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A novel therapeutic antibody screening method using bacterial high-content imaging reveals functional antibody binding phenotypes of Escherichia coli ST131.

ABSTRACT: 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.

SUBMITTER: Maes M

PROVIDER: S-EPMC7382476 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Escherichia coli sequence type 131 (ST131) has emerged rapidly to become the most prevalent extraintestinal pathogenic E. coli clones in circulation today. Previous investigations appeared to exonerate retail meat as a source of human exposure to ST131; however, these studies focused mainly on extensively multidrug-resistant ST131 strains, which typically carry allele 30 of the fimH type 1 fimbrial adhesin gene (ST131-H30). To estimate the frequency of extraintestinal human infections arising from foodborne ST131 strains without bias toward particular sublineages or phenotypes, we conducted a 1-year prospective study of E. coli from meat products and clinical cultures in Flagstaff, Arizona. We characterized all isolates by multilocus sequence typing, fimH typing, and core genome phylogenetic analyses, and we screened isolates for avian-associated ColV plasmids as an indication of poultry adaptation. E. coli was isolated from 79.8% of the 2,452 meat samples and 72.4% of the 1,735 culture-positive clinical samples. Twenty-seven meat isolates were ST131 and belonged almost exclusively (n = 25) to the ST131-H22 lineage. All but 1 of the 25 H22 meat isolates were from poultry products, and all but 2 carried poultry-associated ColV plasmids. Of the 1,188 contemporaneous human clinical E. coli isolates, 24 were ST131-H22, one-quarter of which occurred in the same high-resolution phylogenetic clades as the ST131-H22 meat isolates and carried ColV plasmids. Molecular clock analysis of an international ST131-H22 genome collection suggested that ColV plasmids have been acquired at least six times since the 1940s and that poultry-to-human transmission is not limited to the United States.IMPORTANCEE. coli ST131 is an important extraintestinal pathogen that can colonize the gastrointestinal tracts of humans and food animals. Here, we combined detection of accessory traits associated with avian adaptation (ColV plasmids) with high-resolution phylogenetics to quantify the portion of human infections caused by ST131 strains of food animal origin. Our results suggest that one ST131 sublineage-ST131-H22-has become established in poultry populations around the world and that meat may serve as a vehicle for human exposure and infection. ST131-H22 is just one of many E. coli lineages that may be transmitted from food animals to humans. Additional studies that combine detection of host-associated accessory elements with phylogenetics may allow us to quantify the total fraction of human extraintestinal infections attributable to food animal E. coli strains.

Project description:Uropathogenic Escherichia coli (UPEC) is the cause of ~75% of all urinary tract infections (UTIs) and is increasingly associated with multidrug resistance. This includes UPEC strains from the recently emerged and globally disseminated sequence type 131 (ST131), which is now the dominant fluoroquinolone-resistant UPEC clone worldwide. Most ST131 strains are motile and produce H4-type flagella. Here, we applied a combination of saturated Tn5 mutagenesis and transposon directed insertion site sequencing (TraDIS) as a high throughput genetic screen and identified 30 genes associated with enhanced motility of the reference ST131 strain EC958. This included 12 genes that repress motility of E. coli K-12, four of which (lrhA, ihfA, ydiV, lrp) were confirmed in EC958. Other genes represented novel factors that impact motility, and we focused our investigation on characterisation of the mprA, hemK and yjeA genes. Mutation of each of these genes in EC958 led to increased transcription of flagellar genes (flhD and fliC), increased expression of the FliC flagellin, enhanced flagella synthesis and a hyper-motile phenotype. Complementation restored all of these properties to wild-type level. We also identified Tn5 insertions in several intergenic regions (IGRs) on the EC958 chromosome that were associated with enhanced motility; this included flhDC and EC958_1546. In both of these cases, the Tn5 insertions were associated with increased transcription of the downstream gene(s), which resulted in enhanced motility. The EC958_1546 gene encodes a phage protein with similarity to esterase/deacetylase enzymes involved in the hydrolysis of sialic acid derivatives found in human mucus. We showed that over-expression of EC958_1546 led to enhanced motility of EC958 as well as the UPEC strains CFT073 and UTI89, demonstrating its activity affects the motility of different UPEC strains. Overall, this study has identified and characterised a number of novel factors associated with enhanced UPEC motility.

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A novel therapeutic antibody screening method using bacterial high-content imaging reveals functional antibody binding phenotypes of Escherichia coli ST131.

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