The year in Clinical Microbiology ECCMID 2015, Copenhagen

Transcription

1 The year in Clinical Microbiology ECCMID 2015, Copenhagen Alex W. Friedrich Chair and head of department Department of Medical Microbiology and Infection Prevention University Medical Center Groningen The Netherlands

2 What s at the horizont for clinical microbiology?

3 What s at the horizont for clinical microbiology? Target nature: Complex microbial systems

4 Human microbiome is a complex ecosystem that varies between individuals Identification of demographic and lifehistory characteristics that could be correlated with different community types at each body site. Nature May 15;509(7500): doi: /nature13178 There were strong associations between whether individuals had been breastfed as an infant, their gender, and their level of education with their community types at several body sites. --- Over the course of the sampling period, community types were Most stable: from the gut and the vagina The least stable: from the oral cavity plaques Own Comments 1

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6 Human microbiome is a complex ecosystem that varies between individuals Identification of demographic and lifehistory characteristics that could be correlated with different community types at each body site. Nature May 15;509(7500): doi: /nature13178 There were strong associations between whether individuals had been breastfed as an infant, their gender, and their level of education with their community types at several body sites. --- Over the course of the sampling period, the most stable community types were from the gut and the vagina The least stable from the oral cavity plaques sites within the oral cavity were the least stable, whereas those in Own Comments Understanding the diversity of community types and the mechanisms that result in an individual having a particular type or changing types, New chances for - assessing disease risk and - to personalize diagnostic and therapy Human microbiome influenced by our lifestyle even on long-term 1

7 All domains of life feature diverse molecular clock machineries that synchronize physiological processes to diurnal environmental fluctuations. However, no mechanisms are known to cross-regulate prokaryotic and eukaryotic circadian rhythms in multikingdom ecosystems. The intestinal microbiota exhibits diurnal oscillations that are influenced by food intake rhythms, leading to time-specific compositional and functional profiles over the course of a day. Ablation of host molecular clock components or induction of jet lag leads to aberrant microbiota diurnal fluctuations and dysbiosis, driven by impaired feeding rhythmicity. Jet-lag induced dysbiosis in both mice and humans promotes glucose intolerance and obesity that are transferrable to germfree mice upon fecal transplantation. Volume 159, Issue 3, 23 October 2014, Pages Comments The results presented in this study may prompt future studies to determine the impact of circadian misalignment on factors shaping the microbiota, including immune and metabolic pathways of host, eating patterns, stress hormone levels, and bowel movement 2

8 Creation of a large catalog of the human gut microbial genes by combining 249 newly sequenced samples of the MetaHit project and 1,018 published samples from the Human Microbiome Project (HMP). Integrated gene catalog (IGR) was composed then of 9,879,896 genes. The catalog includes close-to-complete sets of genes for most gut microbes, which are also of considerably higher quality than in previous catalogs. Analyses of a group of samples from Chinese and Danish individuals using the catalog revealed country-specific gut microbial signatures. 3

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10 Comments This expanded catalog can facilitate quantitative characterization of metagenomic, metatranscriptomic and metaproteomic data from the gut microbiome to understand its variation across populations in human health and disease. Merging of global and openly accessible database one of the most important issues Data ownership: network or single cloud

11 What s at the horizont for clinical microbiology? Target nature: Complex microbial systems Technology: Use of NGS

12 In critically ill patients, the development of pneumonia results in significant morbidity and mortality and health care costs. Traditional diagnostics overestimate role of fast-growers and underestimate role of non-culturable microorganisms Application of amplification of nearly full-length bacterial 16S rdna and next-generation sequencing (NGS) was used to characterize the spectrum of bacteria in patients with pulmonary infections. The results from 61 patients demonstrated that 27 samples yielded PCR amplimers suitable for NGS (PacBio technology). Out of the 27 sequenced samples, 20 had bacterial culture growth and only 3 cases showed clear non-concordance between the clinical and NGS results. Confirmation that VAP is caused by polymicrobial co-infection, rather than one single causative agent Comments Technical improvements with real-time sequencing of unmodified DNA via e.g. nanopores, for instance, might significantly shorten diagnostic times and increase the accuracy of pathogenes diagnosis. Other systems (e.g. Illumina MiSeq) can also be used for extended stretches of DNA, but then several independent PCR s covering the fragment of interest need to be performed 4

13 Accurate and rapid diagnostic methods are required to optimize the clinical management of infected patients. The availability of genome sequences obtained using next-generation sequencing (NGS) have not only enabled advances in fundamental biology, helping to understand the pathogenesis of microorganisms and their genomic evolution, but have also had implications for clinical microbiology. Conclusions NGS methods have reached a point, both in terms of cost and speed, where they might enter the routine microbiology laboratory and be used routinely Increasing number of genome sequences will enable the development of new and improved assays. Comments NGS has the potential to replace several existing tests, in particular ID/AST for microorganisms that are difficult to grow. Ad-hoc and on-demand diagnostic tools based on Genomics using WGS for personalized identification of species, AMR, outbreaks Towards personalized Theragnostics 5

14 Classical PCR: - Answers to questions posed Next-generation sequencing (NGS): - Also answers to questions not posed by metagenomics in the clinical microbiology laboratory However, practical deployment of the technology is hindered by the bioinformatics challenge of analyzing results accurately and in a clinically relevant timeframe SURPI ( sequence-based ultrarapid pathogen identification ), a computational pipeline for pathogen identification from complex metagenomics NGS data generated from clinical samples, demonstrated in the analysis of clinical samples. Cloud-based and standalone servers, SURPI leveraged two state-of-the-art aligners for accelerated analyses, SNAP and RAPSearch, which are as accurate as existing bioinformatics tools but orders of magnitude faster in performance. Sensitivity is not decreased Comments Nowadays in BLAST-approach, the computational time for metagenomics is days/weeks SURPI has directly contributed to real-time microbial diagnosis in acutely ill patients (minutes to hours), Metagenomic deep sequencing from clinical specimens will be routinely performable in major labs within 10 years from today Proteomic-analysis will allow therapy follow-up analysis 6

15 Clinical metagenomic analysis, (microbial and host DNA) Still numerous obstacles Read alignment methodology, for analysis of the hundreds of millions of reads per run generated through sequencing technologies Read assignment methodology Database for identification of taxa in complex microbial samples Results and Conclusions 4 CF-samples Detection: Subtraction of human genomic information (33%-90%) Validation (SNP Genotyping), Genomic reconstruction, Microbiol. confirmation Co-infection MSSA/MRSA in simulated reads was not yet successful s Perspectives Comments Identification from clinical specimen as proof of principle Global alignment and expandable databases (PathSeq, MePIC and SURPI ) Clinical metagenomics evolving and progressing driven by (microbial) geneticist 7

16 Substantial bacterial contamination is routinely found in existing human-derived RNA-seq datasets that likely arises from environmental sources. Identical Cell Lines Analyzed in Separate Studies Show Differences in Bacterial Read Profiles Different Bacterial Read Profiles across Sequencing Centers Using Identical RNA Samples and Library Preparation Kits Contamination is less relevant for studies utilizing relatively homogeneous microbial communities, but it can be a confounding factor in the assessment of samples in which the predominant genetic material is human. Comments In NGS everything is amplified! Usage of highly purified metabolic enzymes and other reagents used in sequence library preparation For some cases, selective bioinformatic decontamination possible Quality control remains an nextgeneration issue 8

17 Preventive use What s at the horizont for clinical microbiology? Target nature: Complex microbial systems Technology: Use of NGS

18 Low resolution bacterial genotyping has a very limited role because healthcareassociated MRSA (HA-MRSA) belong to a restricted number of successful lineages worldwide. The purpose of this study was to use WGS to define phylogeny and transmission dynamics of MRSA in a hospital setting (in a 1000-bed hospital in NE-Thailand) where MRSA transmission was common. All MRSA belonged to MLST ST239. Based on WGS (SNP-based analysis) of 79 isolates from 51 individuals, phylogenetic analysis identified a flux of distinct ST239 clades over time in each intensive care unit. In total, five main clades were identified, which varied in the carriage of plasmids. Sequence data confirmed intra- and interwards transmission events and identified individual patients who were colonized by more than one clade. Comments Diversity of one ST can be shown by WGS Higher diversity by extended transmission chains and regionally circulating nosocomial strains with multiple introduction The application of WGS has the potential to provide accurate insights, which could be used to inform low-cost prevention planning and implementation. Dynamics differ on basis of background molecular epidemiology 9

19 Backwards-compatibility of WGS spa typing of methicillin-resistant Staphylococcus aureus (MRSA) has traditionally been done by PCR amplification and Sanger sequencing of the spa repeat region. Evaluate the reliability of MRSA spa typing by 150-bp paired-end Illumina WGS using using the software program Velvet v for genome assembly. This was done by comparing the spa typing results of WGS with Sanger sequencing results. A 97% agreement between spa types obtained by the two methods was observed. The isolates differed in spa types by the two methods, in most cases due to the lack of 24-bp repeats in the whole-genomesequenced isolates. These spa types were mostly related and would have been grouped together No consequences for outbreak investigations. Comments spa typing by WGS can reliably replace Sanger sequencing. The advantage of the additional genomic information gained by WGS is higher than the disadvantage of a small number of inexact spa types. Bletz et al.& Harmsen CMI (2014) confirm results with 99,1% of concordance between spa-ngs and spa-sanger (250 nt read length) Sequence-based typing: data for eternity 10

20 The greater resolution of whole genome sequencing (WGS) data relative to older genotyping methods makes it a potentially powerful method in infectious disease epidemiology The use of whole-genome sequencing (WGS) is highy effective for investigating transmission at different scales: - Direct transmission between individuals - Local outbreak investigations - The worldwide spread of internationally successful bacterial pathogens Limitations of WGS must be still solved- Short read alignment need near-complete, genomes: - Slow mutators, make transmission tracking difficult (M. tuberculosis) - Evolutionary clock changes speed (C. difficile sporeform) - Outbreak analyses have uncovered hypermutators, (MRSA with a disrupted DNA repair gene) Place, Time, Person, Species, Subtype, (vector of direction) More mutations between two anatomical sites than between two persons at the same site 11

21 Whole Genome Sequencing (WGS) was used and state of-the-art analytical techniques to define an alarming outbreak of MDR Klebsiella pneumoniae within a major hospital in Kathmandu, Nepal, during This outbreak occurred in children admitted to neighbouring high-dependency paediatric wards and was characterized by high incidence of bloodstream infections and a case-fatality rate. The WGS analysis permitted the identification of two MDR K. pneumonia lineages causing distinct outbreaks. The strains belonging to Outbreak Lineage 1 were circulating in the hospital for 6 months before the outbreak and had gained genes facilitating infection and antimicrobial resistance. This work allows a comprehensive insight into how hospital outbreaks of drug resistant K. pneumoniae occur in lowincome settings. Comments The power of these data outlines that some real-time genetic characterization should be performed during future healthcare infection control practices in both high- and low-income settings to identify outbreaks and limit onwards transmission. Most important: Capacity building and worldwide collaboration is necessary 12

22 From a public health perspective, the aim of this study was to assess the usefulness of whole-genome sequencing in outbreak investigations in a strictly environmental pathogen for which human infection is a dead-end. From an evolutionary perspective, the s wanted to measure the impact of different evolutionary forces in shaping the genetic diversity in a population of this environmental pathogen over a short period of time. 69 strains linked to recurrent outbreaks in a single location (Alcoy, Spain) over 11 years were analyzed by WGS. There were found some examples where the genome sequences of isolates of the same sequence type and outbreak did not cluster together and were more closely related to sequences from different outbreaks. The analyses identified 16 recombination events responsible for almost 98% of the SNPs detected in the core genome and an apparent acceleration in the evolutionary rate. Comments Recombination (10-fold difference in substitution rates) Recombination already in the environment and not in humans (natural habitat) Not increased selective pressure Typing can be misleading The results obtained in this study will have profound implications for the understanding of microbial populations and for public health interventions in Legionella outbreak investigations 13

23 Although whole-genome sequencing has been found to improve discrimination of outbreak clusters, whether this procedure can be used in real-time in a public health laboratory is not known. Therefore, a retrospective and prospective analysis was conducted. Nearly real-time whole-genome analysis of S. enterica serovar Enteritidis results improved detection of clusters of common PFGE types and outbreak resolution than PFGE PFGE and MLVA overestimate outbreaks (up to 30% of diverse isolates considered identical) This study demonstrates the practical feasibility and benefits of deploying these Comments PFGE is considered to be gld standard for food-borne pathogen NGS better than PFGE, detecting outbreaks by improved resolution (public health importance) Affordable and rapid next-generation sequencing technologies and associated bioinformatics will be potent tools in achieving these goals. 14

24 Monomorrphic microorganisms. Classical typing tools not discriminatry enough To improve discrimination of Francisella tularensis strains, a new gene-by-gene typing approach, MLST+, was developed based on whole genome data of 15 sequenced F. tularensis ssp. holarctica strains. MLST+ was applied to investigate an epidemic of lethal tularemia among nonhuman primates in two animal facilities in Germany. MLST+ might be superior to the current gold standard methods (cansnp or MLVA ) for typing F. tularensis strains. Monomorphic microorganism, MLST+ approach is adaequate Even in spatially and temporary close area MLST+ may lead to a discriminatory power comparable to core genome gene SNPtyping PLoS One Apr 9;10(4):e Comments For clonal species like F. tularensis it might be additionally necessary to extent the genetic analysis beyond the core genome NGS/MLST+ powerful method for typing of microorganisms of public health importance Resolution of the WGS analysis might be increased by incorporating accessory genome genes, repetitive elements, pseudogenes or genes of the Francisella pathogenicity islands. 15

25 Other technologies Preventive use What s at the horizont for clinical microbiology? Target nature: Complex microbial systems Technology: Use of NGS

26 Determination whether shotgun proteomic approaches could be used to identify tuberculosis (TB)-specific biomarkers in the urine of well-characterised patients with active TB versus no TB. SDS-page followed by digestion using trypsin after which HPLC tandem mass-spectrometry used to identify the urinary proteome 10 Mtb. proteins were observed exclusively in the urine of definite TB patients In addition, a gene ontology enrichment analysis identified a panel of 27 human proteins that were significant discriminators (p<0.05) for TB disease compared to no TB disease Identified in urine as markers of active pulmonary TB disease Furthermore, 7 common human proteins were differentially over- or under-expressed in the TB versus the non-tb group. Comments The biomarkers identified in the current study hold promise for the development of new point-of-care diagnostics for TB. Urine (ultrafiltrate of blood representing molecules from all organs) is excellent specimen Very promising, validation studies under way 16

27 Nanotechnology Objectives Miniaturization of growth chambers, such as the nanowell slide, has been demonstrated as a novel tool for single cell analysis. In the current study, the nanowell slide was applied to prokaryotic organisms for rapid MIC determination. This system allows to detect transition of lag-phase to log-phase (due to sensitivity) The nanowell antibiotic susceptibility testing (AST) device delivers precise MIC determinations for clinical uropathogenic Escherichia coli strains against multiple antibiotics within 4 h. Comments In traditional detection systems (micro-titre readers) sensitivity measurements allow to detect gown culters in late log-phase The nanowell AST device has the potential to be broadly applied to determine antimicrobial susceptibility in a variety of bacterial infections, thereby addressing the clinician s need for diagnostic speed and accuracy. 17

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29 Nanotechnology Objectives Miniaturization of growth chambers, such as the nanowell slide, has been demonstrated as a novel tool for single cell analysis. In the current study, the nanowell slide was applied to prokaryotic organisms for rapid MIC determination. Traditional detection systems (micro-titre readers) allow to detect grown cultures in late log-phase This system allows to detect transition of lag-phase to log-phase (due to sensitivity) Using nanotechnology precise MIC determinations for clinical uropathogenic Escherichia coli strains against multiple antibiotics within 4 h. Comments The nanowell AST device has the potential to be broadly applied to determine antimicrobial susceptibility in a variety of bacterial infections, thereby addressing the clinician s need for diagnostic speed and accuracy. First signs of shifting from clinical microbiology towards clinical nano-biology 19

30 Lessons learned from last 12 Months! Human Microbiome seems to is our only exo-organ - It is influenced by our life and seems to have influence on us - the only organ we even share with our contacts Genomic Reference databases (e.g. Gut microbiome) are growing - Open crowd-sharing of all sequence-data is the only way for success Next gen applications entering from early-adopters to pre-routine phase - new understanding of polymicrobial infections (e.g. VAP) - remaining issues: quality of data/data management/capacity building Typing succes is dependent on species-specific evolutionary clock - in microorganisms with high environmental tenacity high genomic plasticity we need to do more research to understand cut-off for heteroclonality Modern techniques fastly evolving (Deep Sequencing, Proteomics, MS, nanotechniques) - Clinical microbiology labs need to be on track for new developments