Louise Pankhurst

The human gut microbiome plays an important role in resisting colonization of the host by pathogens, but we lack the ability to predict which communities will be protective. We studied how human gut bacteria influence colonization of two major bacterial pathogens, both in vitro and in gnotobiotic mice. Whereas single species alone had negligible effects, colonization resistance greatly increased with community diversity. Moreover, this community-level resistance rested critically upon certain species being present. We explained these ecological patterns through the collective ability of resistant communities to consume nutrients that overlap with those used by the pathogen. Furthermore, we applied our findings to successfully predict communities that resist a novel target strain. Our work provides a reason why microbiome diversity is beneficial and suggests a route for the rational design of pathogen-resistant communities.

Background: Bacterial genomes follow a U-shaped frequency distribution whereby most genomic loci are either rare (accessory) or common (core); the union of these is the pan-genome. The alignable fraction of two genomes from a single species can be low (e.g. 50-70%), such that no single reference genome can access all single nucleotide polymorphisms (SNPs). The pragmatic solution is to choose a close reference, and analyse SNPs only in the core genome. Given much bacterial adaptability hinges on the accessory genome, this is an unsatisfactory limitation. Results: We present a novel pan-genome graph structure and algorithms implemented in the software pandora , which approximates a sequenced genome as a recombinant of reference genomes, detects novel variation and then pan-genotypes multiple samples. The method takes fastq as input and outputs a multi-sample VCF with respect to an inferred data-dependent reference genome, and is available at https://github.com/rmcolq/pandora . Constructing a reference graph from 578 E. coli genomes, we analyse a diverse set of 20 E. coli isolates. We show pandora recovers at least 13k more rare SNPs than single-reference based tools, achieves equal or better error rates with Nanopore as with Illumina data, 6-24x lower Nanopore error rates than other tools, and provides a stable framework for analysing diverse samples without reference bias. We also show that our inferred recombinant VCF reference genome is significantly better than simply picking the closest RefSeq reference. Conclusions: This is a step towards comprehensive cohort analysis of bacterial pan-genomic variation, with potential impacts on genotype/phenotype and epidemiological studies.

Sequencing of pathogen DNA directly from clinical samples offers the possibilities of rapid diagnosis, faster antimicrobial resistance prediction and enhanced outbreak investigation. The approach is especially advantageous for infections caused by species which grow very slowly in culture, such as Mycobacteria tuberculosis. Since the pathogen of interest may represent as little as 0.01% of the total DNA, enrichment of the input material for target sequences by specific amplification and, or depletion of non-target DNA (human, other bacteria) is essential for success. Here, we investigated the potential of isothermal multiple displacement amplification by Phi29 polymerase. We directed the amplification reaction towards Mycobacteria DNA in sputum samples by exploiting in our oligonucleotide primer design, their high GC content (approximately 65%) relative to human DNA. Amplified DNA was then sequenced using the Oxford Nanopore Technology MinION. In addition, a model system comprising standardised ‘mock clinical samples’ was designed. Pooled infection negative human sputum samples were spiked with enumerated Mycobacterium bovis (BCG) Pasteur strain at concentrations spanning the typical range at which Mycobacterium tuberculosis is found in human sputum samples (106 - 101 BCG cells/ml). To assess the amount of BCG sequence enrichment achieved, sample DNA was sequenced both before, and after amplification. Reads from amplified samples, which mapped to a BCG reference genome, comprised short repeated sequences - apparently transcribed multiple times from the same fragment of BCG DNA. Therefore post-amplification, the samples were enriched for BCG sequences relative to unamplified sequences (8,101 BCG reference mapped reads, increasing to 28,617 at 106 BCG cells/ml sample), but BCG genome coverage declined markedly (for example 89.4% to 4.1%). In summary, the use of standardised mock clinical samples allowed direct comparison of data from different Mycobacteria enrichment experiments and sequencing runs. However, optimal conditions for multiple displacement amplification of minority Mycobacteria DNAs, remain to be identified.

Carbapenem-resistant Enterobacteriaceae (CRE) are a health threat, but effective control interventions remain unclear. Hospital wastewater sites are increasingly highlighted as important potential reservoirs. We investigated a large Klebsiella pneumoniae carbapenemase (KPC)-producing E. coli (KPC-EC) outbreak and wider CRE incidence trends over eight years in the Central Manchester Foundation NHS Trust (CMFT), UK, to determine the impact of Infection Prevention and Control measures. Bacteriology and patient administration data (2009 to 2017) were linked; a subset of CMFT/regional KPC-EC isolates (n=268) was sequenced. Control interventions followed international guidelines and included cohorting, rectal screening (n=184,539 screens), environmental sampling, enhanced cleaning, and ward closure/plumbing replacement. Segmented regression of time trends of CRE detections was used to evaluate the impact of interventions on CRE incidence. Genomic analysis (n=268 isolates) identified spread of a KPC-EC outbreak clone (ST216, strain-A; n=125) amongst patients and the environment, particularly on two cardiac wards (W3/W4), despite control measures. ST216 strain-A had caused an antecedent outbreak, and shared its KPC plasmids with other E. coli lineages and Enterobacteriaceae. CRE acquisition incidence declined after W3/W4 closure and plumbing replacement, suggesting an environmental contribution. However, W3/W4 wastewater sites were rapidly re-colonised with CRE and patient CRE acquisitions recurred, albeit at lower rates. Patient relocation and plumbing replacement were associated with control of a clonal KPC-EC outbreak; however, environmental contamination with CRE and patient CRE acquisitions recurred rapidly following this intervention. The large numbers of cases and persistence of bla_KPC in E. coli, including pathogenic lineages, is a concern.

Purpose. Speed of bloodstream infection diagnosis is vital to reduce morbidity and mortality. Whole genome sequencing (WGS) performed directly from liquid blood culture could provide single-assay species and antibiotic susceptibility prediction; however, high inhibitor and human cell/DNA concentrations limit pathogen recovery. We develop a method for the preparation of bacterial DNA for WGS-based diagnostics direct from liquid blood culture. Methodology. We evaluate three commercial DNA extraction kits: BiOstic Bacteraemia, Amplex Hyplex and MolYsis Plus. Differential centrifugation, filtration, selective lysis and solid-phase reversible immobilization bead clean-up are tested to improve human cells/DNA and inhibitor removal. Using WGS (Illumina/MinION), we assess human DNA removal, pathogen recovery, and predict species and antibiotic susceptibility inpositive blood cultures of 44 Gram-negative and 54 Staphylococcus species. Results/Key findings. BiOstic kit extractions yield the greatest mean DNA concentration, 94–301 ng µl⁻¹, versus 0–2.5 ng µl⁻¹ using Amplex and MolYsis kits. However, we note higher levels of inhibition (260/280 ratio 0.9–2.1) and human DNA (0.0–4.4×10⁶ copies) in BiOstic extracts. Differential centrifugation (2000 g , 1 min) prior to BiOstic extraction reduces human DNA by 63–89 % with selective lysis minimizing by a further 62 %. Post-extraction bead clean-up lowers inhibition. Overall, 67 % of sequenced samples (Illumina MiSeq) contain 93 % concordance between WGS-based species and susceptibility predictions and clinical diagnosis. If >60 % of sequencing reads are human (7/98 samples) susceptibility prediction becomes compromised. Novel MinION-based WGS (n=9) currently gives rapid species identification but not susceptibility prediction. Conclusion. Our method for DNA preparation allows WGS-based diagnosis direct from blood culture bottles, providing species and antibiotic susceptibility prediction in a single assay.

Preliminary screening of pulmonary tuberculosis (TB) in India still relies on sputum microscopy, which has low sensitivity leading to high rate of false negatives. Moreover, conventional phenotypic drug susceptibility testing (DST) is conducted over a period of weeks leading to delays in correct treatment. Next generation sequencing technologies (Illumina and Oxford Nanopore) have made it possible to sequence miniscule amount of DNA and generate enough data within a day for detecting specific microbes and their DST profile. Sputum samples from two pulmonary TB patients were processed by decontamination and DNA was isolated from the decontaminated sputum sediments. The isolated DNA was used for sequencing by Illumina and by MinION (Oxford Nanopore Technologies). The sequence data was used to diagnose TB and to determine the DST profiles for the first- and second-line drugs by Mykrobe Predictor. Validation was conducted by sequencing DNA (by Illumina) isolated from pure growth culture from both the samples individually. DNA sequencing data (for both, Ilumina and MinION) from one of the sputum samples indicated the presence of Mycobacterium tuberculosis (M. tb) resistant to streptomycin, isoniazid, rifampicin and ethambutol and its lineage was predicted to be Beijing East Asia. The second sample indicated the presence of M. tb sensitive to the first- and second-line drugs by MinION and showed minor resistance call only to rifampicin by Illumina. Lineage of the second sample was predicted to be East Africa Indian Ocean, whereas Illumina data indicated it to be Delhi Central Asia. The two samples were correctly diagnosed for the presence of M. tb in the sputum DNA. Their DST profiles and lineage were also successfully determined from both the sequencing platforms (with minor discrepancies) paving the way towards diagnosis and DST of TB from DNA isolated from sputum samples at point-of-care. Nanopore sequencing currently requires skilled personnel for DNA processing and is a costly method – these drawbacks need to be addressed for successful implementation of Nanopore as a point-of-care diagnostic method in future. Preliminary screening of pulmonary tuberculosis (TB) in India still relies on sputum microscopy, which has low sensitivity leading to high rate of false negatives. Moreover, conventional phenotypic drug susceptibility testing (DST) is conducted over a period of weeks leading to delays in correct treatment. Next generation sequencing technologies (Illumina and Oxford Nanopore) have made it possible to sequence miniscule amount of DNA and generate enough data within a day for detecting specific microbes and their DST profile. Sputum samples from two pulmonary TB patients were processed by decontamination and DNA was isolated from the decontaminated sputum sediments. The isolated DNA was used for sequencing by Illumina and by MinION (Oxford Nanopore Technologies). The sequence data was used to diagnose TB and to determine the DST profiles for the first- and second-line drugs by Mykrobe Predictor. Validation was conducted by sequencing DNA (by Illumina) isolated from pure growth culture from both the samples individually. DNA sequencing data (for both, Ilumina and MinION) from one of the sputum samples indicated the presence of Mycobacterium tuberculosis (M. tb) resistant to streptomycin, isoniazid, rifampicin and ethambutol and its lineage was predicted to be Beijing East Asia. The second sample indicated the presence of M. tb sensitive to the first- and second-line drugs by MinION and showed minor resistance call only to rifampicin by Illumina. Lineage of the second sample was predicted to be East Africa Indian Ocean, whereas Illumina data indicated it to be Delhi Central Asia. The two samples were correctly diagnosed for the presence of M. tb in the sputum DNA. Their DST profiles and lineage were also successfully determined from both the sequencing platforms (with minor discrepancies) paving the way towards diagnosis and DST of TB from DNA isolated from sputum samples at point-of-care. Nanopore sequencing currently requires skilled personnel for DNA processing and is a costly method – these drawbacks need to be addressed for successful implementation of Nanopore as a point-of-care diagnostic method in future.

Routine full characterization of Mycobacterium tuberculosis (TB) is culture-based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near point of care. We demonstrate a low-cost DNA extraction method for TB WGS direct from patient samples. We initially evaluated the method using the Illumina MiSeq sequencer (40 smear-positive respiratory samples, obtained after routine clinical testing, and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction was obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. Using an Illumina MiSeq/MiniSeq the workflow from patient sample to results can be completed in 44/16 hours at a cost of £96/£198 per sample. We then employed a non-specific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis BCG strain (BCG), and to combined culture-negative sputum DNA and BCG DNA. For the latest flowcell, the estimated turnaround time from patient to identification of BCG was 6 hours, with full susceptibility and surveillance results 2 hours later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of the MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis in direct samples.

The recent widespread emergence of carbapenem resistance in Enterobacteriaceae is a major public health concern, as carbapenems are a therapy of last resort in this family of common bacterial pathogens. Resistance genes can mobilize via various mechanisms including conjugation and transposition, however the importance of this mobility in short-term evolution, such as within nosocomial outbreaks, is currently unknown. Using a combination of short- and long-read whole genome sequencing of 281 bla _KPC -positive Enterobacteriaceae isolated from a single hospital over five years, we demonstrate rapid dissemination of this carbapenem resistance gene to multiple species, strains, and plasmids. Mobility of bla _KPC occurs at multiple nested genetic levels, with transmission of bla _KPC strains between individuals, frequent transfer of bla _KPC plasmids between strains/species, and frequent transposition of the bla _KPC transposon Tn 4401 between plasmids. We also identify a common insertion site for Tn 4401 within various Tn 2 -like elements, suggesting that homologous recombination between Tn 2 -like elements has enhanced the spread of Tn 4401 between different plasmid vectors. Furthermore, while short-read sequencing has known limitations for plasmid assembly, various studies have attempted to overcome this with the use of reference-based methods. We also demonstrate that as a consequence of the genetic mobility observed herein, plasmid structures can be extremely dynamic, and therefore these reference-based methods, as well as traditional partial typing methods, can produce very misleading conclusions. Overall, our findings demonstrate that non-clonal resistance gene dissemination can be extremely rapid, presenting significant challenges for public health surveillance and achieving effective control of antibiotic resistance. Importance: Increasing antibiotic resistance is a major threat to human health, as highlighted by the recent emergence of multi-drug resistant “superbugs”. Here, we tracked how one important multi-drug resistance gene spread in a single hospital over five years. This revealed high levels of resistance gene mobility to multiple bacterial species, which was facilitated by various different genetic mechanisms. The mobility occurred at multiple nested genetic levels, analogous to a Russian doll set where smaller dolls may be carried along inside larger dolls. Our results challenge traditional views that drug-resistance outbreaks are due to transmission of a single pathogenic strain. Instead, outbreaks can be “gene-based”, and we must therefore focus on tracking specific resistance genes and their context rather than only specific bacteria.

Rapid and accurate detection of antibiotic resistance in pathogens is an urgent need, affecting both patient care and population-scale control. Microbial genome sequencing promises much, but many barriers exist to its routine deployment. Here, we address these challenges, using a de Bruijn graph comparison of clinical isolate and curated knowledge-base to identify species and predict resistance profile, including minor populations. This is implemented in a package, Mykrobe predictor, for S. aureus and M. tuberculosis, running in under three minutes on a laptop from raw data. For S. aureus, we train and validate in 495/471 samples respectively, finding error rates comparable to gold-standard phenotypic methods, with sensitivity/specificity of 99.3%/99.5% across 12 drugs. For M. tuberculosis, we identify species and predict resistance with specificity of 98.5% (training/validating on 1920/1609 samples). Sensitivity of 82.6% is limited by current understanding of genetic mechanisms. We also show that analysis of minor populations increases power to detect phenotypic resistance in second-line drugs without appreciable loss of specificity. Finally, we demonstrate feasibility of an emerging single-molecule sequencing technique.