Biology subjects

Clarke, P.

Publications and source records attributed to Clarke, P..

12 recordsLinked to original sources

bioRxiv2025

Staphylococcus haemolyticus is a reservoir of antibiotic resistance genes in the preterm infant gut

Among coagulase-negative staphylococci, Staphylococcus haemolyticus is a primary cause of bloodstream infections in preterm infants, with gut colonisation being recognised as a risk factor for subsequent infection. Through a re-analysis of a 16S rRNA gene sequencing dataset (n=497 preterm infants), we found that S. haemolyticus was abundant and prevalent in the gut in the first month of life. To better understand the diversity of S. haemolyticus among preterm infants, we generated genome sequences of S. haemolyticus strains (n=140), which were isolated from 44 stool samples of 22 preterm infants from four different hospitals in the United Kingdom. Core genome phylogenetic analyses, incorporating 126 publicly available S. haemolyticus genome sequences, showed that 85/140 (60.1%) of the isolates, from three different hospitals, formed a clonal group with 79/85 (92.9%) strains being assigned to Multi-Locus Sequence Type (ST) 49. Antibiotic resistance genes were highly prevalent in the genome sequences. Using logistic regression, we found a strong association between the presence of the gene mecA and phenotypic resistance to oxacillin (odds ratio [OR]: 158.00, p<0.0001), and the aacA-aphD gene and phenotypic resistance to gentamicin aacA-aphD (OR: 162.00, p<0.001). None of the strains from the preterm infant cohort had a complete Staphylococcal Cassette Chromosome mec (SCCmec) element. The aacA-aphD gene was associated with the transposon Tn4001. Using hybrid genome assemblies, we found it to be present on the chromosome (54.5% of strains) or on diverse plasmids (27.3%). Four strains (18.2%) had Tn4001 copies on both plasmid and chromosome. Our data suggest the existence of a distinct sub-population of S. haemolyticus that has adapted to colonise the gut of preterm infants. Prevalent resistance to antibiotics is of clinical concern and the diversity of genetic contexts of mecA and Tn4001 suggests widespread horizontal gene transfer and recombination in this species.

microbiology↗

bioRxiv2024

Impact of early life antibiotic and probiotic treatment on gut microbiome and resistome of very-low-birth-weight preterm infants

Preterm infants (<37 weeks gestation) are often administered broad-spectrum antibiotics in hospitals due to their vulnerability to severe morbidity, including necrotising enterocolitis and sepsis. However, antibiotics can disrupt the development of early-life microbiota, potentially impairing gut immunity and colonisation resistance. Evidence shows that probiotics (e.g., certain Bifidobacterium strains) may help restore healthy gut microbiota. In this study, we examined the effects of probiotics and antibiotics on the preterm gut microbiome and resistome in two unique cohorts of 34 very-low-birth-weight, human-milk- fed preterm infants (moderate to very preterm), with one cohort receiving probiotics. Within each group, some infants were treated with antibiotics (benzylpenicillin and/or gentamicin) while others served as non-antibiotic treated controls. We performed shotgun metagenomic sequencing on 93 longitudinal faecal samples from 34 infants, generated >300 metagenome- assembled genomes, and obtained [~]90 isolate genomes through targeted culturomics, enabling analysis of the microbiome/resistome at species and strain levels. Additionally, we investigated in vitro horizontal gene transfer (HGT) capacity of preterm infant-derived multidrug-resistant (MDR) pathogen Enterococcus via neonatal gut models. Overall, probiotic supplementation significantly reduced antibiotic resistance gene prevalence, MDR pathogen load, and helped restore a typical early-life microbiota. However, the persistence of MDR pathogens like Enterococcus, with high HGT potential, highlights the need for ongoing surveillance in neonatal care. Our findings underscore the complex interactions between antibiotics, probiotics, and HGT in shaping the neonatal microbiome and support further research into probiotics for antimicrobial stewardship in preterm populations.

microbiology↗

bioRxiv2022

Characterisation of neonatal Staphylococcus capitis NRCS-A isolates compared with non NRCS-A Staphylococcus capitis from neonates and adults.

Staphylococcus capitis is a frequent cause of Late-Onset Sepsis (LOS) in neonates admitted to Neonatal Intensive Care Units (NICU). The NRCS-A clone of S. capitis has been isolated from NICUs globally although the reasons for the global success of this clone are not understood. We analysed a collection of S. capitis colonising babies admitted to two NICUs, one in the UK and one in Germany as well as corresponding pathological clinical isolates. Genome analysis identified 3 groups; non-NRCS-A isolates, NRCS-A isolates, and a group of proto NRCS-A - isolates closely related to NRCS-A but not associated with neonatal infection. All bloodstream isolates belonged to the NRCS-A group and were indistinguishable from strains carried on the skin or in the gut. NRCS-A isolates showed increased tolerance to chlorhexidine and antibiotics relative to the other S. capitis as well as enhanced ability to grow at higher pH values. Analysis of 138 pangenomes of the clades identified characteristic nsr and tarJ genes in the NRCS-A and proto groups with a CRISPR-cas system only seen in NRCS-A isolates which also showed enrichment of genes for metal acquisition and transport. We found evidence for transmission of S. capitis NRCS-A within NICU, with related isolates shared between babies and multiple acquisitions by some babies. Our data show NRCS-A strains commonly colonise uninfected babies in NICU representing a potential reservoir for potential infection. This work provides more evidence that adaptation to survive in the gut and on skin facilitates spread of NRCS-A, and that metal acquisition and tolerance may be important to the biology of NRCS-A. Understanding how NRCS-A survives in NICUs can help develop infection control procedures against this clone.

genomics↗

bioRxiv2022

Partial refuges from biological control due to intraspecific variation in protective host traits: a case study with the egg parasitoid Trissolcus japonicus

Predicting how much of a host or prey population may be attacked by their natural enemies is fundamental to several subfields of applied ecology, particularly biological control of pest organisms. Hosts or prey can occupy refuges from natural enemy attack, but habitat or ecological refuges are challenging or impossible to predict in a laboratory setting - which is often where efficacy and specificity testing of candidate biological control agents is done. Here we explore how intraspecific variation in continuous traits that confers some protection from natural enemy attack - even after the natural enemy has encountered the prey - could provide partial refuges. The size of these trait-based refuges should depend on the relationship between trait values and host/prey susceptibility to natural enemy attack, and on how common different trait values are within a host/prey population. These can be readily estimated in laboratory testing of natural enemy impact on target or non-target prey or hosts as long as sufficient host material is available. We provide a general framework for how intraspecific variation in protective host traits could be integrated into biological control research, specifically with reference to non-target testing as part of classical biological control programs. As a case study, we exposed different host clutch sizes of target (pest) and non-target (native species) stink bug (Hemiptera: Pentatomidae) species to a well-studied exotic biocontrol agent, the egg parasitoid Trissolcus japonicus (Hymenoptera: Scelionidae). Although we observed several behavioural and reproductive responses to variation in host egg mass size by T. japonicus, they did not translate to increases in predicted refuge size (proportion host survival) large enough to change the conclusions of non-target testing. We encourage researchers to investigate intraspecific variation in a wider variety of protective host and prey traits and their consequences for refuge size.

ecology↗

bioRxiv2022

A novel bacteriocin produced by Bifidobacterium longum subsp. infantis has dual antimicrobial and immunomodulatory activity

Bacteriocins are ribosomally-synthesized antimicrobial peptides produced by bacteria with either narrow or broad spectrum activity. Many genome mining studies have indicated that bacteriocin gene clusters are widespread within certain gut microbiota members. In early life, Bifidobacterium comprise the dominant microbiota genus in vaginally delivered and breast-fed infants, with high levels associated with improved health. However, in many cases the mechanisms underlying these beneficial effects are unknown, although a limited number of studies have suggested that bacteriocin production by Bifidobacterium may represent a key mechanism for preventing pathogen over-growth. Here, we used BAGEL4 and antiSMASH to identify putative bacteriocin sequences in the whole genome sequences of 33 Bifidobacterium strains isolated from infants participating in two clinical studies. We identified a novel non-lantibiotic bacteriocin from Bifidobacterium longum subsp. infantis LH_664, with 40% sequence homology to Lactococcin 972 from Lactococcus lactis subsp. lactis. The putative bacteriocin (Bifidococcin_664) was chemically synthesized and studied for antimicrobial and immune-modulatory activities. We determined it has discrete activity against Clostridium perfringens and it appears to have novel immune stimulatory activities, promoting macrophage phagocytosis and specific cytokine release. These data highlight strain-specific beneficial properties in the early life genus Bifidobacterium, and suggest avenues for development of novel and highly specific dual action antimicrobials, and possible probiotic strains, that are active against clinically important bacterial pathogens. Data summarySamples LH_9 to LH_666 were previously sequenced and deposited to ENA under accession numbers ERS2658025-ERS2658043. Samples LH_986 to LH_1052 are newly sequenced and deposited to NCBI under accession numbers SAMN24838598-SAMN24838611. Additionally, previously assembled publicly available sequences (n=7) were retrieved online from NCBI Genomes database. See Supplementary Table S1 for further details.

microbiology↗

bioRxiv2021

Strain dependent structural effects and in vivo efficacy of enterovirus-D68 inhibitors

Acute flaccid myelitis (AFM) leads to loss of limb control in young children and is likely due to Enterovirus-D68 (EV-D68), for which there is no current treatment. We have developed a lead isoxazole-3-carboxamide analog of pleconaril (11526092) which displayed potent inhibition of the pleconaril-resistant CVB3-Woodruff (IC50 6-20 nM), EV-D68 (IC50 58 nM), and other enteroviruses. A mouse respiratory model of EV-D68 infection, in which pleconaril is inactive, showed decreased viremia of 3 log units as well as statistically significant 1 log reduction in lung titer reduction at day 5 after treatment with 11526092. A cryo-electron microscopy (cryo-EM) structure of EV-D68 in complex with 11526092 suggests that the increased potency may be due to additional hydrophobic interactions. Cryo-EM structures of 11526092 and pleconaril demonstrate destabilization of EV-D68 (MO strain) compared to the previously described stabilization of EV-D68 (Fermon strain) with pleconaril, illustrating clear strain dependent mechanisms of this molecule. 11526092 represents a more potent inhibitor in vitro with in vivo efficacy providing a potential future treatment for EV-D68 and AFM, suggesting an improvement over pleconaril for further optimization. One-Sentence Summary11526092 demonstrates protein destabilization, improved in vitro potency and in vivo efficacy when compared with pleconaril against EV-D68.

pharmacology and toxicology↗

bioRxiv2021

Dissemination and pathogenesis of toxigenic Clostridium perfringens strains linked to neonatal intensive care units and Necrotising Enterocolitis

BackgroundClostridium perfringens is an anaerobic toxin-producing bacterium that has long been associated with intestinal diseases, particularly in neonatal humans and animals. More recently, infant gut microbiome studies have suggested an important link between C. perfringens and the devastating preterm-associated disease Necrotising Enterocolitis (NEC), but in-depth studies on this pathogen (genomics and mechanistic) are lacking. Methods/MaterialsWe isolated and whole-genome sequenced 274 infant-associated C. perfringens isolates from 5 hospitals across the UK between 2011-2016 (including longitudinal samples from 31 individuals). We performed in-depth genomic analyses, phenotypically characterised pathogenic traits of 10 strains (including 4 C. perfringens from NEC patients) and established a novel oral-challenge C57BL/6 mouse infection model for microbe-host studies. ResultsPore-forming toxin encoding genes pfoA and cpb2 were enriched within hypervirulent lineages that exclusively consisted of C. perfringens-associated NEC (CPA-NEC) strains, in addition to overabundance of colonisation factors. Importantly, we identified a circulating C. perfringens variant, eventually linked to a fatal CPA-NEC case. The variant was detected consistently within 6 individuals in two sister hospitals across a 40-day window, demonstrating for the first time the intra- and inter-hospital dissemination of C. perfringens. CPA-NEC isolates were determined phenotypically to be more virulent (linked with overabundance of gene pfoA) than isolates obtained from non-NEC preterm babies. In addition, two pfoA-positive CPA-NEC C. perfringens strains were confirmed to induce clinical inflammatory tissue lesions in vivo. ConclusionsHypervirulent lineages are linked to CPA-NEC, potentially due to the production of pore-forming toxins, coupled with higher metabolic, transmission, and pathogenic capacities. These studies indicate C. perfringens is an important bacterial pathogen in preterm infants and highlights the requirement for further investigation into development of intervention and therapeutic strategies.

microbiology↗

bioRxiv2019

Preterm infants harbour diverse Klebsiella populations, including atypical species that encode and produce an array of antimicrobial resistance- and virulence-associated factors

Klebsiella spp. are frequently enriched in the gut microbiota of preterm neonates, and overgrowth is associated with necrotizing enterocolitis, nosocomial infections and late-onset sepsis. Little is known about the genomic and phenotypic characteristics of preterm-associated Klebsiella as previous studies have focussed on recovery of antimicrobial-resistant isolates or culture-independent molecular analyses. Faecal samples from a UK cohort of healthy and sick preterm neonates (n=109) were screened on MacConkey agar to isolate lactose-positive Enterobacteriaceae. Whole-genome sequences were generated for isolates. Approximately one-tenth of faecal samples harboured Klebsiella spp. (Klebsiella pneumoniae, 7.3 %; Klebsiella quasipneumoniae, 0.9 %; Klebsiella grimontii, 2.8 %; Klebsiella michiganensis, 1.8 %). Isolates recovered from NEC- and sepsis-affected infants and those showing no signs of clinical infection (i.e. healthy) encoded multiple {beta}-lactamases, which may prove problematic when defining treatment regimens for NEC or sepsis, and suggest healthy preterm infants contribute to the resistome. No difference was observed between isolates recovered from healthy and sick infants with respect to in vitro siderophore production (all encoded enterobactin in their genomes). All K. pneumoniae, K. quasipneumoniae, K. grimontii and K. michiganensis faecal isolates tested were able to reside and persist in macrophages, indicating their immune evasion abilities. Using a curated dataset of Klebsiella oxytoca, K. grimontii and K. michiganensis whole-genome sequences, metapangenome analyses of published metagenomic data confirmed our findings regarding the presence of K. michiganensis in the preterm gut, and highlight the importance of refined analyses with curated sequence databases when studying closely related species present in metagenomic data.

microbiology↗

bioRxiv2019

Microbiota supplementation with Bifidobacterium and Lactobacillus modifies the preterm infant gut microbiota and metabolome

Supplementation with members of the early-life microbiota or probiotics is becoming increasingly popular to attempt to beneficially manipulate the preterm gut microbiota. We performed a large longitudinal study comprising two preterm groups; 101 orally supplemented with Bifidobacterium and Lactobacillus (Bif/Lacto) and 133 non-supplemented (Control) matched by age, sex, birth-mode, and diet. 16S rRNA metataxonomic profiling on stool samples (n = 592) indicated a predominance of Bifidobacterium, and a reduction of pathobionts in the Bif/Lacto group. Metabolic phenotyping found a parallel increase in fecal acetate and lactate in the Bif/Lacto group compared to the Control group, which positively correlated with Bifidobacterium abundance consistent with the ability of the supplemented Bifidobacterium strain to metabolize human milk oligosaccharides and reduced gut pH. This study demonstrates that microbiota supplementation can modify the preterm microbiome and the gastrointestinal environment to more closely resemble that of a full-term infant.

microbiology↗

bioRxiv2019

The off-target kinase landscape of clinical PARP inhibitors

Four PARP inhibitors have been approved by the FDA as cancer therapeutics, yet there are several clinical settings where no strong rationale exists to inform clinicians on which to choose in terms of clinical effectiveness and toxicity. The four drugs have largely similar PARP family inhibition profiles, but several differences at the molecular and clinical level have been reported that remain poorly understood. We previously hypothesized that PARP inhibitors could have an inherent capacity to inhibit kinases off-target. Here, we characterise the off-target kinase landscape of four FDA-approved PARP drugs. We demonstrate that all four PARP inhibitors have a unique polypharmacological profile across the kinome. Niraparib and rucaparib inhibit DYRK1s, CDK16 and PIM3 at clinically achievable submicromolar concentrations. These represent the most potently inhibited off-targets of PARP inhibitors identified to date and should be investigated further to clarify their potential implications for efficacy and safety in the clinic, including use of PARP inhibitors in combination with other agents, including immunotherapy.

pharmacology and toxicology↗

bioRxiv2018

The plasma biomarker soluble SIGLEC-1 is associated with the type I interferon transcriptional signature, ethnic background and renal disease in systemic lupus erythematosus

The molecular heterogeneity of autoimmune and inflammatory diseases has been one of the main obstacles to the development of safe and specific therapeutic options. Here we have evaluated the diagnostic and clinical value of a robust, inexpensive, immunoassay detecting the circulating soluble form of the monocyte-specific surface receptor sialic acid binding Ig-like lectin 1 (sSIGLEC-1).\n\nWe developed an immunoassay to measure sSIGLEC-1 in small volumes of plasma/serum from systemic lupus erythematosus (SLE) patients and healthy donors. Plasma concentrations of sSIGLEC-1 strongly correlated with expression of SIGLEC-1 on the surface of blood monocytes and with type I interferon (IFN)-regulated gene (IRG) expression in SLE patients. In addition, we identified marked ancestry-related differences in sSIGLEC-1 concentrations in SLE patients, with patients of non-European ancestry showing higher levels compared to patients of European ancestry. Higher sSIGLEC-1 concentrations were associated with lower serum complement component 3 and increased frequency of renal complications in European patients, but not with the SLEDAI clinical score.\n\nOur sSIGLEC-1 immunoassay provides a specific and easily-assayed marker for monocyte-macrophage activation, and interferonopathy in SLE and other diseases. Further studies can extend its clinical associations and its potential use to stratify patients and as a secondary endpoint in trials.

immunology↗

bioRxiv2017

Rapid MinION metagenomic profiling of the preterm infant gut microbiota to aid in pathogen diagnostics

The Oxford Nanopore MinION sequencing platform offers near real time analysis of DNA reads as they are generated, which makes the device attractive for in-field or clinical deployment, e.g. rapid diagnostics. We used the MinION platform for shotgun metagenomic sequencing and analysis of gut-associated microbial communities; firstly, we used a 20-species human microbiota mock community to demonstrate how Nanopore metagenomic sequence data can be reliably and rapidly classified. Secondly, we profiled faecal microbiomes from preterm infants at increased risk of necrotising enterocolitis and sepsis. In single patient time course, we captured the diversity of the immature gut microbiota and observed how its complexity changes over time in response to interventions, i.e. probiotic, antibiotics and episodes of suspected sepsis. Finally, we performed real-time runs from sample to analysis using faecal samples of critically ill infants and of healthy infants receiving probiotic supplementation. Real-time analysis was facilitated by our new NanoOK RT software package which analysed sequences as they were generated. We reliably identified potentially pathogenic taxa (i.e. Klebsiella pneumoniae and Enterobacter cloacae) and their corresponding antimicrobial resistance (AMR) gene profiles within as little as one hour of sequencing. Antibiotic treatment decisions may be rapidly modified in response to these AMR profiles, which we validated using pathogen isolation, whole genome sequencing and antibiotic susceptibility testing. Our results demonstrate that our pipeline can process clinical samples to a rich dataset able to inform tailored patient antimicrobial treatment in less than 5 hours.

genomics↗