marked by acute, voluminous watery diarrhea, is caused by the gram-negative, aquatic bacterium Vibrio cholerae. All seven cholera pandemics since 1817 were identified as being caused by just 2 of over 155 known V. choleraeserogroups: O1 and O139. The O1 serogroup is divided into two biotypes: classical and El Tor. Classical biotype is associated with pandemics 1 through 6, but the El Tor biotype has since displaced classical as the causative agent of the ongoing 7th cholera pandemic over the past 60 years. The El Tor genome resembles that of classical but has acquired two unique pathogenicity islands known as Vibrio Seventh Pandemic (VSP) -1 and -2. El Tor biotype has been associated with prolonged colonization, infection, and disease both in humans and in the zebrafish natural host model. The zebrafish model allows for complete observation of Vibrio cholerae infection in a system undisrupted by antibiotic use or immune suppression. El Tor strains colonize the zebrafish intestine for up to 10 days longer than classical strains. Preliminary studies demonstrate VSP-2 is required to observe this phenotype, but VSP-1 is not. By creating targeted regional knockouts of the VSP-2 island, the specific gene(s) essential for enabling prolonged colonization will be identified and applied to the understanding of how El Tor interacts with a natural host. By identifying the genes used by El Tor to colonize a natural host for prolonged periods, we gain insight into how this pathogen may persist in the environment and perpetuate disease.
Transcriptional profiles of Streptococcus pneumoniae associated with adaptation to the nasopharynx environment
Authors: Barton Thomas E , Green Angharad E , Neill Daniel R
Published: May 2022

Streptococcus pneumoniae is a major cause of global morbidity and mortality. It behaves as a commensal in the host nasopharynx, but can become pathogenic, invading the lungs, blood, and meninges. As such, identification of pneumococcal virulence and colonisation factors remains a major research objective. We previously described an experimental evolution approach for the identification of pneumococcal genes that make niche-specific contributions to fitness and virulence. Sequential passage of pneumococci through mouse models of nasopharyngeal-carriage and pneumonia was performed, generating bacterial lineages adapted to the nasopharynx and lungs, respectively.

Using RNA-Seq differential gene expression analysis, this study compared the transcriptomic profile of a nasopharynx-evolved pneumococcal lineage that showed evidence of enhanced nasopharyngeal colonisation potential, with a lab-adapted ancestor strain. Here, we describe how the genomic adaptations acquired by this lineage, and which we have demonstrated facilitate survival in the nasopharynx, can influence bacterial gene expression.

One key finding was the identification of five adjacent upregulated genes, representing a putative pneumococcal operon. These poorly characterised genes are predicted to encode a carbohydrate-scavenging pathway. Expression of the operon within the nasopharynx may facilitate sugar acquisition from host glycoproteins. Of note, the nasopharynx evolved pneumococcal lineage carries a single nucleotide insertion mutation immediately adjacent to the -10 element of the operon predicted promotor sequence. This may contribute to increased operon gene expression in the nasopharynx-adapted pneumococcus, thereby enhancing colonisation potential. Confirmatory mechanistic investigations are underway, which will aid the identification of pneumococcal virulence factors involved in the commensal to pathogen switch.
Exploring bacterial diversity via a curated and searchable snapshot of archived DNA
Authors: Blackwell Grace , Hunt Martin , Lima Leandro , Malone Kerri , Horesh Gal , Alako Blaise , Iqbal Zamin , Thomson Nicholas
Published: May 2022

The open sharing of genomic data provides an incredibly rich resource for the study of bacterial evolution and function, and even anthropogenic perturbations such as the widespread use of antimicrobials. Whilst these archives are rich in data, considerable processing is required before a biological question can be addressed. Here, we have assembled, quality controlled and characterised 661,405 bacterial genomes that were in the European Nucleotide Archive (ENA) at the end of November of 2018, using a uniform standardised approach. A searchable index has been produced, facilitating the easy interrogation of the entire dataset for a specific gene or mutation. Our analysis shows how uneven the species composition is within this database, with just 20 of the total 2,336 species making up 90% of the high-quality genomes. The over-represented species tend to be acute/common human pathogens, often aligning with research priorities at different levels from individuals with targeted but focussed research questions, areas of focus for the funding bodies or national public health agencies, to those identified globally as priority pathogens by the WHO for their resistance to front- and last-line antimicrobials. Whilst this is a rich resource which often forms the context or references for multi-‘omic’ studies and supports discovery research in many domains, understanding the actual and potential biases in bacterial diversity depicted in this snapshot, and hence within the data being submitted to the public sequencing archives, is essential if we are to target and fill gaps in our understanding of the bacterial kingdom.
Genomic diversity of Bacillus anthracis in endemic settings: novel approaches and data
Authors: Forde Taya , Dennis Tristan , Hassim Ayesha , Kiwelu Ireen , Medvecky Matej , Mmbaga Blandina T. , Mshanga Deogratius , Heerden Henriette Van , Vogel Adeline , Zadoks Ruth N. , Lembo Tiziana , Biek Roman
Published: May 2022

Anthrax, caused by the spore-forming bacterium Bacillus anthracis, remains endemic in many developing countries where it has significant impacts on the health and livelihoods of livestock-keeping communities. While the global genomic diversity of B. anthracisis is well characterised, few studies have quantified its diversity in endemic settings at more local scales, where this information could be critical for elucidating transmission dynamics and guiding control efforts. We collected samples from 526 anthrax-suspected animal carcasses between 2016 and 2018 in the Ngorongoro Conservation Area in northern Tanzania. Seventy five percent were confirmed positive through qPCR, suggesting that anthrax is responsible for a large proportion of sudden deaths in livestock in this area. A subset of positive samples were cultured for whole genome sequencing (n = 73), including multiple isolates from individual carcasses. All sequenced isolates belonged to the Ancient A lineage of B. anthracis, a common strain in southeastern Africa. No clear spatial clustering was observed, possibly reflecting extensive animal movement related to local nomadic practices. Moreover, high levels of within-host diversity were observed which suggests that cases commonly result from simultaneous infection with multiple strains. Additionally, we trialed a targeted sequence capture approach on 93 samples. This was successful in recovering >80% of the chromosomal genome at > 15X coverage from 60% of samples tested, thus representing a valuable tool for culture-free sequencing of this high-risk bacterium. Our work paves the way for integrating genomic data for B. anthracis into epidemiological studies and monitoring of control programs in endemic areas worldwide.
Immunoproteomic analysis of surface proteins in bovine-adapted lineages of Staphylococcus aureus
Authors: Drumm Shauna D , Cormican Paul , Owens Rebecca , Mitchell Jennifer , Keane Orla M.
Published: May 2022

In Ireland, Staphylococcus aureus is the most frequent cause of bovine intramammary infection (IMI) with the bovine-adapted lineages CC151 and CC97 most commonly found. While bovine mastitis vaccines are available that target S. aureus, this pathogen is still a significant source of infection in dairy cows. Therefore, there’s a clear need for a more effective vaccine against S. aureus. However, S. aureus is considered a clonal organism, therefore identifying new potential protein targets common to all major lineages is an important step for vaccine design.

Two S. aureus strains, one belonging to ST151 and another to ST3170 (CC97), were used to infect two groups of dairy cows. Antibodies raised by individual cows were used to identify immunodominantsurface proteins for each strain. One-dimensional serum blotting determined that the antibody response to S. aureus infection was largely strain specific, and to a lesser extent, animal specific.

Two-dimensional serum blotting followed by mass spectrometry of immunoreactive spots was used to identify potential vaccine candidates that were immunodominant for both strains. These blots showed that proteins to which an antibody response was generated in the ST151 infected cows were generally different to those generated in the ST3170 infected cows. However, mass spectrometry also identified immunoreactive proteins common to both strains including Clumping factor B and Iron-regulated surface determinant protein A. Common immunoreactive secreted proteins are also currently being identified.

This study will identify immunodominant proteins expressed by the bovine-adapted strains ST3170 and ST151 that could potentially be used as candidates in vaccine research.
Non-typeable Haemophilus influenzae biofilm formation on primary human airway epithelia cultured at air-liquid interface
Authors: Horton Katie L , Coles Janice L , Hueppe Jana F , Cleary David W , Jackson Claire L , Lucas Jane S , Allan Raymond N
Published: May 2022

Introduction

Primary ciliary dyskinesia (PCD) is an inherited heterogeneous disorder associated with defective motile cilia on airway epithelia, resulting in impaired mucociliary function and ultimately, bronchiectasis. Non-typeable Haemophilus influenzae (NTHi) is frequently isolated from PCD airways where it resides in biofilms. It is unclear if biological pathways in response to NTHi infection differ in PCD, contributing to increased susceptibility to biofilm-associated infection.

Aim

The development of a biologically representative co-culture model to investigate the interactions between PCD airway epithelia and NTHi biofilms.

Methods

Air-liquid interface (ALI)-cultured non-PCD primary nasal cells were grown in transwells. Barrier properties were assessed by trans-epithelial electrical resistance (TEER), FITC-dextran passage and tight junction staining. Ciliary function was assessed by high-speed video microscopy at 37°C. ALI cultures were infected with a PCD NTHi isolate at multiplicity of infections (MOIs) 10, 50 or 100 and co-cultured to form biofilms over 3 days. TEER was measured pre- and post-infection, NTHi recoverability assessed by conventional culture, and biofilm formation confirmed by scanning electron microscopy (SEM).

Results

Epithelial culture at ALI was successful, with tight barriers being formed and normal ciliary beat pattern and frequency recorded (mean 14Hz). Following co-culture, TEER increased relative to pre-infection at all MOIs but maximum NTHi recoverability was at MOI 50 (median colony forming units, 6.8x106per cm2). SEM confirmed NTHi biofilm formation at MOI 50.

Conclusions

A successful NTHi biofilm co-culture model on human primary epithelia was established. This will be used with PCD epithelia to investigate host-pathogen interactions during NTHi biofilm colonisation.
Development of a CRISPR interference system in Campylobacter jejuni
Authors: Coates Ruby , Grant Andrew , Floto Andres , Parkhill Julian
Published: May 2022

Campylobacter spp. are the leading cause of bacterial food-borne illness in humans worldwide, with Campylobacter jejuniresponsible for 80% of these infections. There is no current vaccine and antibiotic resistance is emerging. There is an urgent need to understand fundamental C. jejuni biology for the development of new strategies to prevent and treat infections. The range of molecular tools available to regulate gene expression in C. jejuni is limited, which impacts studies into the function of essential and conditionally essential genes. My project aims to address this by applying a CRISPR-based interference system known as CRISPRi in C. jejuni as a means to control gene expression and thereby investigate gene function. To validate the CRISPRi system in C. jejuni, I have paired the dCas9 and sgRNA backbone from the Streptococcus pyogenesCRISPRi system with several C. jejuni-derived promoters to develop a series of CRISPRi constructs targeting several genes. Through rigorous sgRNA target design I have successfully targeted and repressed expression of the endogenous arylsulphatase (AstA) enzyme, as well as achieving partial repression of expression of the regulatory flagellar protein FlgR in two clinically relevant C. jejuni strains. This is the first report of a CRISPRi system for Campylobacter.
Biochemical and Biophysical Characterization of Exoribonucleases from the Human Pathogen Mycobacterium tuberculosis
Authors: Costa Vanessa , Archer Margarida , Arraiano Cecília , Brito José , Matos Rute
Published: May 2022

Mycobacterium tuberculosis remains the leading cause of mortality from a single infectious organism, infecting nearly one-third of the global population. The current emergence of multidrug-resistant strains represents a serious health problem nowadays. Moreover, regulation of gene expression through RNA metabolism is a key mechanism for bacterial growth, division and rapid accommodation to environmental conditions. Ribonucleases are enzymes present in all living organisms that play an important role in RNA processing and degradation. Particularly, ribonucleases belonging to the RNB-family are often essential for viability of prokaryotes and are implicated in the establishment of virulence of several pathogens. In the present study we aim to structurally and functionally characterize two putative exoribonucleases from the RNB-family of enzymes inM. tuberculosis. Overexpression and purification of the proteins was performed and further in vitro activity, binding and helicase assays using synthetic RNA substrates were accomplished. In parallel, a biophysical characterization proceeded with several crystallization trials and protein stability tests. We have demonstrated that both RNases are 3’-5’ exoribonucleases with different degradation properties and unravelled the importance of highly conserved residues for catalysis. Moreover, we were able to identify improved buffer formulations that increase protein stability, possibly enhancing their propensity to crystallize. The information regarding RNA metabolism in M. tuberculosis is limited and RNB-family enzymes have not been previously characterized in this important human pathogen. Thus, a complete knowledge of these ribonucleases is an approach to recognize their influence in M. tuberculosis metabolism and to better understand the post-transcriptional control in this pathogen.
Improving phage genome annotation to understand phage biology: the case of Pseudomonas aeruginosa LES prophages
Authors: Gonzalez-Tortuero Enrique , Krishnamurthi Revathy , Goodhead Ian , Allison Heather , James Chloe
Published: May 2022

Pseudomonas aeruginosais an important opportunistic pathogen, causing nosocomial infections. The Liverpool Epidemic Strain (LES), a major cause of mortality and morbidity in cystic fibrosis patients, harbours five prophages associated with increased fitness and survival in models of infection. However, ~76.5% of the LES prophage genes are hypothetical proteins. Also, little is known about the LES prophage interactions with the lysogen and other prophages. In this study, we used the VIral Genome Annotation (VIGA) pipeline to re-annotate the LES prophage genomes and improve the prediction of gene function. The RefSeq viral proteins database was used for homology-based gene prediction and the Prokaryotic Virus Orthologous Genes (PVOGs) and Reference Viral DataBase (RVDB) were used for HMM-based protein function prediction. InterProScan 5.47-5.82 and Infernal 1.1.3 (with Rfam 14.3) were applied to enrich the gene function prediction for all genes and ncRNA elements, respectively. The number of putative coding sequences had increased by 1.17-1.43 times. Multiple genes related to DNA recombination and host cell lysis were identified in this reannotation. Also, we have identified new ncRNA elements in these prophages, such astRNAs in prophages 2 and 5 and a putative regulatory ncRNA, a Hammerhead-II ribozyme, in prophage 4. All this new information will be combined with data from future RNAseq experiments to map the expression profiles of each LES prophage under inducing and non-inducing conditions to characterise interactions between the prophages and their lysogen host.
Amoeba & Biofilms in UK Chlorinated Drinking Water Distributions Systems: Impact on Water Safety
Authors: Olmo Gonzalo Del , Puzon Geoffrey J. , Preciado Carolina Calero , Douterelo Isabel
Published: May 2022

Amoeba-related diseases have been related with the presence of certain amoebas in domestic water, including drinking water. Biofilms in drinking water distribution systems are able to support amoeba growth by providing a food source and protecting them against disinfectants. Additionally, amoeba growth can be favoured by warm temperatures and climate change appears to contribute to its geographic spread.

The presence of amoeba and its association with potential pathogenic bacteria was studied in a real-scale chlorinated DWDS. The test facility comprised three independent pipe loops fed with water from the local water supply and for this study a varied flow hydraulic profile was applied based on daily patterns observed in real UK distribution networks. The daily regime was repeated for a biofilm growth phase of 30 days. Amoeba viability was tested by a culture-based method, non-nutrient agar (NNA)-E. coli plates, and then confirm by qPCR using specific primers to detect species of amoeba including Naegleria and Acanthamoeba. Amplicon sequencing of the 16s rRNA gene was used to characterise the biofilm and planktonic bacterial communities.

Several amoeba species belonging to the genera Acanthamoeba, Vermamoeba and Naegleria were identified in 30-day old biofilm samples. While the bacterial communities in biofilms were dominated by Variovorax, Pseudomonas and Aquabacterium. This study yielded new insights on the dynamics of amoeba and bacterial communities in DWDS. However, more research is required to accurately establish the impact of these inter-kingdom associations on human health.
How to sequence 10,000 bacterial genomes and retain your sanity: an accessible, efficient and global approach
Authors: Perez-Sepulveda Blanca , Heavens Darren , Pulford Caisey , Predeus Alexander , Low Ross , Webster Hermione , Dykes Gregory , Schudoma Christian , Rowe Will , Lipscombe James , Watkins Chris , Kumwenda Benjamin , Shearer Neil , Costigan Karl , Baker Kate , Feasey Nicholas , Hinton Jay , Hall Neil ,   The 10KSG Consortium
Published: May 2022

Non-typhoidal Salmonella(NTS)are typically associated with enterocolitis and linked to the industrialisation of food production. In recent years, NTS has been associated with invasive disease (iNTS disease) causing an estimated 77,000 deaths each year worldwide; 80% of mortality occurs in sub-Saharan Africa. New clades of S. Typhimurium and S. Enteritidis have been identified, which are characterised by genomic degradation, altered prophage repertoires and novel multidrug resistant plasmids. To understand how these clades are contributing to the burden and severity of iNTS disease, it is crucial to expand genome-based surveillance to cover more countries, and incorporate historical isolates to generate an evolutionary timeline of the development of iNTS.

We developedand validateda robust and inexpensive method for large-scale collection and sequencing of bacterial genomes. The “10,000 Salmonella genomes” project established a worldwide research collaboration to generate information relevant to the epidemiology, drug resistance and virulence factors of Salmonellae using a whole-genome sequencing approach. By streamlining collection of isolates and developing an efficient logistics pipeline, we gathered 10,419 clinical and environmental isolates from collections in low and middle-income countries within six months. Genome sequences are now available for isolates from 51 countries/territories dating from 1949 to 2017, with ~80 % representing African and Latin-American datasets. Our method can be applied to other large sample collections that require maximisation of resources within a limited timeframe. Detailed genome analyses are in progress and it is hoped that the resulting data will contribute to public health control strategies in low and middle-income countries.
Regulatory cascade in SaPI activation
Authors: Haag Andreas , Podkowik Magdalena , Ibarra-Chávez Rodrigo , del Sol Francisca Gallego , Ram Geeta , Chen John , Marina Alberto , Novick Richard , Penadés José
Published: May 2022

Staphylococcal pathogenicity islands (SaPIs) are mobile genetic elements encoding superantigens and other toxinsand are induced for excision, replication, packaging and intercell transfer by phage-encoded anti-repressors that counter the SaPI master repressor. Though SaPI induction has heretofore been assumed to be the exclusive province of helper phages, we report here the remarkable discovery that one of the SaPIs, SaPI3, can instead be induced only by a second, co-resident SaPI, which must first be induced by a phage. This induction cascade thus represents intricate regulatory triad; SaPI3, the beneficiary of this intracellular largess, is the prototype of a hitherto uniquely immobile SaPI lineage. We report that members of this lineage are controlled by a novel regulatory module and are induced by a highly conserved but previously uncharacterised SaPI protein. SaPI3 and its cousins are thus SaPI satellites, just as most other SaPIs are phage satellites.
Piperacillin/tazobactam resistance in a clinical isolate of Escherichia colidue to IS26-mediated amplification of blaTEM-1B
Authors: Hubbard Alasdair , Mason Jenifer , Roberts Paul , Parry Christopher , Corless Caroline , Aartsen John van , Howard Alex , Bulgasim Issra , Fraser Alice , Adams Emily , Roberts Adam , Edwards Thomas
Published: May 2022

We identified a clinical isolate of Escherichia coli displaying an unusual, emerging phenotype; piperacillin/tazobactam (TZP)-resistant, 3rd generation cephalosporin-susceptible. Prior to treatment with TZP, a TZP-susceptible E. coli isolate was isolated from the same patient. Hyperproduction of a class A β-lactamase has previously been linked to this phenotype, but the mechanism of hyperproduction in isolates lacking promoter region mutations is not well understood.

Clonality of the two isolates was initially assessed with RFLP, and β-lactamase activity was determined using a nitrocefin assay. Both isolates were sequenced on Illumina and Oxford Nanopore Technology platforms and fitness assessed competitively. A plasmid construct containing the insertion sequence IS26was used to capture a translocatable unit (TU) in vitro.

The two E. coli clinical isolates were confirmed to be clonal, with the TZP-resistant isolate hyperproducing blaTEM-1. However, no promoter region mutations were identified in the TZP-resistant isolate. Hybrid assembly revealed that an ~11kb segment of DNA was excised from a IS26 flanked pseudo-compound transposon in the TZP-resistant isolate, forming a circular TU containing blaTEM-1. Multiple re-insertion events of the TU, mediated by IS26, led to tandem repeats of the TU within the chromosome, increasing the copy number of blaTEM-1. Excision and insertion events were confirmed via capture of the TU. Amplification of the TU in the TZP-resistant isolate incurred no significant change in fitness in different environmental conditions.

This study improves the understanding of the TZP-resistant, 3rd generation cephalosporin-susceptible phenotype in E. coli and antimicrobial resistance prediction of this phenotype from genotypic data.