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Abstract Background Escherichia coli is the most common cause of human bloodstream infections and bacterial sepsis/septic shock. However, translation of preclinical septic shock resuscitative therapies remains limited mainly due to low-fidelity of available models in mimicking clinical illness. To overcome the translational barrier, we sought to replicate sepsis complexity by creating an acutely critically-ill preclinical bacterial septic shock model undergoing active 48-h intensive care management. Aim To develop a clinically relevant large-animal (ovine) live-bacterial infusion model for septic shock. Methods Septic shock was induced by intravenous infusion of the live antibiotic resistant extra-intestinal pathogenic E. coli sequence type 131 strain EC958 in eight anesthetised and mechanically ventilated sheep. A bacterial dose range of 2 × 105–2 × 109 cfu/mL was used for the dose optimisation phase (n = 4) and upon dose confirmation the model was developed (n = 5). Post-shock the animals underwent an early-vasopressor and volume-restriction resuscitation strategy with active haemodynamic management and monitoring over 48 h. Serial blood samples were collected for testing of pro-inflammatory (IL-6, IL-8, VEGFA) and anti-inflammatory (IL-10) cytokines and hyaluronan assay to assess endothelial integrity. Tissue samples were collected for histopathology and transmission electron microscopy. Results The 2 × 107 cfu/mL bacterial dose led to a reproducible distributive shock within a pre-determined 12-h period. Five sheep were used to demonstrate consistency of the model. Bacterial infusion led to development of septic shock in all animals. The baseline mean arterial blood pressure reduced from a median of 91 mmHg (71, 102) to 50 mmHg (48, 57) (p = 0.004) and lactate levels increased from a median of 0.5 mM (0.3, 0.8) to 2.1 mM (2.0, 2.3) (p = 0.02) post-shock. The baseline median hyaluronan levels increased significantly from 25 ng/mL (18, 86) to 168 ng/mL (86, 569), p = 0.05 but not the median vasopressor dependency index which increased within 1 h of resuscitation from zero to 0.39 mmHg−1 (0.06, 5.13), p = 0.065, and. Over the 48 h, there was a significant decrease in the systemic vascular resistance index (F = 7.46, p = 0.01) and increase in the pro-inflammatory cytokines [IL-6 (F = 8.90, p = 0.02), IL-8 (F = 5.28, p = 0.03), and VEGFA (F = 6.47, p = 0.02)]. Conclusions This critically ill large-animal model was consistent in reproducing septic shock and will be applied in investigating advanced resuscitation and therapeutic interventions.

Abstract Infections with Enterobacterales (E) are increasingly difficult to treat due to antimicrobial resistance. After ceftriaxone replaced chloramphenicol (CHL) as empiric therapy for suspected sepsis in Malawi in 2004, extended-spectrum beta-lactamase (ESBL)-E rapidly emerged. Concurrently, resistance to CHL in Escherichia coli and Klebsiella spp. decreased, raising the possibility of CHL re-introduction. However, many phenotypically susceptible isolates still carry CHL acetyltransferase (cat) genes. To understand the molecular mechanisms and stability of this re-emerging CHL susceptibility we use a combination of genomics, phenotypic susceptibility assays, experimental evolution, and functional assays for CAT activity. Here, we show that of 840 Malawian E. coli and Klebsiella spp. isolates, 31% have discordant CHL susceptibility genotype–phenotype, and we select a subset of 42 isolates for in-depth analysis. Stable degradation of cat genes by insertion sequences leads to re-emergence of CHL susceptibility. Our study suggests that CHL could be reintroduced as a reserve agent for critically ill patients with ESBL-E infections in Malawi and similar settings and highlights the ongoing challenges in inferring antimicrobial resistance from sequence data.

Abstract Background Breast screening recommendation based on individual risk assessment is emerging as an alternative approach to improve compliance and efficiency to detect breast cancer (BC) early. In Vietnam, prior knowledge to stratify risk based on genetic factors is currently lacking. Methods This study recruited 892 BC patients and 735 healthy Vietnamese women from 2016 to 2021. DNA from blood samples of BC patients was first analyzed for pathogenic variants associated with hereditary breast and ovarian cancer syndrome (HBOC). For patients with no HBOC and healthy participants, DNA was genotyped for 398 BC susceptibility single-nucleotide polymorphism (SNPs) by next-generation sequencing to identify significantly associated SNPs and construct a polygenic risk score (PRS). Results The prevalence of HBOC predisposition gene mutations in Vietnamese women with BC was 5.4%. HBOC cases were significantly younger and enriched in the age group of 20–39 years old. In patients with no HBOC, we found 36 SNPs significantly associated with BC that were mostly similar to other Asian ethnicities; 34 of them were used to build a PRS model achieving an area under the receiver operating characteristics curve of 0.61 (95% CI: 0.56–0.68). Women in the top 1% PRS percentile had an odds ratio of 5.09 (95% CI: 3.10–7.86) while those in the bottom 1% had an odds ratio of 0.21 (95% CI: 0.09–0.39) to develop BC. Conclusions This study provides the first large datasets for HBOC gene analysis, BC susceptibility SNP association testing, and PRS modeling for Vietnamese women. Together, these data could aid the development of personalized BC screening recommendations for women in Vietnam.

The emergence of multicancer early detection (MCED) tests holds promise for improving early cancer detection and public health outcomes. However, positive MCED test results require confirmation through recommended cancer diagnostic imaging modalities. To address these challenges, we have developed a consultation and work-up protocol for definitive diagnostic results post MCED testing, named SPOT-MAS. Developed through circulating tumor DNA (ctDNA) analysis and in line with professional guidelines and advisory board consensus, this protocol standardizes information to aid general practitioners in accessing, interpreting and managing SPOT-MAS results. Clinical effectiveness is demonstrated through a series of identified cancer cases. Our research indicates that the protocol could empower healthcare professionals to confidently interpret circulating tumor DNA test results for 5 common types of cancer, thereby facilitating the clinical integration of MCED tests.

Escherichia coli exhibit extensive genetic diversity at the genome level, particularly within their accessory genome. The tRNA integrated genomic islands (GIs), a part of the E. coli accessory genome, play an important role in pathogenicity. However, studies examining the evolution of GIs have been challenging due to their large size, considerable gene content variation and fragmented assembly in draft genomes. Here we examined the evolution of the GI integrated at pheV-tRNA (GI-pheV), with a primary focus on uropathogenic E. coli (UPEC) and the globally disseminated multidrug resistant ST131 clone. We show the gene content of GI-pheV is highly diverse and arranged in a modular configuration, with the P4 integrase encoding gene intP4 the only conserved gene. Despite this diversity, the GI-pheV gene content displayed conserved features among strains from the same pathotype. In ST131, GI-pheV corresponding to the reference strain EC958 (EC958_GI-pheV) was found in ~90% of strains. Phylogenetic analyses suggested that GI-pheV in ST131 has evolved together with the core genome, with the loss/gain of specific modules (or the entire GI) linked to strain specific events. Overall, we show GI-pheV exhibits a dynamic evolutionary pathway, in which modules and genes have evolved through multiple events including insertions, deletions and recombination.

In the realm of cancer immunotherapy, the meticulous selection of neoantigens plays a fundamental role in enhancing personalized treatments. Traditionally, this selection process has heavily relied on predicting the binding of peptides to human leukocyte antigens (pHLA). Nevertheless, this approach often overlooks the dynamic interaction between tumor cells and the immune system. In response to this limitation, we have developed an innovative prediction algorithm rooted in machine learning, integrating T cell receptor β chain (TCRβ) profiling data from colorectal cancer (CRC) patients for a more precise neoantigen prioritization. TCRβ sequencing was conducted to profile the TCR repertoire of tumor-infiltrating lymphocytes (TILs) from 28 CRC patients. The data unveiled both intra-tumor and inter-patient heterogeneity in the TCRβ repertoires of CRC patients, likely resulting from the stochastic utilization of V and J segments in response to neoantigens. Our novel combined model integrates pHLA binding information with pHLA-TCR binding to prioritize neoantigens, resulting in heightened specificity and sensitivity compared to models using individual features alone. The efficacy of our proposed model was corroborated through ELISpot assays on long peptides, performed on four CRC patients. These assays demonstrated that neoantigen candidates prioritized by our combined model outperformed predictions made by the established tool NetMHCpan. This comprehensive assessment underscores the significance of integrating pHLA binding with pHLA-TCR binding analysis for more effective immunotherapeutic strategies.

Abstract Bacteria adapt to selective pressure in their immediate environment in multiple ways. One mechanism involves the acquisition of independent mutations that disable or modify a key pathway, providing a signature of adaptation via convergent evolution. Extra-intestinal pathogenic Escherichia coli (ExPEC) belonging to sequence type 95 (ST95) represent a global clone frequently associated with severe human infections including acute pyelonephritis, sepsis, and neonatal meningitis. Here, we analysed a publicly available dataset of 613 ST95 genomes and identified a series of loss-of-function mutations that disrupt cellulose production or its modification in 55.3% of strains. We show the inability to produce cellulose significantly enhances ST95 invasive infection in a rat model of neonatal meningitis, leading to the disruption of intestinal barrier integrity in newborn pups and enhanced dissemination to the liver, spleen and brain. Consistent with these observations, disruption of cellulose production in ST95 augmented innate immune signalling and tissue neutrophil infiltration in a mouse model of urinary tract infection. Mutations that disrupt cellulose production were also identified in other virulent ExPEC STs, Shigella and Salmonella, suggesting a correlative association with many Enterobacteriaceae that cause severe human infection. Together, our findings provide an explanation for the emergence of hypervirulent Enterobacteriaceae clones.

Neonatal meningitis is a devastating disease associated with high mortality and neurological sequelae. Escherichia coli is the second most common cause of neonatal meningitis in full-term infants (herein NMEC) and the most common cause of meningitis in preterm neonates. Here, we investigated the genomic relatedness of a collection of 58 NMEC isolates spanning 1974–2020 and isolated from seven different geographic regions. We show NMEC are comprised of diverse sequence types (STs), with ST95 (34.5%) and ST1193 (15.5%) the most common. No single virulence gene profile was conserved in all isolates; however, genes encoding fimbrial adhesins, iron acquisition systems, the K1 capsule, and O antigen types O18, O75, and O2 were most prevalent. Antibiotic resistance genes occurred infrequently in our collection. We also monitored the infection dynamics in three patients that suffered recrudescent invasive infection caused by the original infecting isolate despite appropriate antibiotic treatment based on antibiogram profile and resistance genotype. These patients exhibited severe gut dysbiosis. In one patient, the causative NMEC isolate was also detected in the fecal flora at the time of the second infection episode and after treatment. Thus, although antibiotics are the standard of care for NMEC treatment, our data suggest that failure to eliminate the causative NMEC that resides intestinally can lead to the existence of a refractory reservoir that may seed recrudescent infection.

ABSTRACT Urinary tract infections (UTIs) are one of the most common bacterial infections in humans, with ~400 million cases across the globe each year. Uropathogenic Escherichia coli (UPEC) is the major cause of UTI and increasingly associated with antibiotic resistance. This scenario has been worsened by the emergence and spread of pandemic UPEC sequence type 131 (ST131), a multidrug-resistant clone associated with extraordinarily high rates of infection. Here, we employed transposon-directed insertion site sequencing in combination with metabolomic profiling to identify genes and biochemical pathways required for growth and survival of the UPEC ST131 reference strain EC958 in human urine (HU). We identified 24 genes required for growth in HU, which mapped to diverse pathways involving small peptide, amino acid and nucleotide metabolism, the stringent response pathway, and lipopolysaccharide biosynthesis. We also discovered a role for UPEC resistance to fluoride during growth in HU, most likely associated with fluoridation of drinking water. Complementary nuclear magnetic resonance (NMR)-based metabolomics identified changes in a range of HU metabolites following UPEC growth, the most pronounced being L-lactate, which was utilized as a carbon source via the L-lactate dehydrogenase LldD. Using a mouse UTI model with mixed competitive infection experiments, we demonstrated a role for nucleotide metabolism and the stringent response in UPEC colonization of the mouse bladder. Together, our application of two omics technologies combined with different infection-relevant settings has uncovered new factors required for UPEC growth in HU, thus enhancing our understanding of this pivotal step in the UPEC infection pathway.IMPORTANCEUropathogenic Escherichia coli (UPEC) cause ~80% of all urinary tract infections (UTIs), with increasing rates of antibiotic resistance presenting an urgent threat to effective treatment. To cause infection, UPEC must grow efficiently in human urine (HU), necessitating a need to understand mechanisms that promote its adaptation and survival in this nutrient-limited environment. Here, we used a combination of functional genomic and metabolomic techniques and identified roles for the metabolism of small peptides, amino acids, nucleotides, and L-lactate, as well as the stringent response pathway, lipopolysaccharide biosynthesis, and fluoride resistance, for UPEC growth in HU. We further demonstrated that pathways involving nucleotide metabolism and the stringent response are required for UPEC colonization of the mouse bladder. The UPEC genes and metabolic pathways identified in this study represent targets for the development of innovative therapeutics to prevent UPEC growth during human UTI, an urgent need given the rapidly rising rates of global antibiotic resistance.

Breast cancer is the leading cause of cancer death in Vietnamese women, but its mutational landscape and actionable alterations for targeted therapies remain unknown. After treatment, a sensitive biomarker to complement conventional imaging to monitor patients is also lacking. In this prospective multi‐center study, 134 early‐stage breast cancer patients eligible for curative‐intent surgery were recruited. Genomic DNA from tumor tissues and paired white blood cells were sequenced to profile all tumor‐derived mutations in 95 cancer‐associated genes. Our bioinformatic algorithm was then utilized to identify top mutations for individual patients. Serial plasma samples were collected before surgery and at scheduled visits after surgery. Personalized assay tracking the selected mutations were performed to detect circulating tumor DNA (ctDNA) in the plasma. We found that the mutational landscape of the Vietnamese was largely similar to other Asian cohorts, showing higher TP53 mutation frequency than in Caucasians. Alterations in PIK3CA and PI3K signaling were dominant, particularly in our triple‐negative subgroup. Using top‐ranked mutations, we detected ctDNA in pre‐operative plasma in 24.6–43.5% of the hormone‐receptor‐positive groups and 76.9–80.8% of the hormone‐receptor‐negative groups. The detection rate was associated with breast cancer subtypes and clinicopathological features that increased the risk of relapse. Interim analysis after a 15‐month follow‐up revealed post‐operative detection of ctDNA in all three patients that had recurrence, with a lead time of 7–13 months ahead of clinical diagnosis. Our personalized assay is streamlined and affordable with promising clinical utility in residual cancer surveillance. We also generated the first somatic variant dataset for Vietnamese breast cancer women that could lay the foundation for precision cancer medicine in Vietnam.

Despite their promise, circulating tumor DNA (ctDNA)-based assays for multi-cancer early detection face challenges in test performance, due mostly to the limited abundance of ctDNA and its inherent variability. To address these challenges, published assays to date demanded a very high-depth sequencing, resulting in an elevated price of test. Herein, we developed a multimodal assay called SPOT-MAS (screening for the presence of tumor by methylation and size) to simultaneously profile methylomics, fragmentomics, copy number, and end motifs in a single workflow using targeted and shallow genome-wide sequencing (~0.55×) of cell-free DNA. We applied SPOT-MAS to 738 non-metastatic patients with breast, colorectal, gastric, lung, and liver cancer, and 1550 healthy controls. We then employed machine learning to extract multiple cancer and tissue-specific signatures for detecting and locating cancer. SPOT-MAS successfully detected the five cancer types with a sensitivity of 72.4% at 97.0% specificity. The sensitivities for detecting early-stage cancers were 73.9% and 62.3% for stages I and II, respectively, increasing to 88.3% for non-metastatic stage IIIA. For tumor-of-origin, our assay achieved an accuracy of 0.7. Our study demonstrates comparable performance to other ctDNA-based assays while requiring significantly lower sequencing depth, making it economically feasible for population-wide screening.

Abstract Background Late detection of hepatocellular carcinoma (HCC) results in an overall 5-year survival rate of less than 16%. Liquid biopsy (LB) assays based on detecting circulating tumor DNA (ctDNA) might provide an opportunity to detect HCC early noninvasively. Increasing evidence indicates that ctDNA detection using mutation-based assays is significantly challenged by the abundance of white blood cell-derived mutations, non-tumor tissue-derived somatic mutations in plasma, and the mutational tumor heterogeneity. Methods Here, we employed concurrent analysis of cancer-related mutations, and their fragment length profiles to differentiate mutations from different sources. To distinguish persons with HCC (PwHCC) from healthy participants, we built a classification model using three fragmentomic features of ctDNA through deep sequencing of thirteen genes associated with HCC. Results Our model achieved an area under the curve (AUC) of 0.88, a sensitivity of 89%, and a specificity of 82% in the discovery cohort consisting of 55 PwHCC and 55 healthy participants. In an independent validation cohort of 54 PwHCC and 53 healthy participants, the established model achieved comparable classification performance with an AUC of 0.86 and yielded a sensitivity and specificity of 81%. Conclusions Our study provides a rationale for subsequent clinical evaluation of our assay performance in a large-scale prospective study.

Severe sepsis has a high mortality rate. Here, the authors provide genomic, transcriptomic, proteomic and metabolomic data across four sepsis-causing pathogens and identify a signature of global increase in fatty acid and lipid biosynthesis as well as cholesterol acquisition.

IntroductionNeoantigen-based immunotherapy has emerged as a promising strategy for improving the life expectancy of cancer patients. This therapeutic approach heavily relies on accurate identification of cancer mutations using DNA sequencing (DNAseq) data. However, current workflows tend to provide a large number of neoantigen candidates, of which only a limited number elicit efficient and immunogenic T-cell responses suitable for downstream clinical evaluation. To overcome this limitation and increase the number of high-quality immunogenic neoantigens, we propose integrating RNA sequencing (RNAseq) data into the mutation identification step in the neoantigen prediction workflow.MethodsIn this study, we characterize the mutation profiles identified from DNAseq and/or RNAseq data in tumor tissues of 25 patients with colorectal cancer (CRC). Immunogenicity was then validated by ELISpot assay using long synthesis peptides (sLP).ResultsWe detected only 22.4% of variants shared between the two methods. In contrast, RNAseq-derived variants displayed unique features of affinity and immunogenicity. We further established that neoantigen candidates identified by RNAseq data significantly increased the number of highly immunogenic neoantigens (confirmed by ELISpot) that would otherwise be overlooked if relying solely on DNAseq data.DiscussionThis integrative approach holds great potential for improving the selection of neoantigens for personalized cancer immunotherapy, ultimately leading to enhanced treatment outcomes and improved survival rates for cancer patients.

Abstract α-Thalassemia is a common inherited blood disorder manifested mainly by the deletions of α-globin genes. In geographical areas with high carrier frequencies, screening of α-thalassemia carrier state is therefore of vital importance. This study presents a novel method for identifying female carriers of common α-thalassemia deletions using samples routinely taken for non-invasive prenatal tests for screening of fetal chromosomal aneuploidies. A total of 68,885 Vietnamese pregnant women were recruited and α-thalassemia statuses were determined by gap-PCR, revealing 5344 women (7.76%) carried deletions including αα/−−SEA (4.066%), αα/−α3.7 (2.934%), αα/−α4.2 (0.656%), and rare genotypes (0.102%). A two-stage model was built to predict these α-thalassemia deletions from targeted sequencing of the HBA gene cluster on maternal cfDNA. Our method achieved F1-scores of 97.14–99.55% for detecting the three common genotypes and 94.74% for detecting rare genotypes (−α3.7/−α4.2, αα/−−THAI, −α3.7/−−SEA, −α4.2/−−SEA). Additionally, the positive predictive values were 100.00% for αα/αα, 99.29% for αα/−−SEA, 94.87% for αα/−α3.7, and 96.51% for αα/−α4.2; and the negative predictive values were 97.63%, 99.99%, 99.99%, and 100.00%, respectively. As NIPT is increasingly adopted for pregnant women, utilizing cfDNA from NIPT to detect maternal carriers of common α-thalassemia deletions will be cost-effective and expand the benefits of NIPT.

Plasmids are major drivers of increasing antibiotic resistance, necessitating an urgent need to understand their biology. Here we describe a detailed dissection of the molecular components controlling the genetics of I-complex plasmids, a group of antibiotic resistance plasmids found frequently in pathogenic Escherichia coli and other Enterobacteriaceae that cause significant human disease. We show these plasmids cluster into four distinct subgroups, with the prototype IncI1 plasmid R64 subgroup displaying low nucleotide sequence conservation to other I-complex plasmids. Using pMS7163B, an I-complex plasmid distantly related to R64, we performed a high-resolution transposon-based genetic screen and defined genes involved in replication, stability, and conjugative transfer. We identified the replicon and a partitioning system as essential for replication/stability. Genes required for conjugation included the type IV secretion system, relaxosome, and several uncharacterised genes located in the pMS7163B leading transfer region that exhibited an upstream strand-specific transposon insertion bias. The overexpression of these genes severely impacted host cell growth or reduced fitness during mixed competitive growth, demonstrating that their expression must be controlled to avoid deleterious impacts. These genes were present in >80% of all I-complex plasmids and broadly conserved across multiple plasmid incompatibility groups, implicating an important role in plasmid dissemination. Author summary Antimicrobial resistance is one of the greatest threats to human health. Left unchecked, we risk a rapid escalation of untreatable infections. Plasmids are one of the most important vehicles for resistance gene carriage and transmission between bacteria, and thus an understanding of plasmid biology is crucial to controlling the spread antimicrobial resistance. Here, we combine advanced bioinformatics and a state-of-the-art genetic screen to understand the molecular mechanisms involved in the maintenance and spread of a group of plasmids strongly associated with antibiotic resistance among bacteria that cause human infection. We characterised genes involved in the replication and maintenance of these plasmids, and experimentally demonstrated that plasmid spread is dependent on a well-conserved secretion system. Our genetic screen also discovered a set of broadly conserved, uncharacterised genes that adversely impact host fitness and plasmid spread under dysregulation. Taken together, these findings describe a molecular blueprint for the biology of a group of clinically relevant antibiotic resistance plasmids found frequently in Gram-negative bacterial pathogens.

IntroductionBreast cancer causes the most cancer-related death in women and is the costliest cancer in the US regarding medical service and prescription drug expenses. Breast cancer screening is recommended by health authorities in the US, but current screening efforts are often compromised by high false positive rates. Liquid biopsy based on circulating tumor DNA (ctDNA) has emerged as a potential approach to screen for cancer. However, the detection of breast cancer, particularly in early stages, is challenging due to the low amount of ctDNA and heterogeneity of molecular subtypes.MethodsHere, we employed a multimodal approach, namely Screen for the Presence of Tumor by DNA Methylation and Size (SPOT-MAS), to simultaneously analyze multiple signatures of cell free DNA (cfDNA) in plasma samples of 239 nonmetastatic breast cancer patients and 278 healthy subjects.ResultsWe identified distinct profiles of genome-wide methylation changes (GWM), copy number alterations (CNA), and 4-nucleotide oligomer (4-mer) end motifs (EM) in cfDNA of breast cancer patients. We further used all three signatures to construct a multi-featured machine learning model and showed that the combination model outperformed base models built from individual features, achieving an AUC of 0.91 (95% CI: 0.87-0.95), a sensitivity of 65% at 96% specificity.DiscussionOur findings showed that a multimodal liquid biopsy assay based on analysis of cfDNA methylation, CNA and EM could enhance the accuracy for the detection of early- stage breast cancer.

Abstract Targeted therapy with tyrosine kinase inhibitors (TKI) provides survival benefits to a majority of patients with non-small cell lung cancer (NSCLC). However, resistance to TKI almost always develops after treatment. Although genetic and epigenetic alterations have each been shown to drive resistance to TKI in cell line models, clinical evidence for their contribution in the acquisition of resistance remains limited. Here, we employed liquid biopsy for simultaneous analysis of genetic and epigenetic changes in 122 Vietnamese NSCLC patients undergoing TKI therapy and displaying acquired resistance. We detected multiple profiles of resistance mutations in 51 patients (41.8%). Of those, genetic alterations in EGFR, particularly EGFR amplification (n = 6), showed pronounced genome instability and genome-wide hypomethylation. Interestingly, the level of hypomethylation was associated with the duration of response to TKI treatment. We also detected hypermethylation in regulatory regions of Homeobox genes which are known to be involved in tumor differentiation. In contrast, such changes were not observed in cases with MET (n = 4) and HER2 (n = 4) amplification. Thus, our study showed that liquid biopsy could provide important insights into the heterogeneity of TKI resistance mechanisms in NSCLC patients, providing essential information for prediction of resistance and selection of subsequent treatment.

Abstract Background Several inherited metabolic diseases are underreported in Vietnam, namely glucose‐6‐phosphate dehydrogenase deficiency (G6PDd), phenylketonuria (PKU) and galactosemia (GAL). Whilst massively parallel sequencing (MPS) allows researchers to screen several loci simultaneously for pathogenic variants, no screening programme uses MPS to uncover the variant spectra of these diseases in the Vietnamese population. Methods Pregnant women (mean age of 32) from across Vietnam attending routine prenatal health checks agreed to participate and had their blood drawn. MPS was used to detect variants in their G6PD, PAH and GALT genes. Results Of 3259 women screened across Vietnam, 450 (13.8%) carried disease‐associated variants for G6PD, PAH and GALT. The prevalence of carriers was 8.9% (291 of 3259) in G6PD and 4.6% (152 of 3259) in PKU, whilst GAL was low at 0.2% (7 of 3259). Two GALT variants, c.593 T > C and c.1034C > A, have rarely been reported. Conclusion This study highlights the need for routine carrier screening, where women give blood whilst receiving routine prenatal care, in Vietnam. The use of MPS is suitable for screening multiple variants, allowing for identifying rare pathogenic variants. The data from our study will inform policymakers in constructing cost‐effective genetic metabolic carrier screening programmes.

Abstract Background Oxford Nanopore Technology (ONT) long-read sequencing has become a popular platform for microbial researchers due to the accessibility and affordability of its devices. However, easy and automated construction of high-quality bacterial genomes using nanopore reads remains challenging. Here we aimed to create a reproducible end-to-end bacterial genome assembly pipeline using ONT in combination with Illumina sequencing. Results We evaluated the performance of several popular tools used during genome reconstruction, including base-calling, filtering, assembly, and polishing. We also assessed overall genome accuracy using ONT both natively and with Illumina. All steps were validated using the high-quality complete reference genome for the Escherichia coli sequence type (ST)131 strain EC958. Software chosen at each stage were incorporated into our final pipeline, MicroPIPE. Further validation of MicroPIPE was carried out using 11 additional ST131 E. coli isolates, which demonstrated that complete circularised chromosomes and plasmids could be achieved without manual intervention. Twelve publicly available Gram-negative and Gram-positive bacterial genomes (with available raw ONT data and matched complete genomes) were also assembled using MicroPIPE. We found that revised basecalling and updated assembly of the majority of these genomes resulted in improved accuracy compared to the current publicly available complete genomes. Conclusions MicroPIPE is built in modules using Singularity container images and the bioinformatics workflow manager Nextflow, allowing changes and adjustments to be made in response to future tool development. Overall, MicroPIPE provides an easy-access, end-to-end solution for attaining high-quality bacterial genomes. MicroPIPE is available at https://github.com/BeatsonLab-MicrobialGenomics/micropipe .

Extra-intestinal pathogenic Escherichia coli (ExPEC) belong to a critical priority group of antibiotic resistant pathogens. ExPEC establish gut reservoirs that seed infection of the urinary tract and bloodstream, but the mechanisms of gut colonisation remain to be properly understood. Ucl fimbriae are attachment organelles that facilitate ExPEC adherence. Here, we investigated cellular receptors for Ucl fimbriae and Ucl expression to define molecular mechanisms of Ucl-mediated ExPEC colonisation of the gut. We demonstrate differential expression of Ucl fimbriae in ExPEC sequence types associated with disseminated infection. Genome editing of strains from two common sequence types, F11 (ST127) and UTI89 (ST95), identified a single nucleotide polymorphism in the ucl promoter that changes fimbriae expression via activation by the global stress-response regulator OxyR, leading to altered gut colonisation. Structure-function analysis of the Ucl fimbriae tip-adhesin (UclD) identified high-affinity glycan receptor targets, with highest affinity for sialyllacto-N-fucopentose VI, a structure likely to be expressed on the gut epithelium. Comparison of the UclD adhesin to the homologous UcaD tip-adhesin from Proteus mirabilis revealed that although they possess a similar tertiary structure, apart from lacto-N-fucopentose VI that bound to both adhesins at low-micromolar affinity, they recognize different fucose- and glucose-containing oligosaccharides. Competitive surface plasmon resonance analysis together with co-structural investigation of UcaD in complex with monosaccharides revealed a broad-specificity glycan binding pocket shared between UcaD and UclD that could accommodate these interactions. Overall, our study describes a mechanism of adaptation that augments establishment of an ExPEC gut reservoir to seed disseminated infections, providing a pathway for the development of targeted anti-adhesion therapeutics.

Antibiotic-resistant bacteria are an urgent threat to human health. Here, Roberts et al. characterise and monitor an ongoing hospital outbreak of carbapenemase-producing Enterobacter hormaechei by integrating several technologies for whole-genome sequencing and shotgun metagenomics.

ABSTRACT Many antibiotic resistant uropathogenic Escherichia coli (UPEC) strains belong to clones defined by their multilocus sequence type (ST), with ST131 being the most dominant. Although we have a good understanding of resistance development to fluoroquinolones and third-generation cephalosporins by ST131, our understanding of the virulence repertoire that has contributed to its global dissemination is limited. Here we show that the genes encoding Afa/Dr fimbriae, a group of adhesins strongly associated with UPEC that cause gestational pyelonephritis and recurrent cystitis, are found in approximately one third of all ST131 strains. Sequence comparison of the AfaE adhesin protein revealed a unique allelic variant carried by 82.9% of afa-positive ST131 strains. We identify the afa regulatory region as a hotspot for the integration of insertion sequence (IS) elements, all but one of which alter afa transcription. Close investigation demonstrated that the integration of an IS1 element in the afa regulatory region leads to increased expression of Afa/Dr fimbriae, promoting enhanced adhesion to kidney epithelial cells and suggesting a mechanism for altered virulence. Finally, we provide evidence for a more widespread impact of IS1 on ST131 genome evolution, suggesting that IS dynamics contribute to strain level microevolution that impacts ST131 fitness. IMPORTANCE E. coli ST131 is the most common antibiotic resistant UPEC clone associated with human urinary tract and bloodstream infections. Understanding the features of ST131 that have driven its global dissemination remains a critical priority if we are to counter its increasing antibiotic resistance. Here, we utilized a large collection of ST131 isolates to investigate the prevalence, regulation, and function of Afa/Dr fimbriae, a well-characterized UPEC colonization and virulence factor. We show that the afa genes are found frequently in ST131 and demonstrate how the integration of IS elements in the afa regulatory region modulates Afa expression, presenting an example of altered virulence capacity. We also exploit a curated set of ST131 genomes to map the integration of the antibiotic resistance-associated IS1 element in the ST131 pangenome, providing evidence for its widespread impact on ST131 genome evolution.

ABSTRACT Acinetobacter baumannii causes high mortality in ventilator-associated pneumonia patients, and antibiotic treatment is compromised by multidrug-resistant strains resistant to β-lactams, carbapenems, cephalosporins, polymyxins, and tetracyclines. Among COVID-19 patients receiving ventilator support, a multidrug-resistant A. baumannii secondary infection is associated with a 2-fold increase in mortality. Here, we investigated the use of the 8-hydroxyquinoline ionophore PBT2 to break the resistance of A. baumannii to tetracycline class antibiotics. In vitro, the combination of PBT2 and zinc with either tetracycline, doxycycline, or tigecycline was shown to be bactericidal against multidrug-resistant A. baumannii, and any resistance that did arise imposed a fitness cost. PBT2 and zinc disrupted metal ion homeostasis in A. baumannii, increasing cellular zinc and copper while decreasing magnesium accumulation. Using a murine model of pulmonary infection, treatment with PBT2 in combination with tetracycline or tigecycline proved efficacious against multidrug-resistant A. baumannii. These findings suggest that PBT2 may find utility as a resistance breaker to rescue the efficacy of tetracycline-class antibiotics commonly employed to treat multidrug-resistant A. baumannii infections. IMPORTANCE Within intensive care unit settings, multidrug-resistant (MDR) Acinetobacter baumannii is a major cause of ventilator-associated pneumonia, and hospital-associated outbreaks are becoming increasingly widespread. Antibiotic treatment of A. baumannii infection is often compromised by MDR strains resistant to last-resort β-lactam (e.g., carbapenems), polymyxin, and tetracycline class antibiotics. During the on-going COVID-19 pandemic, secondary bacterial infection by A. baumannii has been associated with a 2-fold increase in COVID-19-related mortality. With a rise in antibiotic resistance and a reduction in new antibiotic discovery, it is imperative to investigate alternative therapeutic regimens that complement the use of current antibiotic treatment strategies. Rescuing the efficacy of existing therapies for the treatment of MDR A. baumannii infection represents a financially viable pathway, reducing time, cost, and risk associated with drug innovation.

BackgroundHereditary cancer syndromes (HCS) are responsible for 5-10% of cancer cases. Genetic testing to identify pathogenic variants associated with cancer predisposition has not been routinely available in Vietnam. Consequently, the prevalence and genetic landscape of HCS remain unknown.Methods1165 Vietnamese individuals enrolled in genetic testing at our laboratory in 2020. We performed analysis of germline mutations in 17 high- and moderate- penetrance genes associated with HCS by next generation sequencing.ResultsA total of 41 pathogenic variants in 11 genes were detected in 3.2% individuals. The carrier frequency was 4.2% in people with family or personal history of cancer and 2.6% in those without history. The percentage of mutation carriers for hereditary colorectal cancer syndromes was 1.3% and for hereditary breast and ovarian cancer syndrome was 1.6%. BRCA1 and BRCA2 mutations were the most prevalent with the positive rate of 1.3% in the general cohort and 5.1% in breast or ovarian cancer patients. Most of BRCA1 mutations located at the BRCA C-terminus domains and the top recurrent mutation was NM_007294.3:c.5251C>T (p.Arg1751Ter). One novel variant NM_000038.6(APC):c.6665C>A (p.Pro2222His) was found in a breast cancer patient with a strong family history of cancer. A case study of hereditary cancer syndrome was illustrated to highlight the importance of genetic testing.ConclusionThis is the first largest analysis of carrier frequency and mutation spectrum of HCS in Vietnam. The findings demonstrate the clinical significance of multigene panel testing to identify carriers and their at-risk relatives for better cancer surveillance and management strategies.

Recurrent urinary tract infections occur in ~ 25% of women. Here, Beatson and colleagues use whole genome sequencing to track the dynamics of an E. coli ST131 clone in a single patient over a 5-year period. This study provides unique insights into pathogen evolution during recurrent urinary infection.

Autotransporter proteins are localised to the bacterial surface and promote colonisation of host epithelial surfaces. Here, the authors present the crystal structure of autotransporter UpaB and show evidence for distinct binding sites for glycosaminoglycans and host fibronectin.

Population-specific profiling of mutations in cancer genes is of critical importance for the understanding of cancer biology in general as well as the establishment of optimal diagnostics and treatment guidelines for that particular population. Although genetic analysis of tumor tissue is often used to detect mutations in cancer genes, the invasiveness and limited accessibility hinders its application in large-scale population studies. Here, we used ultra-deep massive parallel sequencing of plasma cell free DNA (cfDNA) to identify the mutation profiles of 265 Vietnamese patients with advanced non-small cell lung cancer (NSCLC). Compared to a cohort of advanced NSCLC patients characterized by sequencing of tissue samples, cfDNA genomic testing, despite lower mutation detection rates, was able to detect major mutations in tested driver genes that reflected similar mutation composition and distribution pattern, as well as major associations between mutation prevalence and clinical features. In conclusion, ultra-deep sequencing of plasma cfDNA represents an alternative approach for population-wide genetic profiling of cancer genes where recruitment of patients is limited to the accessibility of tumor tissue site.

Uropathogenic E. coli (UPEC) causes the majority of urinary tract infections (UTIs), which are a major global public health concern. UPEC uses numerous mechanisms to subvert the innate immune system, including targeting macrophage functions. We recently showed that some UPEC strains rapidly kill human macrophages via an NLRP3-independent pathway, and also trigger NLRP3-dependent IL-1β processing. In this study, we used random transposon mutagenesis in the reference strain CFT073 to identify UPEC genes that mediate human macrophage cell death. Our approach revealed that the hemolysin A (HlyA) toxin is essential for triggering both cell death and NLRP3 inflammasome-mediated IL-1β release in human macrophages. Random transposon mutagenesis also identified the cof gene, which encodes a poorly characterized phosphatase, as a novel hemolysin regulator; a CFT073 mutant deleted for the cof gene secreted significantly reduced levels of HlyA, had diminished hemolytic activity, and was impaired in its capacity to trigger human macrophage cell death and IL-1β release. Together, our findings reveal that Cof fine-tunes production of hemolysin, an important determinant of both UPEC-mediated inflammasome activation and human macrophage cell death.

Abstract Background Familial adenomatous polyposis (FAP) is an autosomal dominant hereditary syndrome characterised by the development of hundreds to thousands of adenomatous colonic polyps during the second decade of life. FAP is caused by germ line mutations in the adenomatous polyposis coli (APC) gene located on chromosome 5q21–22. Case presentation A 36-year-old female was presented with 100–1000 adenomatous colonic polyps, typical of classic FAP symptoms. Genetic testing using massively parallel sequencing identified a 5-bp deletion (c.3927_3931delAAAGA) which causes frameshift (p.Glu1309Aspfs) and creates a premature stop codon, resulting in the replacement of the last 1535 amino acids of APC by five incorrect amino acids. Two of the proband’s four siblings also exhibited classic FAP symptoms and carried the same 5-bp heterozygous deletion in the APC gene. One of the proband’s two nephews also tested positive for this mutation but has not been examined by endoscopy due to his young age. Conclusions We reported here for the first time the use of massively parallel sequencing (MPS)-based genetic testing to identify a germline mutation within a three-generation Vietnamese family. This mutation is most likely responsible for the development of FAP.

ABSTRACT Uropathogenic Escherichia coli (UPEC) is the major cause of urinary tract infections. Nearly half of all UPEC strains secrete hemolysin, a cytotoxic pore-forming toxin. Here, we show that the prevalence of the hemolysin toxin gene (hlyA) is highly variable among the most common 83 E. coli sequence types (STs) represented on the EnteroBase genome database. To explore this diversity in the context of a defined monophyletic lineage, we contextualized sequence variation of the hlyCABD operon within the genealogy of the globally disseminated multidrug-resistant ST131 clone. We show that sequence changes in hlyCABD and its newly defined 1.616-kb-long leader sequence correspond to phylogenetic designation, and that ST131 strains with the strongest hemolytic activity belong to the most extensive multidrug-resistant sublineage (clade C2). To define the set of genes involved in hemolysin production, the clade C2 strain S65EC was completely sequenced and subjected to a genome-wide screen by combining saturated transposon mutagenesis and transposon-directed insertion site sequencing with the capacity to lyse red blood cells. Using this approach, and subsequent targeted mutagenesis and complementation, 13 genes were confirmed to be specifically required for production of active hemolysin. New hemolysin-controlling elements included discrete sets of genes involved in lipopolysaccharide (LPS) inner core biosynthesis (waaC, waaF, waaG, and rfaE) and cytoplasmic chaperone activity (dnaK and dnaJ), and we show these are required for hemolysin secretion. Overall, this work provides a unique description of hemolysin sequence diversity in a single clonal lineage and describes a complex multilevel system of regulatory control for this important toxin. IMPORTANCE Uropathogenic E. coli (UPEC) is the major cause of urinary tract infections and a frequent cause of sepsis. Nearly half of all UPEC strains produce the potent cytotoxin hemolysin, and its expression is associated with enhanced virulence. In this study, we explored hemolysin variation within the globally dominant UPEC ST131 clone, finding that strains from the ST131 sublineage with the greatest multidrug resistance also possess the strongest hemolytic activity. We also employed an innovative forward genetic screen to define the set of genes required for hemolysin production. Using this approach, and subsequent targeted mutagenesis and complementation, we identified new hemolysin-controlling elements involved in LPS inner core biosynthesis and cytoplasmic chaperone activity, and we show that mechanistically they are required for hemolysin secretion. These original discoveries substantially enhance our understanding of hemolysin regulation, secretion and function.

ABSTRACT Proteins secreted by the type V secretion system possess multiple functions, including the capacity to mediate adhesion, aggregation, and biolfilm formation. The type V secretion system can be divided into five subclasses, one of which is the type Ve system. Proteins of the type Ve secretion system are also referred to as inverse autotransporters (IATs). In this study, we performed an in silico analysis of 126 completely sequenced Escherichia coli genomes available in the NCBI database and identified several distinct IAT-encoding gene families whose distribution varied throughout the E. coli phylogeny. The genes included three characterized IATs (intimin, fdeC, and yeeJ) and four uncharacterized IATs (here named iatA, iatB, iatC, and iatD). The four iat genes were cloned from the completely sequenced environmental E. coli strain SMS-3-5 and characterized. Three of these IAT proteins (IatB, IatC, and IatD) were expressed at the cell surface and possessed the capacity to mediate biofilm formation in a recombinant E. coli K-12 strain. Further analysis of the iatB gene, which showed a unique association with extraintestinal E. coli strains, suggested that its regulation is controlled by the LeuO global regulator. Overall, this study provides new data describing the prevalence, sequence variation, domain structure, function, and regulation of IATs found in E. coli. IMPORTANCE Escherichia coli is one of the most prevalent facultative anaerobes of the human gut. E. coli normally exists as a harmless commensal but can also cause disease following the acquisition of genes that enhance its pathogenicity. Adhesion is an important first step in colonization of the host and is mediated by an array of cell surface components. In E. coli, these include a family of adhesins secreted by the type V secretion system. Here, we identified and characterized new proteins from an emerging subclass of the type V secretion system known as the inverse autotransporters (IATs). We found that IAT-encoding genes are present in a wide range of strains and showed that three novel IATs were localized on the E. coli cell surface and mediated biofilm formation. Overall, this study provides new insight into the prevalence, function, and regulation of IATs in E. coli.

The identification and quantification of actionable mutations are of critical importance for effective genotype-directed therapies, prognosis and drug response monitoring in patients with non-small-cell lung cancer (NSCLC). Although tumor tissue biopsy remains the gold standard for diagnosis of NSCLC, the analysis of circulating tumor DNA (ctDNA) in plasma, known as liquid biopsy, has recently emerged as an alternative and noninvasive approach for exploring tumor genetic constitution. In this study, we developed a protocol for liquid biopsy using ultra-deep massively parallel sequencing (MPS) with unique molecular identifier tagging and evaluated its performance for the identification and quantification of tumor-derived mutations from plasma of patients with advanced NSCLC. Paired plasma and tumor tissue samples were used to evaluate mutation profiles detected by ultra-deep MPS, which showed 87.5% concordance. Cross-platform comparison with droplet digital PCR demonstrated comparable detection performance (91.4% concordance, Cohen's kappa coefficient of 0.85 with 95% CI = 0.72-0.97) and great reliability in quantification of mutation allele frequency (Intraclass correlation coefficient of 0.96 with 95% CI = 0.90-0.98). Our results highlight the potential application of liquid biopsy using ultra-deep MPS as a routine assay in clinical practice for both detection and quantification of actionable mutation landscape in NSCLC patients.

ABSTRACT The World Health Organization reports that antibiotic-resistant pathogens represent an imminent global health disaster for the 21st century. Gram-positive superbugs threaten to breach last-line antibiotic treatment, and the pharmaceutical industry antibiotic development pipeline is waning. Here we report the synergy between ionophore-induced physiological stress in Gram-positive bacteria and antibiotic treatment. PBT2 is a safe-for-human-use zinc ionophore that has progressed to phase 2 clinical trials for Alzheimer’s and Huntington’s disease treatment. In combination with zinc, PBT2 exhibits antibacterial activity and disrupts cellular homeostasis in erythromycin-resistant group A Streptococcus (GAS), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE). We were unable to select for mutants resistant to PBT2-zinc treatment. While ineffective alone against resistant bacteria, several clinically relevant antibiotics act synergistically with PBT2-zinc to enhance killing of these Gram-positive pathogens. These data represent a new paradigm whereby disruption of bacterial metal homeostasis reverses antibiotic-resistant phenotypes in a number of priority human bacterial pathogens. IMPORTANCE The rise of bacterial antibiotic resistance coupled with a reduction in new antibiotic development has placed significant burdens on global health care. Resistant bacterial pathogens such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus are leading causes of community- and hospital-acquired infection and present a significant clinical challenge. These pathogens have acquired resistance to broad classes of antimicrobials. Furthermore, Streptococcus pyogenes, a significant disease agent among Indigenous Australians, has now acquired resistance to several antibiotic classes. With a rise in antibiotic resistance and reduction in new antibiotic discovery, it is imperative to investigate alternative therapeutic regimens that complement the use of current antibiotic treatment strategies. As stated by the WHO Director-General, “On current trends, common diseases may become untreatable. Doctors facing patients will have to say, Sorry, there is nothing I can do for you.”

ABSTRACT Curli are bacterial surface-associated amyloid fibers that bind to the dye Congo red (CR) and facilitate uropathogenic Escherichia coli (UPEC) biofilm formation and protection against host innate defenses. Here we sequenced the genome of the curli-producing UPEC pyelonephritis strain MS7163 and showed it belongs to the highly virulent O45:K1:H7 neonatal meningitis-associated clone. MS7163 produced curli at human physiological temperature, and this correlated with biofilm growth, resistance of sessile cells to the human cationic peptide cathelicidin, and enhanced colonization of the mouse bladder. We devised a forward genetic screen using CR staining as a proxy for curli production and identified 41 genes that were required for optimal CR binding, of which 19 genes were essential for curli synthesis. Ten of these genes were novel or poorly characterized with respect to curli synthesis and included genes involved in purine de novo biosynthesis, a regulator that controls the Rcs phosphorelay system, and a novel repressor of curli production (referred to as rcpA). The involvement of these genes in curli production was confirmed by the construction of defined mutants and their complementation. The mutants did not express the curli major subunit CsgA and failed to produce curli based on CR binding. Mutation of purF (the first gene in the purine biosynthesis pathway) and rcpA also led to attenuated colonization of the mouse bladder. Overall, this work has provided new insight into the regulation of curli and the role of these amyloid fibers in UPEC biofilm formation and pathogenesis. IMPORTANCE Uropathogenic Escherichia coli (UPEC) strains are the most common cause of urinary tract infection, a disease increasingly associated with escalating antibiotic resistance. UPEC strains possess multiple surface-associated factors that enable their colonization of the urinary tract, including fimbriae, curli, and autotransporters. Curli are extracellular amyloid fibers that enhance UPEC virulence and promote biofilm formation. Here we examined the function and regulation of curli in a UPEC pyelonephritis strain belonging to the highly virulent O45:K1:H7 neonatal meningitis-associated clone. Curli expression at human physiological temperature led to increased biofilm formation, resistance of sessile cells to the human cationic peptide LL-37, and enhanced bladder colonization. Using a comprehensive genetic screen, we identified multiple genes involved in curli production, including several that were novel or poorly characterized with respect to curli synthesis. In total, this study demonstrates an important role for curli as a UPEC virulence factor that promotes biofilm formation, resistance, and pathogenesis.

Escherichia coli ST131 is a recently emerged and globally disseminated multidrug resistant clone associated with urinary tract and bloodstream infections in both community and clinical settings. The most common group of ST131 strains are defined by resistance to fluoroquinolones and possession of the type 1 fimbriae fimH30 allele. Here we provide an update on our recent work describing the globally epidemiology of ST131. We review the phylogeny of ST131 based on whole genome sequence data and highlight the important role of recombination in the evolution of this clonal lineage. We also summarize our findings on the virulence of the ST131 reference strain EC958, and highlight the use of transposon directed insertion-site sequencing to define genes associated with serum resistance and essential features of its large antibiotic resistance plasmid pEC958.

ABSTRACT Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract and bloodstream infections and possesses an array of virulence factors for colonization, survival, and persistence. One such factor is the polysaccharide K capsule. Among the different K capsule types, the K1 serotype is strongly associated with UPEC infection. In this study, we completely sequenced the K1 UPEC urosepsis strain PA45B and employed a novel combination of a lytic K1 capsule-specific phage, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing (TraDIS) to identify the complement of genes required for capsule production. Our analysis identified known genes involved in capsule biosynthesis, as well as two additional regulatory genes (mprA and lrhA) that we characterized at the molecular level. Mutation of mprA resulted in protection against K1 phage-mediated killing, a phenotype restored by complementation. We also identified a significantly increased unidirectional Tn5 insertion frequency upstream of the lrhA gene and showed that strong expression of LrhA induced by a constitutive Pcl promoter led to loss of capsule production. Further analysis revealed loss of MprA or overexpression of LrhA affected the transcription of capsule biosynthesis genes in PA45B and increased sensitivity to killing in whole blood. Similar phenotypes were also observed in UPEC strains UTI89 (K1) and CFT073 (K2), demonstrating that the effects were neither strain nor capsule type specific. Overall, this study defined the genome of a UPEC urosepsis isolate and identified and characterized two new regulatory factors that affect UPEC capsule production. IMPORTANCE Urinary tract infections (UTIs) are among the most common bacterial infections in humans and are primarily caused by uropathogenic Escherichia coli (UPEC). Many UPEC strains express a polysaccharide K capsule that provides protection against host innate immune factors and contributes to survival and persistence during infection. The K1 serotype is one example of a polysaccharide capsule type and is strongly associated with UPEC strains that cause UTIs, bloodstream infections, and meningitis. The number of UTIs caused by antibiotic-resistant UPEC is steadily increasing, highlighting the need to better understand factors (e.g., the capsule) that contribute to UPEC pathogenesis. This study describes the original and novel application of lytic capsule-specific phage killing, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing to define the entire complement of genes required for capsule production in UPEC. Our comprehensive approach uncovered new genes involved in the regulation of this key virulence determinant.

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.

ABSTRACT Escherichia coli is a versatile pathogen capable of causing intestinal and extraintestinal infections that result in a huge burden of global human disease. The diversity of E. coli is reflected by its multiple different pathotypes and mosaic genome composition. E. coli strains are also a major driver of antibiotic resistance, emphasizing the urgent need for new treatment and prevention measures. Here, we used a large data set comprising 1,700 draft and complete genomes to define the core and accessory genome of E. coli and demonstrated the overlapping relationship between strains from different pathotypes. In combination with proteomic investigation, this analysis revealed core genes that encode surface-exposed or secreted proteins that represent potential broad-coverage vaccine antigens. One of these antigens, YncE, was characterized as a conserved immunogenic antigen able to protect against acute systemic infection in mice after vaccination. Overall, this work provides a genomic blueprint for future analyses of conserved and accessory E. coli genes. The work also identified YncE as a novel antigen that could be exploited in the development of a vaccine against all pathogenic E. coli strains—an important direction given the high global incidence of infections caused by multidrug-resistant strains for which there are few effective antibiotics. IMPORTANCE E. coli is a multifaceted pathogen of major significance to global human health and an important contributor to increasing antibiotic resistance. Given the paucity of therapies still effective against multidrug-resistant pathogenic E. coli strains, novel treatment and prevention strategies are urgently required. In this study, we defined the core and accessory components of the E. coli genome by examining a large collection of draft and completely sequenced strains available from public databases. This data set was mined by employing a reverse-vaccinology approach in combination with proteomics to identify putative broadly protective vaccine antigens. One such antigen was identified that was highly immunogenic and induced protection in a mouse model of bacteremia. Overall, our study provides a genomic and proteomic framework for the selection of novel vaccine antigens that could mediate broad protection against pathogenic E. coli.

Plasmids of incompatibility group (Inc) HI1 are important vectors of antibiotic resistance in both of the major causal agents of enteric fever: Salmonella enterica subspecies enterica serovar Typhi and S. Paratyphi A. In S. Typhi, IncHI1 plasmids appeared in the 1970s and spread globally. In some circumstances they are maintained within the bacterial population even in the absence of selection from antibiotics. The low cost associated with IncH plasmids in Salmonella is due, in part, to the presence of a plasmid gene encoding an H-NS-like global regulator which acts co-operatively with chromosomally encoded H-NS. Very recently, IncHI1 plasmids have crossed from S. Typhi into S. Paratyphi A; the acquisition of drug resistance and possibly other phenotypic traits encoded by IncHI1 plasmids has increased the virulence potential of this neglected pathogen. There is no vaccine for S. Paratyphi A and resistance to the current drugs of choice, the fluoroquinolones, is also spreading rapidly. There is a conserved backbone to all IncH plasmids but variation occurs in regions of the plasmids associated with antibiotic resistance. These IncHI1 plasmids are allowing major human pathogens to sample genes available in their environment, the human gut, and will be maintained by enhancing the competitive advantage of the bacterial host. Therefore competition between closely related resistance plasmids will probably increase the transmission of enteric fever by enhancing the fitness of their bacterial hosts.

ABSTRACT Escherichia coli sequence type 131 (ST131) is a clone of uropathogenic E. coli that has emerged rapidly and disseminated globally in both clinical and community settings. Members of the ST131 lineage from across the globe have been comprehensively characterized in terms of antibiotic resistance, virulence potential, and pathogenicity, but to date nothing is known about the methylome of these important human pathogens. Here we used single-molecule real-time (SMRT) PacBio sequencing to determine the methylome of E. coli EC958, the most-well-characterized completely sequenced ST131 strain. Our analysis of 52,081 methylated adenines in the genome of EC958 discovered three m6A methylation motifs that have not been described previously. Subsequent SMRT sequencing of isogenic knockout mutants identified the two type I methyltransferases (MTases) and one type IIG MTase responsible for m6A methylation of novel recognition sites. Although both type I sites were rare, the type IIG sites accounted for more than 12% of all methylated adenines in EC958. Analysis of the distribution of MTase genes across 95 ST131 genomes revealed their prevalence is highly conserved within the ST131 lineage, with most variation due to the presence or absence of mobile genetic elements on which individual MTase genes are located. IMPORTANCE DNA modification plays a crucial role in bacterial regulation. Despite several examples demonstrating the role of methyltransferase (MTase) enzymes in bacterial virulence, investigation of this phenomenon on a whole-genome scale has remained elusive until now. Here we used single-molecule real-time (SMRT) sequencing to determine the first complete methylome of a strain from the multidrug-resistant E. coli sequence type 131 (ST131) lineage. By interrogating the methylome computationally and with further SMRT sequencing of isogenic mutants representing previously uncharacterized MTase genes, we defined the target sequences of three novel ST131-specific MTases and determined the genomic distribution of all MTase target sequences. Using a large collection of 95 previously sequenced ST131 genomes, we identified mobile genetic elements as a major factor driving diversity in DNA methylation patterns. Overall, our analysis highlights the potential for DNA methylation to dramatically influence gene regulation at the transcriptional level within a well-defined E. coli clone.

Escherichia coli ST131 is now recognised as a leading contributor to urinary tract and bloodstream infections in both community and clinical settings. Here we present the complete, annotated genome of E. coli EC958, which was isolated from the urine of a patient presenting with a urinary tract infection in the Northwest region of England and represents the most well characterised ST131 strain. Sequencing was carried out using the Pacific Biosciences platform, which provided sufficient depth and read-length to produce a complete genome without the need for other technologies. The discovery of spurious contigs within the assembly that correspond to site-specific inversions in the tail fibre regions of prophages demonstrates the potential for this technology to reveal dynamic evolutionary mechanisms. E. coli EC958 belongs to the major subgroup of ST131 strains that produce the CTX-M-15 extended spectrum β-lactamase, are fluoroquinolone resistant and encode the fimH30 type 1 fimbrial adhesin. This subgroup includes the Indian strain NA114 and the North American strain JJ1886. A comparison of the genomes of EC958, JJ1886 and NA114 revealed that differences in the arrangement of genomic islands, prophages and other repetitive elements in the NA114 genome are not biologically relevant and are due to misassembly. The availability of a high quality uropathogenic E. coli ST131 genome provides a reference for understanding this multidrug resistant pathogen and will facilitate novel functional, comparative and clinical studies of the E. coli ST131 clonal lineage.

Urinary tract infection (UTI) is one of the most common bacterial infections in humans, with uropathogenic Escherichia coli (UPEC) the leading causative organism. UPEC has a number of virulence factors that enable it to overcome host defenses within the urinary tract and establish infection. The O antigen and the capsular polysaccharide are two such factors that provide a survival advantage to UPEC. Here we describe the application of the rpsL counter selection system to construct capsule (kpsD) and O antigen (waaL) mutants and complemented derivatives of three reference UPEC strains: CFT073 (O6:K2:H1), RS218 (O18:K1:H7) and 1177 (O1:K1:H7). We observed that while the O1, O6 and O18 antigens were required for survival in human serum, the role of the capsule was less clear and linked to O antigen type. In contrast, both the K1 and K2 capsular antigens provided a survival advantage to UPEC in whole blood. In the mouse urinary tract, mutation of the O6 antigen significantly attenuated CFT073 bladder colonization. Overall, this study contrasts the role of capsule and O antigen in three common UPEC serotypes using defined mutant and complemented strains. The combined mutagenesis-complementation strategy can be applied to study other virulence factors with complex functions both in vitro and in vivo.

Escherichia coli ST131 is a globally disseminated, multidrug resistant clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with antibiotic resistance; however, this phenotype alone is unlikely to explain its dominance amongst multidrug resistant uropathogens circulating worldwide in hospitals and the community. Thus, a greater understanding of the molecular mechanisms that underpin the fitness of E. coli ST131 is required. In this study, we employed hyper-saturated transposon mutagenesis in combination with multiplexed transposon directed insertion-site sequencing to define the essential genes required for in vitro growth and the serum resistome (i.e. genes required for resistance to human serum) of E. coli EC958, a representative of the predominant E. coli ST131 clonal lineage. We identified 315 essential genes in E. coli EC958, 231 (73%) of which were also essential in E. coli K-12. The serum resistome comprised 56 genes, the majority of which encode membrane proteins or factors involved in lipopolysaccharide (LPS) biosynthesis. Targeted mutagenesis confirmed a role in serum resistance for 46 (82%) of these genes. The murein lipoprotein Lpp, along with two lipid A-core biosynthesis enzymes WaaP and WaaG, were most strongly associated with serum resistance. While LPS was the main resistance mechanism defined for E. coli EC958 in serum, the enterobacterial common antigen and colanic acid also impacted on this phenotype. Our analysis also identified a novel function for two genes, hyxA and hyxR, as minor regulators of O-antigen chain length. This study offers novel insight into the genetic make-up of E. coli ST131, and provides a framework for future research on E. coli and other Gram-negative pathogens to define their essential gene repertoire and to dissect the molecular mechanisms that enable them to survive in the bloodstream and cause disease.

Escherichia coli strains causing urinary tract infection (UTI) are increasingly recognized as belonging to specific clones. E. coli clone O25b:H4-ST131 has recently emerged globally as a leading multi-drug resistant pathogen causing urinary tract and bloodstream infections in hospitals and the community. While most molecular studies to date examine the mechanisms conferring multi-drug resistance in E. coli ST131, relatively little is known about their virulence potential. Here we examined E. coli ST131 clinical isolates from two geographically diverse collections, one representing the major pathogenic lineages causing UTI across the United Kingdom and a second representing UTI isolates from patients presenting at two large hospitals in Australia. We determined a draft genome sequence for one representative isolate, E. coli EC958, which produced CTX-M-15 extended-spectrum β-lactamase, CMY-23 type AmpC cephalosporinase and was resistant to ciprofloxacin. Comparative genome analysis indicated that EC958 encodes virulence genes commonly associated with uropathogenic E. coli (UPEC). The genome sequence of EC958 revealed a transposon insertion in the fimB gene encoding the activator of type 1 fimbriae, an important UPEC bladder colonization factor. We identified the same fimB transposon insertion in 59% of the ST131 UK isolates, as well as 71% of ST131 isolates from Australia, suggesting this mutation is common among E. coli ST131 strains. Insertional inactivation of fimB resulted in a phenotype resembling a slower off-to-on switching for type 1 fimbriae. Type 1 fimbriae expression could still be induced in fimB-null isolates; this correlated strongly with adherence to and invasion of human bladder cells and bladder colonisation in a mouse UTI model. We conclude that E. coli ST131 is a geographically widespread, antibiotic resistant clone that has the capacity to produce numerous virulence factors associated with UTI.

Complementation remains a foundation for demonstrating molecular Koch's postulates. While this is frequently achieved using plasmids, limitations such as increased gene copy number and the need for antibiotic supplementation to avoid plasmid loss can restrict their use. Chromosomal integration systems using the Tn7 transposon provide an alternative to plasmids for complementation and facilitate the stable insertion of genes at the chromosomal attTn7 site without the need for selection pressure. Here, we enhanced the utility of mini-Tn7 insertion vectors by the addition of inducible (Pcym) and constitutive (PcL and PrpsM) promoters, allowing different ial transcriptional control of genes integrated into the chromosome. We validated the utility of these promoters by cloning the gfp gene, encoding green fluorescent protein, downstream of each promoter and integrating a mini-Tn7 construct harboring these elements into the attTn7 site on the chromosome of the Escherichia coli K-12 strain MG1655. The PcL and PrpsM promoters provided equivalent levels of GFP expression and offered flexibility based on the target host strain. Activation of the tightly regulated Pcym promoter with its inducer cumate resulted in tunable expression of GFP in a dose-dependent manner. We further demonstrated the tight control of the Pcym promoter using the toxic impCAB genes, and the expression of which is detrimental to E. coli viability. Together, these modified mini-Tn7 vectors allowing differential control of genes integrated into the chromosome at a conserved site offer an efficient system for complementation where plasmid use is restricted. IMPORTANCE Chromosomal integration using mini-Tn7 vectors provides an efficient means to insert genes into the chromosome of many gram-negative bacteria. Insertion occurs at a conserved site and allows for the stable integration of genes in single copy. While this system has multiple benefits for enabling complementation, a cornerstone for fulfilling molecular Koch's postulates, greater flexibility for controlled gene expression would enhance its utility. Here, we have added to the function of mini-Tn7 vectors by the addition of inducible and constitutive promoters and demonstrated their capacity to drive the controlled expression of target genes integrated into the chromosome. In addition to complementation, these modified vectors offer broad application for other approaches including chromosomal tagging, in vivo expression, metabolic engineering, and synthetic biology. [ABSTRACT FROM AUTHOR]