Biology subjects
Publications and source records attributed to Baker, S..
We report the persistent circulation of third-generation cephalosporin resistant Salmonella Typhi in Mumbai, linked to the acquisition and maintenance of a previously characterized IncX3 plasmid carrying the ESBL gene blaSHV-12 and the fluoroquinolone resistance gene qnrB7 in the genetic context of a triple mutant also associated with fluoroquinolone resistance.
Cancer-associated mutations in genes encoding histones dramatically reshape chromatin and support tumorigenesis. Lysine to methionine substitution of residue 27 on histone H3 (K27M) is a driver mutation in high-grade pediatric gliomas, known to abrogate Polycomb Repressive Complex 2 (PRC2) activity. We applied single-molecule systems to image individual nucleosomes and delineate the combinatorial epigenetic patterns associated with H3-K27M expression. We found that chromatin marks on H3-K27M-mutant nucleosomes are dictated both by their incorporation preferences and by intrinsic properties of the mutation. Mutant nucleosomes not only preferentially bind PRC2, but also directly interact with MLL1, thus leading to genome-wide redistribution of H3K4me3. H3-K27M-mediated deregulation of both repressive and active chromatin marks leads to unbalanced bivalent chromatin, which may support a poorly differentiated cellular state. This study provides evidence for a direct effect of H3-K27M oncohistone on the MLL1-H3K4me3 pathway and highlights the capability of single-molecule tools to reveal mechanisms of chromatin deregulation in cancer.
Prevention of SARS-CoV-2 entry in cells through the modulation of viral host receptors, such as ACE2, could represent a new therapeutic approach complementing vaccination. However, the mechanisms controlling ACE2 expression remain elusive. Here, we identify the farnesoid X receptor (FXR) as a direct regulator of ACE2 transcription in multiple COVID19-affected tissues, including the gastrointestinal and respiratory systems. We demonstrate that FXR antagonists, including the over-the-counter compound z-guggulsterone (ZGG) and the off-patent drug ursodeoxycholic acid (UDCA), downregulate ACE2 levels, and reduce susceptibility to SARS-CoV-2 infection in lung, cholangiocyte and gut organoids. We then show that therapeutic levels of UDCA downregulate ACE2 in human organs perfused ex situ and reduce SARS-CoV-2 infection ex vivo. Finally, we perform a retrospective study using registry data and identify a correlation between UDCA treatment and positive clinical outcomes following SARS-CoV-2 infection, including hospitalisation, ICU admission and death. In conclusion, we identify a novel function of FXR in controlling ACE2 expression and provide evidence that this approach could be beneficial for reducing SARS-CoV-2 infection, thereby paving the road for future clinical trials.
Antimicrobial resistance (AMR) is a pressing global health crisis, which has been fuelled by the sustained use of certain classes of antimicrobials, including fluoroquinolones. While the genetic mutations responsible for decreased fluoroquinolone (ciprofloxacin) susceptibility are known, the implications of ciprofloxacin exposure on bacterial growth, survival, and interactions with host cells are not well described. Aiming to understand the influence of inhibitory concentrations of ciprofloxacin in vitro, we subjected three clinical isolates of S. Typhimurium to differing concentrations of ciprofloxacin, dependent on their minimum inhibitory concentrations (MIC), and assessed the impact on bacterial growth, morphology, and transcription. We further investigated the differential morphology and transcription that occurred following ciprofloxacin exposure and measured the ability of ciprofloxacin-treated bacteria to invade and replicate in host cells. We found that ciprofloxacin-exposed S. Typhimurium are able to recover from inhibitory concentrations of ciprofloxacin, and that the drug induces specific morphological and transcriptional signatures associated with the bacterial SOS response, DNA repair, and intracellular survival. In addition, ciprofloxacin-treated S. Typhimurium have increased capacity for intracellular replication in comparison to untreated organisms. These data suggest that S. Typhimurium undergoes an adaptive response under ciprofloxacin perturbation that promotes cellular survival, a consequence that may justify more measured use of ciprofloxacin for Salmonella infections. The combination of multiple experimental approaches provides new insights into the collateral effects that ciprofloxacin and other antimicrobials have on invasive bacterial pathogens. ImportanceAntimicrobial resistance is a critical concern in global health. In particular, there is rising resistance to fluoroquinolones, such as ciprofloxacin, a first-line antimicrobial for many Gram-negative pathogens. We investigated the adaptive response of clinical isolates of Salmonella Typhimurium to ciprofloxacin, finding that the bacteria adapt in short timespans to high concentrations of ciprofloxacin in a way that promotes intracellular survival during early infection. Importantly, by studying three clinically relevant isolates, we were able to show that individual isolates respond differently to ciprofloxacin, and for each isolate, there was a heterogeneous response under ciprofloxacin treatment. The heterogeneity that arises from ciprofloxacin exposure may drive survival and proliferation of Salmonella during treatment and lead to drug resistance.
Bifidobacterium pseudocatenulatum is a member of the human gut microbiota, and has previously been used as a probiotic to improve gut integrity and reduce inflammatory responses. We showed previously that B. pseudocatenulatum was significantly depleted during dysenteric diarrhea, suggesting the organism may aid in recovery from diarrhea. Here, in order to investigate its probiotic potential, we aimed to assess the genomic diversity and predicted metabolic profiles of B. pseudocatenulatum found colonizing the gut of healthy Vietnamese adults and children. We found that the population of B. pseudocatenulatum from each individual was distinct and highly diverse, with intra-clonal variation attributed to gain or loss of carbohydrate utilizing enzymes. The B. pseudocatenulatum genomes were enriched with glycosyl hydrolases that target plant-based non-digestible carbohydrates (GH13, GH43), but not host-derived glycans. Notably, the exopolysaccharide biosynthesis region from organisms isolated from healthy children showed greater genetic diversity, and was subject to a high degree of genetic modification. Antimicrobial susceptibility testing revealed that the Vietnamese B. pseudocatenulatum were uniformly susceptible to beta-lactams, but exhibited variable resistance to azithromycin, tetracycline, ciprofloxacin and metronidazole. The genomic presence of ermX and tet variants conferred resistance against azithromycin and tetracycline, respectively; ciprofloxacin resistance was associated with mutation(s) in the quinolone resistance determining region (GyrA, S115 and/or D119). Our work provides the first detailed genomic and antimicrobial resistance characterization of B. pseudocatenulatum found in the Vietnamese population, which could inform the next phase of rational probiotic design. ImportanceBifidobacterium pseudocatenulatum is a probiotic candidate with potential applications in several health conditions, but its efficacy is largely strain-dependent and associated with distinct genomic and biochemical features. However, most commercial probiotics have been developed by Western institutions, which may not have ideal efficacy when administered in developing countries. This study taps into the underexplored diversity of the organism in Vietnam, and provides more understanding to its lifestyles and antimicrobial susceptibility. These data are key for selecting an optimal probiotic candidate, from our established collection, for downstream investigations and validation. Thus, our work represents a model in identifying and characterizing bespoke probiotics from an indigenous population in a developing setting.
High-content imaging (HCI) is technique for screening multiple cells in high resolution to detect subtle morphological and phenotypic variation. The method has been commonly deployed on model eukaryotic cellular systems, often for screening new drugs and targets. HCI is not commonly utilised for studying bacterial populations but may be powerful tool in understanding and combatting antimicrobial resistance. Consequently, we developed a high-throughput method for phenotyping bacteria under antimicrobial exposure at the scale of individual bacterial cells. Imaging conditions were optimised on an Opera Phenix confocal microscope (Perkin-Elmer) and novel analysis pipelines were established for both Gram-negative bacilli and Gram-positive cocci. The potential of this approach was illustrated using isolates of Klebsiella pneumoniae, Salmonella enterica serovar Typhimurium, and Staphylococcus aureus. HCI enabled the detection and assessment of subtle morphological characteristics, undetectable through conventional phenotypical methods, that could reproducibly distinguish between bacteria exposed to different classes of antimicrobials with distinct modes of action (MOA). In addition, distinctive responses were observed between susceptible and resistant isolates. By phenotyping single bacterial cells, we observed intra-population differences, which may be critical in identifying persistence or emerging resistance during antimicrobial treatment. The work presented here outlines a comprehensive method for investigating morphological changes at scale in bacterial populations under specific perturbation. ImportanceHigh-content imaging (HCI) is a microscopy technique that permits the screening of multiple cells simultaneously in high resolution to detect subtle morphological and phenotypic variation. The power of this methodology is that is can generate large datasets comprised of multiple parameters taken from individual cells subjected to range of different conditions. We aimed to develop novel methods for using HCI to study bacterial cells exposed to a range of different antibiotic classes. Using an Opera Phenix confocal microscope (Perkin-Elmer) and novel analysis pipelines we created a method to study the morphological characteristics of Klebsiella pneumoniae, Salmonella enterica serovar Typhimurium, and Staphylococcus aureus when exposed to antibacterial drugs with differing modes of action. By imaging individual bacterial cells at high resolution and scale, we observed intra-population differences associated with different antibiotics. The outlined methods are highly relevant for how we begin to better understand and combat antimicrobial resistance.
Hypervirulent K. pneumoniae (HvKp) is typically associated with ST23 clone; however, hvKp is also emerging from clones ST11, ST15 and ST147, which are also multi-drug resistant (MDR). Here, we aimed to characterise nine novel MDR hvKp isolates harbouring mosaic plasmids simultaneously carrying antimicrobial resistance (AMR) and virulence genes. Nine HvKp isolates obtained from hospitalised patients in southern India were characterized for antimicrobial susceptibility and hypervirulence phenotypes. All nine hvKp isolates were subjected to whole genome sequencing (WGS) using Ilumina HiSeq2500 and a subset of four were sequenced using Oxford Nanopore MinION. Among the nine isolates, seven were carbapenem-resistant, two of which carried blaNDM-5 on an IncFII plasmid and five carried blaOXA-232 on a ColKP3 plasmid. The virulence determinants were encoded in a mosaic plasmid ([~]320 Kbp) that formed as a result of its insertion in a IncFIB-IncHI1B plasmid co-integrate. The mosaic plasmid carried AMR genes (aadA2, armA, blaOXA-1, msrE, mphE, sul1 and dfrA14) in addition to rmpA2, iutA and iucABCD virulence genes. Interestingly the mosaic plasmid carried its own type IV-A3 CRISPR-cas system that is likely able to target the acquisition of IncF plasmid with the help of a traL spacer. The convergence of virulence and AMR is the biggest threat among invasive K. pneumoniae infections. However, increasing reports of the presence of mosaic plasmid carrying both AMR and virulence genes suggests MDR-hvKp isolates are no longer confined to selected clones and the containment of such isolates is very challenging. IMPORTANCEKlebsiella pneumoniae is an opportunistic pathogen that commonly associated with hospital-acquired infections in the urinary tract, respiratory tract, lung, wound sites. The organism has gained notoriety by acquiring additional genetic traits to become either hypervirulent (HV) phenotype or multidrug resistant (MDR) phenotype. Though the infections by both these phenotypes were very challenging to treat, the MDR K. pneumonia (MDR-Kp) were remained in the hospital settings while HV K. pneumonia (hvKp) strains were mostly originated from the community settings. In a recent turn of events, the evolution of MDR-Kp and hvKp has converged as both clones found to carry both MDR plasmids and virulence plasmid. These convergent strains are challenging to treat and is associated with higher mortality rate. As the recent hvKp isolates harbour mosaic plasmid encoding both AMR and virulence determinants there is a need to investigate the evolution of these pathogens. The significance of our research is in characterising the novel mosaic plasmid identified in MDR-hvKp isolates that belong sequence type (ST) 2096. Tracking the possible evolution pathway of MDR-hvKPs would greatly help in the proper surveillance and management of this superbugs. RepositoriesThe whole genome sequences of the present study isolates have been deposited in GenBank, NCBI, with accession numbers CP053765 - CP053770, CP053771 - CP053780, CP058798-CP058806, JAARNO010000001.1 - JAARNO010000005.1, JAAQSG000000000, JAARNJ000000000, JAARMH000000000 and JAAQTC000000000
Shigella sonnei is the most common agent of shigellosis in high-income countries, and causes a significant disease burden in low- and middle-income countries. Antimicrobial resistance is increasingly common in all settings. Whole genome sequencing (WGS) is increasingly utilised for S. sonnei outbreak investigation and surveillance, but comparison of data between studies and labs is challenging. Here, we present a genomic framework and genotyping scheme for S. sonnei to efficiently identify genotype and resistance determinants from WGS data. The scheme is implemented in the software package Mykrobe and tested on thousands of genomes. Applying this approach to analyse >4,000 S. sonnei isolates sequenced in public health labs in three countries identified several common genotypes associated with increased rates of ciprofloxacin resistance and azithromycin resistance, confirming intercontinental spread of highly-resistant S. sonnei clones and demonstrating the genomic framework can facilitate monitoring of the emergence and spread of resistant clones at local and global scales.
Interstitial cells of Cajal (ICC) generate pacemaker activity responsible for phasic contractions in colonic segmentation and peristalsis. ICC along the submucosal border (ICC-SM) contributing to mixing and more complex patterns of colonic motility. We show the complex patterns of Ca2+ signaling in ICC-SM and the relationship between ICC-SM Ca2+ transients and activation of SMCs using optogenetic tools. ICC-SM displayed rhythmic firing of Ca2+ transients [~]15 cpm and paced adjacent SMCs. The majority of spontaneous activity occurred in regular Ca2+ transients clusters (CTCs) that propagated through the network. CTCs were organized and dependent upon Ca2+ entry through voltage-dependent Ca2+ conductances, L- and T-type Ca2+ channels. Removal of Ca2+ from the external solution abolished CTCs. Ca2+ release mechanisms reduced the duration and amplitude of Ca2+ transients but did not block CTCs. These data reveal how colonic pacemaker ICC-SM exhibit complex Ca2+ firing patterns and drive smooth muscle activity and overall colonic contractions. SynopsisHow Ca2+ signaling in colonic submucosal pacemaker cells couples to smooth muscle responses is unknown. This study shows how ICC modulate colonic motility via complex Ca2+ signaling and defines Ca2+ transients sources using optogenetic techniques.
Colistin is extensively used in animal production in many low- and middle-income countries. There is a need to develop methodologies to benchmark and monitor changes in resistance in commensal bacterial populations in farms. We aimed to evaluate the performance of a broth microdilution method based on culturing a pooled Escherichia coli suspension (30-50 organisms) from each sample. In order to confirm the biological basis and sensitivity of the method, we prepared 16 standard suspensions containing variable ratios of colistin-susceptible and mcr-1 encoded colistin-resistant E. coli which were grown in 2mg/L colistin. The optical density (OD600nm) readings over time were used to generate a growth curve, and were adjusted to the values obtained in the absence of colistin. The median limit of detection of the method was 1 colistin-resistant in 104 susceptible colonies [1st - 3rd quartile, 1:102 - 1:105]. We applied this method to 108 pooled faecal samples from 36 chicken flocks in the Mekong Delta (Vietnam) over the production cycle. The correlation between this method and the prevalence of colistin resistance in individual colonies harvested from field samples, determined by the Minimum Inhibitory Concentration (MIC), was established. The overall prevalence of colistin resistance at sample and isolate level was 38.9% and 19.4%, respectively. Increased colistin resistance was associated with recent (2 weeks) use of colistin and other, non-colistin antimicrobials (OR=3.67 and OR=1.84, respectively). Our method is a sensitive and affordable approach to monitor changes in colistin resistance in pooled E. coli populations from faecal samples over time. IMPORTANCEColistin (polymyxin E) is an antimicrobial with poor solubility properties, and therefore broth microdilution is the only appropriate method for testing colistin resistance. However, estimating colistin resistance in commensal mixed Escherichia coli populations is laborious since it requires individual colony isolation, identification and susceptibility testing. We developed a growth-based microdilution method suitable for pooled faecal samples. We validated the method by comparing it with results from individual MIC testing of 909 E. coli isolates. We used the method to investigate phenotypic colistin resistance in 108 pooled faecal samples from 36 healthy chicken flocks, each sampled three times over the production cycle. A higher level of resistance was seen in flocks recently supplemented with colistin in drinking water, although the observed generated resistance was short-lived. Our method is affordable, and may potentially be integrated into surveillance systems aiming at estimating the prevalence of resistance at colony level in flocks/herds. Furthermore, it may also be adapted to other complex biological systems, such as farms and abattoirs.
BackgroundThe emergence and spread of antimicrobial resistance (AMR) pose a major threat to the effective treatment and control of typhoid fever. The ongoing outbreak of extensively drug resistant (XDR) Salmonella Typhi (S. Typhi) in Pakistan has left azithromycin as the only remaining broadly efficacious oral antimicrobial for typhoid in South Asia. Ominously, azithromycin resistant S. Typhi organisms have been subsequently reported in Bangladesh, Pakistan, and Nepal. MethodsHere, we aimed to understand the molecular basis of AMR in 66 S. Typhi isolated in a cross-sectional study performed in a suburb of Chandigarh in Northern India using whole genome sequencing (WGS) and phylogenetic analysis. ResultsWe identified seven S. Typhi organisms with the R717Q mutation in the acrB gene that was recently found to confer resistance to azithromycin in Bangladesh. Six out of the azithromycin-resistant S. Typhi isolates also exhibited triple mutations in gyrA (S83F and D87N) and parC (S80I) genes and were resistant to ciprofloxacin. These contemporary ciprofloxacin/azithromycin-resistant isolates were phylogenetically distinct from each other and from those reported from Bangladesh, Pakistan, and Nepal. ConclusionsThe independent emergence of azithromycin resistant typhoid in Northern India reflects an emerging broader problem across South Asia and illustrates the urgent need for the introduction of typhoid conjugate vaccines (TCVs) in the region. Key pointsWe identified ciprofloxacin/azithromycin-resistant Salmonella Typhi (S. Typhi) in Chandigarh in Northern India. The independent emergence of ciprofloxacin/azithromycin-resistant typhoid in Bangladesh, Pakistan, Nepal, and India and the continued spread of extensively-drug resistant (XDR) typhoid in Pakistan highlight the limitations of licensed oral treatments for typhoid fever in South Asia.
In presymptomatic Alzheimers disease (AD), beta-amyloid plaques (A{beta}) and tau tangles accumulate in distinct spatiotemporal patterns within the brain, tracking closely with episodic memory decline. Here, we tested whether age-related changes in the segregation of the brains functional episodic memory networks - anterior-temporal (AT) and posterior-medial (PM) networks - are associated with the accumulation of A{beta}, tau and memory decline using fMRI and PET. We found that AT and PM networks were less segregated in older than younger adults and this reduced specialization was associated with more tau and A{beta} in the same regions. The effect of network dedifferentiation on memory depended on the amount of A{beta} and tau, with low segregation and pathology associated with better performance at baseline and low segregation and high pathology related to worse performance over time. This pattern suggests a compensation phase followed by a degenerative phase in the early, preclinical phase of AD.
The earliest stages of Alzheimers disease (AD) involve interactions between multiple pathophysiological processes. Although these processes are well studied, we still lack robust tools to predict individualised trajectories of disease progression. Here, we employ a robust and interpretable machine learning approach to combine multimodal biological data and predict future tau accumulation, translating predictive information from deep phenotyping cohorts at early stages of AD to cognitively normal individuals. In particular, we use machine learning to quantify interactions between key pathological markers ({beta}-amyloid, medial temporal atrophy, tau and APOE 4) at early and asymptomatic stages of AD. We next derive a predictive index that stratifies individuals based on future pathological tau accumulation, highlighting two critical features for optimal clinical trial design. First, future tau accumulation provides a better outcome measure compared to changes in cognition. Second, stratification based on multimodal data compared to {beta}-amyloid alone reduces the sample size required to detect a clinically meaningful change in tau accumulation. Further, we extend our machine learning approach to derive individualised trajectories of future pathological tau accumulation in early AD patients and accurately predict regional future rate of tau accumulation in an independent sample of cognitively unimpaired individuals. Our results propose a robust approach for fine scale stratification and prognostication with translation impact for clinical trial design at asymptomatic and early stages of AD. One Sentence SummaryOur machine learning approach combines baseline multimodal data to make individualised predictions of future pathological tau accumulation at prodromal and asymptomatic stages of Alzheimers disease with high accuracy and regional specificity.
Two drought-tolerant wheat cultivars, TAM 111 and TAM 112, have been widely grown in the Southern Great Plains of the U.S. and used as parents in many wheat breeding programs worldwide. This study aimed to reveal genetic control of yield and yield components in the two cultivars under both dryland and irrigated conditions. A mapping population containing 124 F5:7 recombinant inbred lines (RILs) was developed from the cross of TAM 112/TAM 111. A set of 5,948 SNPs from the wheat 90K iSelect array and double digest restriction-site associated DNA sequencing was used to construct high-density genetic maps. Data for yield and yield components were obtained from 11 environments. QTL analyses were performed based on 11 individual environments, across all environments, within and across mega-environments. Thirty-six unique consistent QTL regions were distributed on 13 chromosomes including 1A, 1B, 1D, 2A, 2D, 3D, 4B, 4D, 6A, 6B, 6D, 7B, and 7D. Ten unique QTL with pleiotropic effects were identified on four chromosomes and eight were in common with the consistent QTL. These QTL increased dry biomass grain yield by 16.3 g m-2, plot yield by 28.1 g m-2, kernels spike-1 by 0.7, spikes m-2 by 14.8, thousand kernel weight by 0.9 g with favorable alleles from either parent. TAM 112 alleles mainly increased spikes m-2 and thousand kernel weight while TMA 111 alleles increased kernels spike-1, harvest index and grain yield. The saturated genetic map and markers linked to significant QTL from this study will be very useful in developing high throughput genotyping markers for tracking the desirable haplotypes of these important yield-related traits in popular parental cultivars.
BackgroundMicrobial whole-genome sequencing (WGS) is now increasingly used to inform public health investigations of infectious disease. This approach has transformed our understanding of the global population structure of Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever. WGS has been particularly informative for understanding the global spread of multi-drug resistant (MDR) typhoid. As WGS capacity becomes more decentralised, there is a growing opportunity for collaboration and sharing of surveillance data within and between countries to inform disease control policies. This requires freely available, community driven tools that reduce the barriers to access genomic data for public health surveillance and that deliver genomic data on a global scale. MethodsHere we present the Pathogenwatch (https://pathogen.watch/styphi) scheme for S. Typhi, a web application enabling the rapid identification of genomic markers of antimicrobial resistance (AMR) and contextualization with public genomic data to identify high-risk clones at a population level. Data are delivered in single genome reports or in collections of genomes combined with geographic and other data using trees, maps and tables. ResultsWe show that the clustering of S. Typhi genomes in Pathogenwatch is comparable to established bioinformatics methods, and that genomic predictions of AMR are largely concordant with phenotypic drug susceptibility data. We demonstrate the public health utility of Pathogenwatch with examples selected from over 4,300 public genomes available in the application. ConclusionsPathogenwatch democratises genomic epidemiology of S. Typhi by providing an intuitive entry point for the analysis of WGS and linked epidemiological data, enabling international public health monitoring of the emergence and spread of high risk clones.
The spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is the dominant target of the immune system. S exhibits substantial conformational flexibility. It transitions from closed to open conformations to expose its receptor binding site, and subsequently from prefusion to postfusion conformations to mediate fusion of viral and cellular membranes. S protein derivatives are components of vaccine candidates and diagnostic assays, as well as tools for research into the biology and immunology of SARS-CoV-2. Here we have designed mutations in S which allow production of thermostable, crosslinked, S protein trimers that are trapped in the closed, pre-fusion, state. We have determined the structures of crosslinked and non-crosslinked proteins, identifying two distinct closed conformations of the S trimer. We demonstrate that the designed, thermostable, closed S trimer can be used in serological assays. This protein has potential applications as a reagent for serology, virology and as an immunogen.
Despite recent gains in typhoid fever control, asymptomatic carriage of Salmonella Typhi in the gallbladder remains an enigma. Aiming to understand if S. Typhi in the gallbladder are vital for transmission and/or adapted for long-term colonisation we performed whole genome sequencing on a collection of S. Typhi isolated from the gallbladders of typhoid carriers. These sequences were compared to contemporaneous sequences from organisms isolated from the blood of acute patients.S. Typhi carriage was not restricted to any particular genotype or conformation of antimicrobial resistance genes but reflective of the general population. However, gallbladder isolates had a higher genetic variability than acute isolates, with median pairwise SNP distances of 21 and 13 SNPs (p=2.8x10-9), respectively. This variation was associated with a higher prevalence of nonsense mutations in the gallbladder isolates in the predominant genotype. Notably, gallbladder isolates displayed a higher frequency of non-synonymous mutations in genes encoding hypothetical proteins, membrane lipoproteins, transport/binding proteins, surface antigens, and carbohydrate degradation. Particularly, we identified several gallbladder-specific non-synonymous mutations involved in LPS synthesis and modification, with some isolates lacking the Vi capsular polysaccharide vaccine target due to a 134Kb deletion. Long-term gallbladder carriage of S. Typhi results in atypically long branch lengths that can distinguish between carriage and acute infection. Our data strongly suggests typhoid carriers are unlikely to play a principal role in disease transmission in endemic settings, but that the hostile environment of the human gallbladder may generate new antigenic variants through immune selection.
The increase of antimicrobial resistance (AMR), and lack of new classes of licensed antimicrobials, have made alternative treatment options for AMR pathogens increasingly attractive. Recent studies have demonstrated anti-bacterial efficacy of a humanised monoclonal antibody (mAb) targeting the O25b O-antigen of Escherichia coli ST131. To evaluate the phenotypic effects of antibody binding to diverse clinical E. coli ST131 O25b bacterial isolates in high-throughput, we designed a novel mAb screening method using high-content imaging (HCI) and image-based morphological profiling to screen a mAb targeting the O25b O-antigen. Screening the antibody against a panel of 86 clinical E. coli ST131 O25:H4 isolates revealed 4 binding phenotypes: no binding (18.60%), weak binding (4.65%), strong binding (69.77%) and strong agglutinating binding (6.98%). Impaired antibody binding could be explained by the presence of insertion sequences or mutations in O-antigen or lipopolysaccharide core biosynthesis genes, affecting the amount, structure or chain length of the O-antigen. The agglutinating binding phenotype was linked with lower O-antigen density, enhanced antibody-mediated phagocytosis and increased serum susceptibly. This study highlights the need to screen candidate mAbs against large panels of clinically relevant isolates, and that HCI can be used to evaluate mAb binding affinity and potential functional efficacy against AMR bacteria.