43:3 August 2016 43:3 August Nanoscale imaging infrared spectroscopyTwo-dimensional microscopy electron Cryogenic CRISPR-Cas Phage therapy Future Tech Microbiology TODAY

Microbiology Today 43:3 August 2016 Future Tech

Editorial

Science is a broad church and the designation of scientific disciplines fascinates me. Four overarching categories map the sciences: formal science, social science, applied science and natural science. The life sciences, considered the study of any living organism, are a strand of the natural sciences, and microbiology, the study of micro-organisms, is one of more than 20 sub-branches found under the ‘life sciences’ umbrella. As technologies and scientific knowledge grew, the number of branches and sub-branches expanded in tandem and continues to today. The definition of each branch and sub-branch stakes a claim to a unique selling point that gives it an identity and a focus, but, nevertheless, with overlap.

dvances and tools from the Hoskisson’s article describes how true resistance. One approach to tackle this study of microbiology underpin collaborations between microbiologists issue is to find alternatives to antibiotics Aseveral of the branches of life and physicists can lead to fascinating used to combat pathogens. In this sciences. Restriction enzymes and insight into biological systems, especially article I describe how the increasing cloning technologies, cloning vectors when applying exciting new techniques. understanding of bacteriophage and their and overexpression vectors, heat-stable They describe how fast, dynamic lifecycles might offer an alternative to DNA polymerases and DNA sequencing, structure–function relationships can be new antibiotic discovery. and the polymerase chain reaction studied using ultrafast two dimensional Finally, Justin O’Grady has written (PCR) are cornerstone tools of many infrared spectroscopy (2D IR) that is a Comment article that highlights the other branches including biochemistry, uniquely sensitive to structural dynamics need for a paradigm shift in diagnostics molecular biology, genetics, genomics, and is capable of extending the range of technology, to allow for the development immunology… The list goes on. information available to microbiologists of a rapid universal diagnostic that Mining microbes for molecular tools studying molecular interactions. This is can detect any pathogen or resistance is an on-going process and sea-change followed by David Bhella’s fascinating for the effective treatment of life- technologies continue to emerge. The article on the emerging technology of threatening infections. He describes relative ease with which microbes can cryo-electron microscopy. how next-generation sequencing be manipulated has also enabled the All organisms are exposed to technology, particularly rapid nanopore development of new technologies that parasites, and evolved a range of sequencing and Oxford Nanopore can be applied to the study of life sciences different immune mechanisms to defend Technologies’ MinION device, has the and present potential solutions to global themselves. Edze Westra describes how potential to drive this technology gear- problems. This issue celebrates some of scientists have discovered that bacteria shift forward by combining rapidity with these significant tools, techniques and and archaea have a sophisticated adaptive comprehensiveness beyond simple technologies from microbes. immune system, known as CRISPR-Cas microbial culture or PCR. First, S. Andrea Gazze, Lewis (clustered regularly interspaced short Microbiology has transformed Francis and Geertje van Keulen describe palindromic repeats). The insights gained the life sciences. Microbiologists and how exciting novel microbial properties about this system have led to several microbes continue to be at the forefront and behaviours can be revealed by applications in industry to protect bacterial of new invention and innovation, and applying nanoscale analytical techniques, species against their viral parasites. In and are pioneering the technology of the such as atomic force microscopy, to pure addition, CRISPR-Cas has been turned into future. cultures and in situ ecosystems such a versatile genome editing method that as the soil. has the potential to treat human genetic Laura Bowater One of the central themes of biology diseases. Editor is that structure is linked to function. I have written the last article on [email protected] Niall Simpson, Neil T. Hunt and Paul A. the growing global problem of antibiotic

Microbiology Today Aug 16 | www.microbiologysociety.org 97 Contents

Microbiology TODAY Articles The integration of nanoscale imaging and 106 quantitation of nanomechanical properties in microbiology S. Andrea Gazze, Lewis Francis & Geertje van Keulen Applying novel nanoscale analytical techniques.

Adding dynamics to structure/function 110 studies in microbiology: two-dimensional infrared spectroscopy Niall Simpson, Neil T. Hunt & Paul A. Hoskisson Exploring molecular dynamics in relation to structure and function.

Cryogenic electron microscopy – 114 beyond the resolution revolution David Bhella New horizons in structural biology research.

CRISPR-Cas: from mechanism to 118 applications Edze Westra Innovative genetic editing changing the biological landscape.

Phage therapy 122 Laura Bowater Rethinking medical treatments using phage. 43:3 August 2016 Features Regulars 130 Legionella turns 40 97 Editorial A symposium four decades after the first outbreak. 100 Council 2016 132 Microbial Genomics: One-Year 101 From the President Anniversary! 102 From the Chief Executive The Society’s newest journal and its extraordinary first year. 103 News 134 Diagnosing and sequencing 126 Annual Conference Ebola in Sierra Leone Member Ian Goodfellow tells us about his experience. 128 Focused Meetings 136 Developing microbiology around the 146 Reviews world Editor Dr Laura Bowater Recipients of the Society’s International Development Fund. Managing Editor Ruth Paget 137 Membership Editorial Board Phil Aldridge, David Bhella, Helen Brown, Emma Denham, Lorena Fernández-Martínez, Paul Hoskisson, James Redfern, Alison Sinclair, The review is underway; find out how you can feed back. Nicola Stonehouse 138 Schoolzone – Old School, New School Address Microbiology Society, Charles Darwin House, 12 Roger Street, London WC1N 2JU T +44 (0)20 7685 2683 E [email protected] Determining phylogenetic relationships and evolutionary history using Design Ian Atherton, Corbicula Design (www.corbiculadesign.co.uk) bioinformatics. Printed by Charlesworth Press, Wakefield 140 Outreach – Light at the bottom of the sea © 2016 Microbiology Society Jenny Search’s outreach work using bioluminescence. ISSN 1464-0570 The views expressed by contributors do not necessarily reflect official policy of the Society; nor can 142 Science policy update the claims of advertisers be guaranteed. The latest from the Policy team. 143 Best of the blog From the history of vaccines to the International Space Station. 144 Membership Q&A Martin Cole looks back at his career. 147 Comment – Moving microbial diagnostics into the 21st century P2 Bacteriophages. Dr Harry Fisher, Justin O’Grady Visuals Unlimited / Science Photo Comprehensive and rapid diagnostic testing. Library Council 2016

Executive Officers President – Professor Neil Gow College of Life Sciences and Medicine, Institute of Medical Sciences, Foresterhill, University of Aberdeen, Aberdeen AB25 2ZD; [email protected] General Secretary – Dr Evelyn M. Doyle School of Biology and Environmental Science, Science Centre West, University College Dublin, Belfield Dublin 4, Ireland; [email protected] Treasurer – Professor Chris Thomas School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT; [email protected]

Elected Members Professor Andrew Davison MRC-University of Glasgow Centre for Virus Research, Church Street, Glasgow G11 5JR; [email protected] Dr Stephen Diggle School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD; [email protected] Professor Stephen Oliver Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA Professor David Pearce Faculty of Health and Life Sciences, Northumbria University, Northumberland Road, Newcastle-upon-Tyne NE1 8ST; [email protected] Dr Mike Skinner Section of Virology, Imperial College London, Faculty of Medicine, St Mary’s Campus, Norfolk Place, London W2 1PG; [email protected] Professor Nicola Stonehouse School of Molecular and Cellular Biology and Astbury Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT; [email protected]

Chairs of Committees Communications Committee – Dr Paul A. Hoskisson Strathclyde Institute of Pharmacy & Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE; [email protected] Finance Committee – Professor Chris Thomas See ‘Treasurer’ above Professional Development Committee – Dr David Whitworth Institute of Biological, Environmental and Rural Sciences Room S22, Cledwyn Building, Aberystwyth University, Ceredigion SY23 3FG; [email protected] Policy Committee – Dr Pat Goodwin C3 Collaborating for Health, c/o Microbiology Society, Charles Darwin House, 12 Roger Street, London WC1N 2JU Publishing Committee – Professor Charles Dorman Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College Dublin, College Green, Dublin 2, Ireland; [email protected] Scientific Conferences Committee – Dr Karen Robinson Nottingham Digestive Diseases Centre, University of Nottingham, Nottingham NG7 2RD

100 Microbiology Today Aug 16 | www.microbiologysociety.org From the President

As the summer months arrive and the academic terms have been wound up, more time may be available for us to complete manuscripts and grant applications. For Microbiology Society staff, Executive Officers and Committee members this is also a period of intense planning for the future. We are looking forward to seeing our plans from last year transform into action in the coming months. For example the Early Career Microbiologists’ Forum will shortly be formed.

pplications for the recently environmental microbiome research box of methods to facilitate cell biology advertised vacancies in our and its relevance to public policy. The and structural biology. Collectively, the ACommittees will soon be reviewed Society will be running workshops to sum of these new methods are so potent and I am pleased to hear that we have inform the development of the report. and broadly applicable that interesting had a particularly encouraging number Our Society headquarters at microbes previously recalcitrant to of members applying for the places in Charles Darwin House in London is detailed analysis are now becoming our Early Career Microbiologists’ Forum. shared with other professional societies accessible to detailed reductionist The ECM Forum is our way to capture – the Biochemical Society, the British investigations. Our publications and the voice and insights of active scientists Ecology Society, the Royal Society of meetings make an important contribution who, for the most part, will still be at the Biology – and a great deal of thought towards showcasing these advances. bench, and many will yet to have been is going into ways in which we can Can I remind you that our operational appointed to a permanent post. This achieve new collaborations due to our structures allow our members to promote is the group within our membership neighbourly proximities. We will update their own vision of what is important and who are still generating data and doing you of news about how the different what defines the horizons of microbiology. experiments, and we are eager to gain societies might help each other while For example, if you feel that our portfolio input from microbiologists who are promoting our own agendas in the of meetings may be missing a trick on the front line of research. There coming months. in developing an understanding of a will be an ECM Forum representative This ‘Future Tech’ issue of particular aspect or area of microbiology, on virtually all of our administrative Microbiology Today is packed with useful you have the opportunity to present committees, meaning that we will articles exploring current methodologies your idea for a session at our Annual absorb the perspective of early career that are transforming microbiology and Conference or as a Focused Meeting researchers across the whole aspect of other biological disciplines. This is the through our Committees. We really running the Society. They will be given age when next-generation sequencing do wish to remain responsive to our serious resource and have significant is synergising with powerful genome members providing ideas for meetings influence to shape the things to come. editing methods such as the CRISPR- and scientific sessions. This will help make us more nimble, and Cas9 system, making the analysis and I hope that you have enjoyed some better informed and engaged to enable manipulation of microbial genes superbly summer sun and that you have had an our organisation to feel confident that its efficient. We are using the CRISPR-Cas9 opportunity for a break to help inspire decision-making is truly representative system in my own laboratory to analyse your scientific imaginations. across the entire membership. multigene families in Candida, and the The Microbiology Society is also acceleration in progress that makes Neil Gow developing an accessible, evidence- complex genetic constructs possible is President based report for policy- and decision- extraordinary. In addition, new imaging [email protected] makers exploring human, animal and technologies have opened a Pandora’s

Microbiology Today Aug 16 | www.microbiologysociety.org 101 From the Chief Executive

At this year’s Annual Conference in March, we kicked off a major project to find out directly from you, the microbiology community, what more the Microbiology Society can do to support your careers, to connect and empower communities, so that the science of microbiology can make the greatest possible contribution to society. A rewarding scientific career always faces big challenges, and our job is to help you to tackle them. The more you tell Council, me and the rest of the staff about those challenges, the more we can do together to advance microbiology.

e know that many of the them. In policy, for example, we can see you, we are listening to what you tell us, activities the Society carries out the impact we have on your behalf every and we also use more formal methods Won your behalf have important time a politician quotes our briefings, or like surveys and focus groups. impacts because you tell us so. After incorporates one of our recommendations These methods will all give us the Annual Conference this year, you into an official document. The ways extremely valuable information that let us know about new collaborations of assessing some of what we do use allows us to build a picture of how and grants that you will undertake quantitative benchmarks, such as the we can be most helpful. In the end, with colleagues you met, about new number of times the papers in our there is no substitute for face-to-face sponsorship for your research that scientific journals are cited by others conversations. So if you think there are came directly from contacts you made, or the number of hits on the website – ways in which the Society could do even and about technical problems you fixed Microbiology Online received very nearly more for your career, please take the by drawing on the expertise of other a million visitors last year. time to tell us. There are many ways delegates you encountered. All of these are valuable indicators you can help to shape the future of your We know that some of the Society’s that Council, the Committees and Society. You could think about standing other activities are important because Divisions, supported by the staff, are for election to serve on Council or a of the demand for them. The Scientific investing in programmes that help Committee or Division. You could Conferences Committee, for example, you to advance both knowledge about come to an occasion like one of our has to make hard choices about which microbiology and also its application conferences or the series of events on Focused Meetings we are able to run, into useful processes relevant to the the day of the Annual General Meeting, because you are submitting so many environment, the economy, policymaking which will be on 8 September. You excellent proposals. Our journals only or our culture. could invite me to come and visit your publish a proportion of the articles that But the Society’s resources are finite, laboratory. Or you could just email are submitted to them because the and we need to be constantly vigilant to me and tell me what you think the Editors and referees work hard to identify the possibility that we could use them Microbiology Society does well, what the most interesting and scientifically even more effectively. So we are using a it could do more of, and how we can challenging work. And grant schemes like range of different techniques to consult work together to advance the science of the Harry Smith Vacation Studentships you about the greatest needs of the microbiology. reluctantly have to disappoint some microbiology community. At the Annual deserving candidates because the demand Conference, we asked you to jot down on Peter Cotgreave outstrips our capacity to fund them. Post-it notes what you thought we should Chief Executive Other activities are judged by the be doing. Every time Council or staff [email protected] extent to which external audiences rate members come into contact with any of

102 Microbiology Today Aug 16 | www.microbiologysociety.org News

Focused Meetings 2016 Annual Conference 2017 The Society has already hosted two Focused Meetings this year and has a Next year’s Annual Conference will bring us to further three taking place in September. These include: the historic city of Edinburgh, UK. The date is The Dynamic Fungus confirmed as 3–6 April and the event will take place at the Edinburgh International Conference 5–7 September – Mercure Exeter Rougemont Hotel, Exeter, UK Centre (EICC). More information on planning Molecular Biology and Pathogenesis of Avian Viruses your trip can be found on pp. 126–127. 27–29 September – Charles Darwin House, London, UK Irish Division: Exploring the Microbe–Immune System Deaths Interface It is with sadness that the Society announces 1–2 September – Rochestown Park Hotel, Cork, Ireland the death of Mr Michael Carlile, who joined the For more information on these events, visit pp. 128–129. Society in 1956.

New online correction tool for Society journals Authors publishing in the Microbiology Society’s journals This will remove the need for an operator to input the author can now benefit from an online correction tool available via corrections, so we hope that this will reduce the potential for our new production partner, Exeter Premedia. Authors will errors to be introduced and speed up the production process. be able to view their typeset PDF online, then add corrections directly to the article on the cloud-based production platform. To find out more about the Microbiology Society’s journals Once they have finished, authors can rebuild the PDF to view and to submit your next article to one of them, visit their changes in the final PDF before approving the proof. www.microbiologyresearch.org

Microbiology Today Aug 16 | www.microbiologysociety.org 103 Parliamentary events link Upcoming grant deadlines

scientists and policy-makers Date Grant Notes In May, the Society attended Science 1 September 2015 Travel Grants For conferences and courses and the Assembly 2016 at the National from 1 October onwards* Assembly for Wales in Cardiff. The event, which aims to strengthen ties between 15 September 2015 Microbiology in Schools For School Members to receive the Assembly and the Welsh STEM Fund funding for microbiology teaching community, was themed ‘Planetary initiatives taking place on or after and Space Science’. Speakers included 1 November scientists, representatives from the Royal 1 October 2015 Research Visit Grants Astronomical Society and the UK Space Agency, and the Chief Scientific Adviser International For visits and events from for Wales, Professor Julie Williams Development Fund 1 December onwards CBE. Attendees had the opportunity to Education and Outreach hear from and network with Assembly Grants Members. At Parliamentary Links Day in June, Rolling application scientists and policy-makers came together at the UK Houses of Parliament Local Microbiology Event Sponsorship for a very topical discussion about All members can apply for funds to support microbiology-related events, what was next for science after the e.g. sponsored talks. referendum on the UK’s membership of *Please note, you do not need to have received confirmation of abstract acceptance the European Union. Society members to apply for these grants as conditional offers will be made. In this case, evidence of and staff also attended a luncheon in acceptance is required to claim your grant. the House of Lords, where they were joined by Jim Dowd MP. The Society also Annual General Meeting under the AGM and Celebration of the highlighted its science policy work at Society’s Work page: an afternoon networking session with and Showcase of the www.microbiologysociety.org/agm Members of Parliament and Peers. Society’s Achievements The Microbiology Society Annual Contributions and feedback General Meeting (AGM) will be held as The Society welcomes contributions and part of a wider Showcase of the Society’s feedback from members. Please contact Achievements event on 8 September 2016. [email protected] with This will include the Young Microbiologist your ideas. of the Year final, a presentation from our Microbiology Outreach Prize winner and a Benjamin Thompson Special Lecture. Head of Communications The notice of the AGM and all associated [email protected] papers are now available to members

Get the latest updates, follow the Society on: +

104 Microbiology Today Aug 16 | www.microbiologysociety.org Could you be the next “ Editor of Microbiology Today?

After a successful three-year term of office, Laura Bowater stands down as Editor of Microbiology Today at the end of 2016. The Society is looking to appoint a new member who is interested in being the next Editor of our prestigious and highly regarded magazine. The role will begin in January 2017 and is open to any Full, Full Concessionary or Honorary Member of the Society. There will be a handover period from November through to December 2016.

My role as Editor of Microbiology Today has provided me with a unique opportunity to develop a worldwide network of contacts and to be introduced to the incredible variety of research being undertaken by committed and dedicated microbiologists. It has been a real privilege and a pleasure to move Microbiology Today forward, and it is one of the few roles I have had that garners unsolicited, positive feedback on a regular basis. Laura Bowater

As the Editor you are responsible for the scientific content and integrity of the magazine. You will be required to: • attend and chair two Editorial Board meetings per year • read, assess and edit all submitted feature and Comment articles • liaise with the Managing Editor, Head of Communications and Chief Executive at the Microbiology Society’s main offices withegard r to any matters arising that involve the content or development of Microbiology Today • represent and promote the magazine outside the Society

“In addition, you will sit on the Communications Committee and will be required to attend two meetings for the Committee each year.

Desirable qualities for the position include: • an interest in a wide range of subjects across the whole of microbiology • an ability to assess contributions for their suitability for publication • excellent communication skills, including the ability to deal with sensitive issues • enthusiasm about the communication of microbiology to key stakeholders, including other scientists, parliamentarians, policy-makers, educators and the general public

You do not need to have been involved with the Editorial Board or the Society previously to apply, but you do need to be a Full, Full Concessionary or Honorary Member. If you are interested, please read a full description of the role at www.microbiologysociety.org/MTEditor and send your CV with the names of two referees to [email protected]. The closing date for applications is Monday 12 September 2016. The appointment will be subject to approval by the Communications Committee.

The Microbiology Society is committed to creating an inclusive culture that supports equality and diversity, and fully reflects both its membership and the global microbiology community. The integration of nanoscale imaging and quantitation of nanomechanical properties in microbiology

S. Andrea Gazze, Lewis Francis & Geertje van Keulen Recent developments in nanotechnology enable the imaging, quantification and manipulation of materials at the near-atomic level. The number of applications of atomic- force microscopy (AFM) in the life sciences is increasing, now allowing the integrated study of topological and quantitative nanoscale mechanical characterization of living cells and their interactions with their environments, which was inconceivable until recently.

FM is a surface-sensitive reconstruct a 3D topological technique whereby a sample is image of the sample. Forces between Ascanned with a sharp tip mounted the surface and cantilever tip govern on a micrometre-sized flexible cantilever. the cantilever deflections differentially Irregularities in surface topography will when moving towards or away from the deflect the cantilever while scanning the sample (Fig. 1b), from which a range surface (Fig. 1a). These deflections are of mechanical properties can be detected, processed and then used to quantified at the nanoscale, such

106 Microbiology Today Aug 16 | www.microbiologysociety.org as surface stiffness, adhesion and 1 deformation. 2 Individual microbial cells, cells 4 Detector

within a microbial community, and their Laser source 3 Surface stiffness Surface 123 subcellular components can readily 4 be imaged by well-known techniques 6 such as confocal laser scanning 5 Sample microscopy (CSLM) and scanning on the sample Applied force 5 = Surface 6 adhesiveness electron microscopy (SEM). These (a) (b) techniques require staining, labelling and/or other sample preparations, which Fig. 1. Basic working principles of AFM: cantilever deflections leading to determination of (a) surface may introduce artefacts. Most excitingly, topography, and (b) force-curve measurements for quantification of nanomechanical properties. microbial materials can be analysed S. Andrea Gazze quantitatively by AFM under physiological conditions without the need for prior (a) (b) sample preparation. EPS ‘halo’

Nanomechanical properties of microbial cell surfaces and Hypha extracellular (polymeric) substances in pure cultures and biofilms Extracellular polymeric substances (EPS) form a composite biomaterial of water, proteins, nucleic acids and polysaccharides in which microbial cells 500 nm EPS ‘halo’ 150 nm are embedded to construct a biofilm that favours microbial growth and survival Fig. 2. Topology of a filamentous bacterial hypha surrounded by a thin film of EPS (a) and rodlet-structured by permitting the local accumulation aerial cell surface at higher magnification (b).S. Andrea Gazze of nutrients and other metabolites. Biofilms form the main method of its usefulness in studying different stages affect surfaces in a similar way. The next microbial colonisation of natural and of biofilm and other types of multicellular stage of cellular differentiation can also artificial substrates, with potential to development. AFM has revealed that be followed with AFM: chaplin and rodlin cause significant risk to health and the cells are first surrounded by a thin ‘halo’ proteins are excreted on the aerial cell economy, e.g. by biofouling medical and of secreted EPS (Fig. 2a), which can be surface of Streptomyces bacteria, which industrial materials such as implants up to a cell’s diameter away from the then typically self-assemble into thicker and membranes. Studying the properties cell surface and a few tens of nanometre rodlet-shaped amyloidal aggregates, of biofilms and their development is in thickness. Importantly, the microbial conferring a concomitant change in therefore of paramount importance, not community can produce overlapping mechanical properties from wettable to only for the advancement of microbiology EPS layers with different mechanical water-repellent (Fig. 2b). This change in general, but also to control detrimental properties. Interestingly, both filamentous in surface properties can be visualised microbial effects on specific applications. bacteria (Fig. 2a) and fungi secrete EPS and quantified with AFM, providing High-resolution topographical in a similar way, showing that organisms quantitative nanomechanical insights imaging and mechanical force analysis from different kingdoms with similar into morphological differentiation. of composite biomaterials in their natural cellular morphology (growing by hyphal AFM can also be co-applied with state by AFM has already demonstrated elongation from the tip) can colonise and other analytical techniques such as

Microbiology Today Aug 16 | www.microbiologysociety.org 107 CSLM, nanoscale secondary ion mass soil particles, such as etch pits, Nanoscale detection of microbial spectrometry (NanoSIMS), and several secondary mineral formation and processes in situ: soil others. Such hybrid instrumentation can hyphal-shaped channels (Fig. 3a). Microbial activities greatly affect soil reveal subcellular localisation of labelled Inductively-coupled plasma atomic properties and soil ecosystem processes intra- and extracellular molecules, with emission spectroscopy (ICP-AES) such as the formation and breakdown overlying bionanomechanical properties, can be applied to give the overall of soil organic matter (SOM). Different leading to deep mechanistic insights dissolution rate of minerals, e.g. techniques exist to evaluate macroscale such as those demonstrated for the released mineral cations in solution soil properties such as composition, lateral expansion of antimicrobial pores at parts-per-billion (ppb) levels. AFM wettability, bulk density, porosity, soil in lipid bilayers. can complement ICP-AES data by hardness and deformation. In turn, these detecting in real time the release of properties may affect the severity and Nanoscale detection of microbial mineral components, the specific impact of runoff, flood risk, soil stability processes in situ: bio-weathering of geometry of bioweathering features, to tillage, and other agricultural and minerals and the different reactivity of mineral anthropogenic activities. The application Terrestrial microbes play a pivotal surfaces resulting from exposure to of AFM to soils allows nanoscale imaging role in mineral weathering, soil formation fungal exuded substances such as and quantification of the nanophysical and the generation of essential growth organic acids (Fig. 3b). Furthermore, the properties of soil building blocks, i.e. nutrients. This process is especially specific nanoscale geometries of such single particles and soil aggregates, evident in forested ecosystems microbial signatures alongside AFM to understand how these ultimately dominated by ectomycorrhizal symbioses characterisation of mineral components influence bulk soil properties. with tree roots. The role of these fungi refractive to dissolution have also For example, the presence of SOM in mineral weathering can be assessed provided a novel molecular mechanism strongly affects the physical properties in situ by evaluating the presence for mineral bond hydrolysis and of soil aggregates. By applying AFM, of microbial signatures in rocks and dissolution. high-resolution topological maps can

500 nm 200 nm (a) (b)

Fig. 3. Bioweathering of silicate minerals by microbial signatures: (a) fungal hypha-created channel and (b) etch pits resulting from fungal organic acid exudates. S. Andrea Gazze

108 Microbiology Today Aug 16 | www.microbiologysociety.org Soil topography Stiffness visual map

7,715 MPa

3,139 MPa

100 nm 2 µm (a) (b)

Biomolecules on soil 20

15

10 Soil roughness (nm) Soil roughness 5

0 100 nm (c) (d) Soil without SOM Natural soil

Fig. 4. Nanoscale natural soil characterisations: (a) topography of soil particle, (b) mineral surface with different stiffness properties (unit MPa), (c) biomolecules on a soil particle, (d) quantitative exemplar of the nano-roughness of natural and organic-matter-deprived soils. S. Andrea Gazze be obtained for mineral surfaces of soil Microbiology: crossing scales Further reading particles and the presence, distribution Exciting novel microbial properties and Gazze, S. A. & others (2013). Nanoscale and nature of biological and other organic behaviours can be revealed by applying observations of extracellular polymeric matter that is covering soil minerals nanoscale analytical techniques per se to substances deposition on phyllosilicates by (Fig. 4). How the nanomechanical pure cultures and in situ ecosystems. The an ectomycorrhizal fungus. Geomicrobiol J 30, properties are affected by SOM can be combined application of biomolecular, 721–730. measured, for instance, by the change nanoscale and macroscopic techniques Gazze, S. A. & others (2014). Chlorite in elastic modulus (Young’s modulus) in has the potential to stepwise fill the gaps topography and dissolution of the interlayer the presence and absence of microbial in understanding microbial(-influenced) studied with atomic force microscopy. Am or plant exudates (Fig. 4b), while the processes in the real world across Mineral 99, 128–138. surface roughness of single aggregates molecular to global scales. Matthews, G. P. & others (2016). Soil appears to increase with the presence of hydrophobicity – relating effects at atomic, bio-organic matter (Fig. 4d). It is therefore S. Andrea Gazze, Lewis Francis & molecular, core and national scales. Geophys very timely to integrate nanoscopic Geertje van Keulen Res Abstr 18, 8478. profiling and bulk soil characterisations Institute of Life Science, Swansea Rakowska, P. D. & others (2013). Nanoscale with functional expression studies, such University, Swansea SA2 8PP, Wales, UK imaging reveals laterally expanding as metaproteomics, to generate deep [email protected] antimicrobial pores in lipid bilayers. Proc Natl mechanistic understanding of the role of [email protected] Acad Sci U S A 110, 8918–8923. microbes in soil ecosystems. [email protected]

Microbiology Today Aug 16 | www.microbiologysociety.org 109 Adding dynamics to structure/function studies in microbiology: two-dimensional infrared spectroscopy

Niall Simpson, Neil T. Hunt & Paul A. Hoskisson Dr Neil Hunt operating a 2D IR spectrometer at the University of Strathclyde. Tim Briggs

ne of the central themes of biology Microbiology is a pioneering discipline. Many of the is that structure is linked to function. fundamental processes of biology were first elucidated OAt the molecular level, however, biology is dominated by intermolecular in microbial systems – deciphering the genetic code, the interactions, such as those between protein–ligand, protein–protein, protein– one gene–one enzyme hypothesis, the mRNA hypothesis, nucleic acid and nucleic acid–nucleic acid. As microbiologists, we often look the first genome sequences, and many more key biological for ways to characterise these kinds of breakthroughs were all the result of microbiologists! In an interactions with a view to understanding our favourite (micro)biological system. age where interdisciplinary projects are encouraged it is These chemical interactions are underpinned by hydrogen bonding, and easy to be cynical. However, true collaborations between electrostatic or hydrophobic processes, occurring between the molecular biologists and physicists can lead to fascinating insight into partners. As we know, proteins are not biological systems, especially when exciting new techniques static entities, and these intermolecular interactions are generally controlled by can be applied to biology. rapid structural fluctuations. For example,

110 Microbiology Today Aug 16 | www.microbiologysociety.org Adding dynamics to structure/function studies in microbiology: two-dimensional infrared spectroscopy

Dr Neil Hunt operating a 2D IR spectrometer at the University of Strathclyde. Tim Briggs

the typical lifetime of a hydrogen bond called ultrafast two-dimensional infrared fundamental to our understanding in solution is around 1 picosecond (to spectroscopy (2D IR). 2D IR is uniquely of globally relevant microbiological put this into context – 1 picosecond is sensitive to structural dynamics and problems such as antimicrobial to 1 second as 1 second is to 31,710 it is capable of extending the range of resistance and climate change. years). To fully understand and exploit information available to microbiologists Infrared (IR) spectroscopy is useful the biology, therefore, it is necessary to studying molecular interactions in a for studying proteins because the C=O complete the structure–function puzzle complementary way to established tools, bonds found in peptide linkages have by incorporating dynamics. This kind of such as macromolecular crystallography, stretching vibrational modes (so-called approach has proved very useful in terms two-dimensional nuclear magnetic ‘amide-I modes’) that are intense and of understanding protein–ligand binding, resonance (2D NMR), surface plasmon sensitive to the inter- and intra-molecular such as antibiotic molecules binding to resonance (SPR) and isothermal processes (think, school ‘mass on a their target proteins, or post-translational titration calorimetry (ITC). In terms of spring’ type experiments) that underpin modifications of proteins. microbiological applications, 2D IR is a biological function, such as protein In order to observe fast processes, technique in its infancy and we are only folding, ligand binding and enzymatic you need a ‘camera’ with a fast shutter now beginning to discover its potential. reactions. These are most commonly speed. One way to approach the We feel 2D IR is capable of making studied via IR absorption spectroscopy, understanding of protein dynamics with a novel contribution to microbiology which gives rise to a ‘one-dimensional’ sub-picosecond temporal resolution is research areas, at a time when studies plot of absorbance versus vibrational a relatively new experimental technique of protein–ligand interactions are frequency (See Fig. 1a). However, the

Microbiology Today Aug 16 | www.microbiologysociety.org 111 (a) Absorbance

Vibrational frequency (cm–1) )

–1 (b)

benefits of amide-I mode spectroscopy thought of as a map of the vibrational (cm 1 are restricted because the absorptions modes within a molecule and how they from individual amino acid residues in interact. This off-diagonal region of the proteins overlap, or couple strongly to spectrum is richly structured, revealing form delocalised vibrations that are due patterns that are unique to secondary to protein secondary structure elements structural elements of the protein, but Pump frequency, ω Pump frequency, such as α-helices or β-sheets. also reports on interactions between Multidimensional spectroscopy them. Most usefully, in terms of providing Probe frequency, (cm–1) methods such as 2D IR measure the complementary information to established ω3 same molecular response as an IR microbiological techniques, the anti- absorption experiment, but spread the diagonal direction of the 2D IR spectrum Fig. 1. (a) Representation of a ‘one-dimensional’ IR spectrum out over a 2nd frequency axis reports on the flexibility of molecules, spectrum with two vibrational modes and (b) 2D in order to uncover features that are as structural motion allows vibrations to IR counterpart to the same spectrum, illustrating not resolved in 1D. Practically, rather undergo small frequency changes during coupling patterns between the two vibrational than simply measuring absorption, the time delay between laser pulses modes. These two coupled vibrations will generate a 2D IR experiment consists of two (Fig. 2). In turn, more flexible molecules off-diagonal patterns, at coordinates indicated by IR laser pulses that hit the sample give rise to greater anti-diagonal line the blue arrows. Niall Simpson with a precisely controlled time delay broadening. These dynamics have been complementary fashion. For example, the between them. The first pulse excites shown to vary sensitively with ligand fast dynamics extracted by 2D IR bridge the sample at a particular frequency binding and provide fundamental insight the gap between the atomic resolution (makes it vibrate) and the second laser into this fundamental intermolecular of crystallography and the slower pulse measures the effect of this on the contact. Thus, 2D IR provides a large (µs–ms) dynamics accessible by NMR. molecule via an absorption spectrum. By quantity of new information but the Importantly, 2D IR measures protein progressively changing the wavelength difficulty lies in interpreting this in terms dynamics on the exact timescale that of the laser for the first (pump) pulse, of biological function. computational simulations like molecular one builds up a 2D plot of excitation An important tool in this is to dynamics predict. These methods are (pump) versus detection (probe) link 2D IR data to other methods in a used extensively to infer biological frequency. The time delay between the Pulse delay time 1 Pulse delay time 2 laser pulses allows us to examine a protein’s structure and its dynamics on the timescale of the pulse duration (~100 fs). This means that the IR absorption

spectrum lies along the diagonal of the ) –1 2D IR plot (a surface response plot) while (cm new information is found in the off- 1 diagonal region of the spectrum (Fig. 1b). It is these off-diagonal regions that report on interactions between the structural components of our protein molecules Pump frequency, ω Pump frequency, – interactions between vibrations on

specific amino acid residues or energy –1 Probe frequency, ω3 (cm ) transfer processes within the molecule – essentially informing us on the structural Fig. 2. 2D IR spectra depicting the effect of molecular flexibility on the anti-diagonal width of vibrational interactions within proteins. modes. With longer pulse delay times, it becomes decreasingly likely to find flexible molecules in the same An example of a 2D IR spectrum position or orientation as that at the first pulse. Correspondingly, their excited vibrational frequencies will also is shown in Fig. 1(b). These can be evolve with time, resulting in anti-diagonal line-broadening. Niall Simpson

112 Microbiology Today Aug 16 | www.microbiologysociety.org Catalase–NO, pulse Catalase–NO, pulse delay = 0.5 ps delay = 15 ps

) 1900 1900 –1 (cm 1 1880 1880

1860 1860 Paul A. Hoskisson

Pump frequency, ω Pump frequency, 1840 1840 Strathclyde Institute of Pharmacy and Biomedical Sciences, University 1840 1860 1880 1900 1840 1860 1880 1900 of Strathclyde, 161 Cathedral Street, –1 Probe frequency, ω3 (cm ) Glasgow G4 0RE, UK [email protected] His71 W1 Phe156 @paulhoskisson Fig. 3. 2D IR depicting the anti-diagonal line- Asn143 broadening of a nitric oxide molecule bound at the Further reading active site of catalase. Rapid broadening of the NO Adamczyk, K. & others (2012). Measuring vibration indicates the presence of a network of protein dynamics with ultrafast two- mobile water molecules in the distal cavity that Tyr353 dimensional infrared spectroscopy. Meas Sci are not resolved in the crystal structure, shown Technol 23, 062001. below. Niall Simpson Adamczyk, K. & others (2015). Ultrafast function, but until now experimental and enzymology to augment the 2D infrared spectroscopy reveals water-mediated confirmation of the results has not been IR studies and develop a clear picture coherent dynamics in an enzyme active site. achievable. of the active site structure–function– Chem Sci 6, 505–516. We have used 2D IR to study a dynamic relationship. We have been able Bagchi, S. & others (2010). Conformational range of systems, to shed light on the to recreate mutations within the InhA switching between protein substates studied role dynamics plays in understanding protein that lead to clinical resistance with 2D IR vibrational echo spectroscopy and microbiological processes. Recently, and study how these behave when molecular dynamics simulations. J Phys Chem we applied 2D IR to an investigation of exposed to isoniazid and also natural B 114, 17187–17193. protein structural dynamics of the active enzymatic substrates. Understanding the Hunt, N. T. (2009). 2D-IR spectroscopy: site of catalase from the bacterium role of molecular dynamics in relation to ultrafast insights into biomolecule structure Corynebacterium glutamicum, and how structure and function can aid the design and function. Chem Soc Rev 38, 1837–1848. this enzyme is inhibited by nitric oxide. By of novel anti-TB drugs in the future and Ishikawa, H. & others (2007). Neuroglobin recording the dependence of anti-diagonal will hopefully lead to the recognition dynamics observed with ultrafast 2D-IR peak width of the NO group as a function that 2D IR has a key role to play in vibrational echo spectroscopy. Proc Natl Acad of the time delay between laser pulses microbiology. Sci U S A 104, 16116–16121. (Fig. 3a), we were able to show for the Currently, 2D IR is not a bench-top Shaw, D. J. & others (2015). Multidimensional first time that mobile water molecules experiment, with each spectrometer infrared spectroscopy reveals the vibrational are present in the active site of the being built by the users. However, this and solvation dynamics of isoniazid. J Chem enzyme and that these are responsible was the situation that NMR was in 40 Phys 142, 212401–212409. for large-scale vibrational modes of the years ago and now it is a standard Simpson, N. & Hunt, N. T. (2015). Ultrafast haem pocket that plays a key catalytic experimental technique. 2D IR has 2D-IR spectroscopy of haemoproteins. Int Rev role in the enzymatic reaction. great potential for microbiology and Phys Chem 34, 361–383. Currently, we are using 2D IR biochemistry; there are many problems Simpson, N. & others (2013). Infrared to investigate antimicrobial drug in microbiology that can be assisted spectroscopy of nicotinamide adenine resistance, specifically the inhibition with such techniques – so be open dinucleotides in one and two dimensions. of the essential enoyl reductase (InhA) to collaboration and befriend a tame J Phys Chem B 117, 16468–16478. from Mycobacterium tuberculosis by the physicist! Thielges, M. C. & others (2011). Protein frontline anti-TB drug isoniazid. We have dynamics in cytochrome P450 molecular studied the components of the active Niall Simpson, Neil T. Hunt recognition and substrate specificity using 2D isoniazid-NADH complex and are now Department of Physics, SUPA, University IR vibrational echo spectroscopy. J Am Chem combining standard molecular biology, of Strathclyde, 107 Rottenrow East, Soc 133, 3995–4004. such as site-directed mutagenesis Glasgow G4 0NG, UK

Microbiology Today Aug 16 | www.microbiologysociety.org 113 Cryogenic electron microscopy – beyond the resolution revolution

David Bhella

Cryo-electron microscopy (cryoEM) has recently emerged as a structural biology technique to rival X-ray crystallography. Technological developments in both instrumentation and software have allowed researchers to determine the structures of macromolecular assemblies at close to atomic resolution. Moreover, assemblies that would be intractable by X-ray methods owing to heterogeneity or disordered regions have become viable targets for structure determination, prompting the wide uptake of a method that, until comparatively recently, was sometimes considered ‘niche’. So, what does the future hold for structural electron microscopy and what does it offer the microbiologist?

The long history of a rapidly emerging limited the achievable resolution in integrity of biological assemblies, and method in structural biology biology. Classical biological transmission high-resolution features are lost. Since its invention in the 1930s, the electron microscopy preserves In the 1980s, researchers working transmission electron microscope (TEM) cells and tissue by chemical fixation in the laboratory of Jacques Dubochet, has been an invaluable tool to investigate and dehydration, followed by resin at the University of Lausanne, pioneered the shapes of macromolecular embedding and ultra-thin sectioning. methods for the preparation of biological assemblies and cellular ultrastructure. Purified macromolecules are likewise material in a frozen hydrated state, by Although the TEM has long been capable imaged in a dehydrated state, in negative freezing suspensions of macromolecules of imaging materials at the atomic scale, stain. Drying, fixation and staining so rapidly that there is no time for ice specimen preparation requirements severely compromise the structural crystals to form – a process known

114 Microbiology Today Aug 16 | www.microbiologysociety.org as vitrification. This led to a landmark publication describing cryogenic TEM imaging of viruses (Nature paper in Further reading). Images recorded in the TEM are two-dimensional (2D) projections of the three-dimensional (3D) object. For Fig. 1. Cryomicroscopy of a calicivirus gave 2D projection views of randomly oriented particles (background). structure analysis, images of identical These were processed to produce a 3D density map (bottom-right – central slice through the 3D map), which objects viewed in different orientations can be represented as a coloured 3D surface. Michaela Conley and David Bhella, University of Glasgow may be computationally processed to determine a 3D density map (3D reconstruction – Fig. 1). Methods to recover 3D structure data from negative-stain TEM images were first developed by David DeRosier and Aaron Klug in the late 1960s, working at the Medical Research Council Laboratory of Molecular Biology in Cambridge. In the late 1980s, the application of computational 3D reconstruction methods to cryoEM images led to the publication of the first low-resolution structures for unstained icosahedral viruses and the birth of a new discipline Fig. 2. Specimen drift can lead to a blurred image that is unsuitable for structure analysis (left). The high in the field of structural biology. frame rate of DDD cameras means that this artefact can be computationally corrected (right), yielding sharp images suitable for 3D reconstruction. Diana Alves and Toby Jenkins, University of Bath; David Bhella, University of Glasgow From blob-ology to atoms These early reconstructions provided insights into the quaternary arrangements of larger assemblies and allowed the visualisation of domains in protein structures. Technological innovations in the intervening years led to a steady increase in attainable resolution. Improvements in microscope design and the development of algorithms to correct an imaging artefact known as the contrast-transfer function allowed researchers to determine structures at better than 10 Å (1 nm) resolution in the late 1990s. For the first time this produced reconstructions of sufficient detail to permit the modelling of protein folds under certain optimal conditions. At resolutions around 8–9 Å, Fig. 3. The 2.2 Å structure of -galactosidase. Science 348, 1147–1151 (2015), EMDB 2984 and PDB 5a1a β α-helices are seen as tubes of density.

Microbiology Today Aug 16 | www.microbiologysociety.org 115 350

Structures better than 10 Å 300 Structures better than 4 Å

250

200

150

At 5 Å, β-strands start to resolve and the 100 larger amino acid side-chains become

visible. 50 A step change in the capacity of

cryoEM to solve protein structures came 0 about as a consequence of several 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 technological developments over the past five years. First and foremost, the Fig. 4. A plot to show the number of structures deposited in the EM databank each year that are better than development of second-generation 10 (blue) and 4 (orange) Å resolution. This illustrates the recent explosion of high-resolution structures digital cameras for TEM that directly determined by cryoEM. detect electrons as they pass through the sensor. Direct detection devices (DDD) record high-resolution information far more efficiently than the previous CCD-based cameras or photographic film. DDDs also operate at very high frame rates, between 20 and 400 fps. Thus, rather than recording a single frame, micrographs are recorded as movies of many tens of images. At the highest frame rates, DDD cameras can count individual electrons passing through the camera, greatly reducing noise. DDDs have not only vastly improved the quality of images recorded in the TEM, they have also informed our understanding of specimen behaviour under the electron beam. Specimen drift had been a major problem in cryoEM, Fig. 5. The 3.8 Å structure of Zika virus. Science 352, 467–470 (2016), EMDB 8116 which would severely degrade image quality. Images recorded as movies can stable lenses and automation, have from images recorded in the cryoEM be aligned and averaged to compensate improved data quality and throughput. was solved by analysis of 2D crystals of for specimen drift during the exposure Finally, modern software algorithms bacteriorhodopsin as early as 1990. It (Fig. 2). DDD movies have, however, allow researchers to perform in was nearly 20 years before comparable revealed that specimen movement silico purification: classification of resolutions were achieved for particulate during imaging is much more complex heterogeneous datasets to solve multiple assemblies – helical and icosahedral than previously thought. In addition to structures or exclude damaged particles objects were solved in 2007 and 2008, lateral drift of the specimen, particles from the 3D reconstruction. Taken respectively. The lower symmetry 20S also rotate and move relative to each together, these technological advances proteasome (a 14-mer) and asymmetric other as the ice layer ‘domes’ under the have extended attainable resolution to ribosome were solved in 2013 using DDD electron beam, an effect that can also 2–4 Å, a point at which protein models image data. More recently, astonishing now be corrected in the reconstruction can be built with a high degree of resolutions have been achieved such as process. confidence. the 2.2 Å structure of β-galactosidase Recent developments in electron The first de novo structure deposited (Fig. 3). The improved contrast of DDD microscope design, including more in the protein data bank (PDB) derived cameras operated in electron counting

116 Microbiology Today Aug 16 | www.microbiologysociety.org This remains challenging, as the size of objects that may be imaged in the TEM is limited by the penetrating power of the electron beam. Several approaches have been taken to overcome this; firstly, it is possible to propagate viruses in cells that have thin regions and may Fig. 6. A central slice through a tomogram of a neuronal cell that is infected with herpes simplex virus type 1, be imaged directly (Fig. 6). Many of the capsids (black arrows) are seen travelling along microtubules (white arrows). Rebecca Lauder and Frazer Rixon, most interesting processes occur at the University of Glasgow. heart of the cell and in large organelles. This requires the cell to be sectioned in modes means that smaller assemblies are beginning to allow researchers to a frozen-hydrated state. While this is far are also becoming viable targets for characterise dynamic processes. from trivial, tantalising first steps are analysis, as illustrated by the recent The TEM is a versatile instrument, being taken to achieve this. structure of the ~170 kDa human however. In addition to high-resolution CryoEM has opened new horizons γ-secretase. analysis of purified assemblies, cryo- in structural biology research that Technological advances in cryoEM electron tomography (cryoET) allows us will have broad application to address have led to a seismic shift in the field to investigate the structure of unique fundamental questions in microbiology. of structural biology, bringing about a entities. This is achieved by rotating Rapid calculation of high-resolution rapid expansion in the number of close the specimen in the electron beam and structures for purified macromolecular to atomic-resolution structures in the recording an image every 1–3 degrees. assemblies, as well as the developing EM data bank (Fig. 4). Such structures The resulting tilt-series of images capacity to determine structures in situ, can be determined swiftly, often only is then processed to compute a 3D are tremendously powerful additions requiring one or two days of microscope reconstruction or tomogram. Repeated to the microbiologist’s toolbox that will time followed by several weeks of features within the tomogram may be increasingly inform our understanding of computation. This is illustrated by the extracted, brought into common register molecular structure and function in the speed with which structures of Zika and averaged – subtomogram averaging. coming years. virus have been produced (Fig. 5). As well as providing insights into the structure of pleomorphic structures David Bhella Taking structural biology into such as enveloped viruses, cryoET Centre for Virus Research, Sir Michael the cell raises the tantalising prospect of in situ Stoker Building, 464 Bearsden Road, Electron microscopy as a tool in structure determination: the capacity Glasgow G61 1QH, UK structural biology might be said to to solve the structure of an object [email protected] have come of age in recent years, functioning in the cellular environment. @DavidBhella finally realising its potential as a high- Assemblies such as flagellar motors resolution method. In my view, the or bacteriophages can be investigated Further reading cryomicroscopy revolution has just in situ through tomography of frozen- Adrian, M. & others (1984). Cryo-electron begun. Single particle reconstruction hydrated intact bacteria, albeit presently microscopy of viruses. Nature 308, 32–36. still has considerable potential for at lower resolution. In situ structure Bartesaghi, A. & others (2015). 2.2 Å improvement; minimising specimen analysis of macromolecular assemblies resolution cryo-EM structure of movement should lead to improved within eukaryotic cells is also becoming β-galactosidase in complex with a cell- resolution. Larger, faster DDD cameras a possibility. In virology, many aspects of permeant inhibitor. Science 348, 1147–1151. with improved imaging performance will the replication cycle, for example viral Sirohi, D. & others (2016). The 3.8 Å resolution increase throughput and data quality, attachment, entry, trafficking through cryo-EM structure of Zika virus. Science 352, while software methods to deal with cellular organelles and morphogenesis, 467–470. domain movement within assemblies are becoming open to investigation.

Microbiology Today Aug 16 | www.microbiologysociety.org 117 CRISPR- Cas: from mechanism to applications

Edze Westra Coloured scanning electron micrograph of Lactobacillus bacteria. Science Photo Library

118 Microbiology Today Aug 16 | www.microbiologysociety.org All organisms are exposed to parasites, so they evolved a range of different immune mechanisms to defend themselves. About 10 years ago, scientists discovered that bacteria and archaea, generally regarded as primitive organisms, have a sophisticated adaptive immune system, known as CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats). The insights gained about this system led to applications in industry to protect bacterial species against their viral parasites. In addition, CRISPR-Cas has been turned into a versatile genome editing method that has the potential to treat human genetic diseases.

What is adaptive immunity? components. The first is the so-called The three stages of CRISPR-Cas There are many ways to categorise CRISPR locus, which is located on the How do CRISPR-Cas systems work? immune systems. One common bacterial (or archaeal) genome. CRISPR In a nutshell, CRISPR-Cas systems distinction is whether immune systems loci function as a genetic memory of capture pieces of DNA from a parasite are pre-programmed (innate) or re- previous infections. They are composed (for example, a virus), integrate this programmable (adaptive). The best of repeating sequences that are typically into the CRISPR locus, and use this known example of adaptive immunity around 30 nucleotides in length. information to destroy the parasite when is the antibody response in vertebrates. These repeating sequences (repeats) it infects again. This process is generally Unlike innate immunity, adaptive are separated by unique sequences described as having three (more or immunity is acquired during the lifetime of similar length. It is these unique less) independent stages. The first is of the host and always results in a sequences that matter most. These the ‘Adaptation’ stage, which occurs response that is highly specific towards are the memory sequences, derived immediately upon infection with a novel the parasite that the host interacts with. from parasite genomes, and they are parasite. During ‘Adaptation’, dedicated For a long time it was presumed that referred to as ‘spacers’. Spacers in the Cas proteins capture a piece of parasite adaptive immunity was restricted to CRISPR locus confer immunity against DNA and integrate this into the CRISPR higher organisms. Adaptive immunity in any parasites that carry a matching locus, where it forms a novel spacer. prokaryotes has been one of the most sequence. The sequence information Next, the ‘Expression’ stage takes place, bewildering discoveries in microbiology. stored in the CRISPR locus is used by during which the entire CRISPR locus is the second key element of CRISPR- copied into an RNA molecule. This long The architecture of a CRISPR-Cas Cas systems, the Cas proteins. Cas (for RNA transcript is cleaved by dedicated adaptive immune system CRISPR-associated) proteins are often ribonucleases to generate small crRNA CRISPR-Cas systems consist of two key encoded next to a CRISPR locus. molecules that contain only a single

Microbiology Today Aug 16 | www.microbiologysociety.org 119 “ organism.“ Some first studies indicate The interaction between CRISPR-Cas and antibiotic resistance that this technology can be used to cure human diseases that are caused by is unclear; some studies support the hypothesis that CRISPR- specific genetic mutations. What makes CRISPR-Cas9 systems so suitable for Cas blocks the spread of resistance genes, whereas other genome editing? Firstly, a key feature studies found no evidence for this. Understanding the short- of crRNA-Cas9 complexes is that they will specifically interact with DNA and long-term implications of CRISPR-Cas is key if we are to sequences that are complementary to the crRNA. Secondly, this specificity predict and manipulate the spread of antibiotic resistance. can be readily adjusted by changing the crRNA sequence, which provides extreme control over the crRNA-Cas9/ spacer sequence. A single crRNA forms of CRISPR-Cas systems has facilitated DNA interaction. Thirdly, Cas9 will cleave a complex with one or multiple Cas the development of a CRISPR-Cas9- the corresponding sequence, resulting proteins. This complex is one of the based genome editing technology that in a DNA break, which is an essential key functional modules of the CRISPR- makes it much more straightforward step in the genome editing process. The “Cas immune system, and functions to introduce any mutation of interest double-stranded break will be repaired analogous to an antibody in parasite into the genome of our favourite study by a DNA repair pathway. Depending detection. In the event of re-infection Guide RNA with the same parasite, the ‘Interference’ stage is triggered. During this stage, Cas- crRNA complexes bind and cleave the Matching Cas9 genomic genome of the parasite, which results in sequence host immunity.

Two classes of CRISPR-Cas systems Genomic DNA CRISPR-Cas systems are highly diverse, and have been classified into two main classes, and several types and subtypes. One of the most obvious differences is that Class 1 systems encode many more Donor DNA Cas proteins compared with Class 2 systems. Unlike Class 1 systems, Class Repair 2 Cas-crRNA complexes contain only a Targeted single Cas protein – the most famous genome example being Cas9 – and because of editing their relatively low complexity, Class 2 systems are uniquely suited for genome engineering applications.

Genome engineering Diagram of the process that occurs within the CRISPR-Cas9 gene editing complex. This protein (purple) is used Introducing specific mutations into in genome engineering to cut DNA. It uses a guide RNA sequence (green) to cut DNA (blue) at a complementary a genome has long been extremely site. The cut area of DNA is shown in yellow. The process of inserting a fragment of donor DNA (red) is shown at challenging. Our detailed understanding lower centre, with a genetically modified laboratory mouse.Gunilla Elam / Science Photo Library

120 Microbiology Today Aug 16 | www.microbiologysociety.org benefits of CRISPR-Cas across different environments will help to design optimal defence strategies for bacterial species.

CRISPR-Cas and antibiotic resistance Antibiotic resistance genes often move horizontally between bacterial species, for example through plasmid conjugation. Bacterial immune systems may limit such spread, since they can form a barrier for incoming DNA. The interaction between CRISPR-Cas and antibiotic resistance is unclear; some studies support the hypothesis that CRISPR-Cas blocks the spread of resistance genes, whereas other studies found no evidence for this. Understanding the short- and long-term implications of CRISPR-Cas is key if we are to predict and manipulate the spread of antibiotic resistance. The discovery of the CRISPR-Cas system led to a sea of change in the microbiological sciences. Adjusting to the concept that ‘primitive organisms’, such as bacteria and archaea, had sophisticated adaptive immune systems CRISPR-Cas9 gene editing complex from Streptococcus pyogenes. Molekuul / Science Photo Library was just the start. Since then, scientific on the method that is used, DNA will be infections during the fermentation, which endeavour has seen the number of repaired through the non-homologous leads to product downgrading or loss. In potential uses for this primitive immune end joining (NHEJ) pathway, or the the food industry, there is great interest system, including genetic engineering homology-directed repair (HDR) pathway. in bacterial strains with naturally with higher organisms, increase rapidly. NHEJ will result in higher efficiency of evolved resistance, and CRISPR provides In turn, the potential applications for this mutation incorporation, but with little an ideal system to generate strains that primitive microbial system are providing control over the mutations that are are more robust. However, these applied ethical questions that need to be introduced, while HDR provides full aspects of bacterial culture protection addressed by society as the implications control over the mutations but with naturally lead to questions concerning for the CRISPR-Cas system continue lower efficiency. the evolution and ecology of CRISPR-Cas to emerge, especially within clinical systems. It was recently demonstrated settings. CRISPR-Cas to protect bacterial that mounting a CRISPR-Cas immune species in industry response is associated with a fitness Edze Westra Much of the initial interest in CRISPR- cost for the host. This may help to University of Exeter, Penryn Campus, Cas came from the industry working explain why only half of all bacteria have Penryn, Cornwall TR10 9EZ, UK with lactic acid bacteria. For example, these sophisticated adaptive immune [email protected] yoghurt production can fail due to virus systems. Understanding the costs and

Microbiology Today Aug 16 | www.microbiologysociety.org 121 Phage therapy

Laura Bowater

Coloured transmission electron micrograph of bacteriophage particles (turquoise) attacking a bacterial cell (red). AMI Images / Science Photo Library

122 Microbiology Today Aug 16 | www.microbiologysociety.org As early as 1945, in his Nobel Prize Lecture titled ‘Penicillin’ Alexander Fleming declared, “There is the danger that the ignorant man may easily underdose himself and by exposing his microbes to non-lethal quantities of the drug make them resistant”.

leming’s speech was prescient. In specificity to target particular bacterial May of this year (2016) headlines species. Faround the world reported the case of the first known US patient to be Phage therapies from the past infected with Escherichia coli bacteria In 1915, more than 10 years before that possess resistance to a ‘last resort’ Fleming’s article on penicillin, an antibiotic, colistin. This was not the first English doctor called Frederick Twort time that ominous stories about colistin- (1877–1950) published an article resistant bacteria hit the headlines; entitled ‘The Infectious Disease of the colistin-resistant bacteria have been Micrococcus’ in The Lancet. It described found in China, Canada and Europe, how a phage infection of colonies of including the UK. The implications are Staphylococcus gave rise to clear zones clear: we are running out of effective of lysis as the phages appeared to antibiotics as the number of antibiotic- destroy the bacteria. In 1917, the French resistant pathogens continues to Canadian microbiologist, Félix d’Herelle increase and even the antibiotics of last (1873–1949) described these infectious resort are losing their efficacy. particles as bacteriophage (as they ‘eat’ Tackling this growing crisis is an bacteria). The name has stuck to this urgent priority and attention is focusing day. In 1919, shortly after the end of the on protecting and preserving the First World War, d’Herelle used phages antibiotics that we have at our disposal, to treat dysentery for the first time in the as well as hunting for new antimicrobial Hôpital des Enfants-Malades in Paris. products. Antibiotics work by targeting The phage preparation was administered and destroying bacteria. However, to a 12-year-old boy suffering with they are not unique in that respect. severe dysentery. The patient’s Bacteriophages, or phages for short, can symptoms improved immediately and also target and destroy bacterial cells. the boy recovered fully within a few Phages are ubiquitous; they are found in days. Encouraged by the success of seawater, freshwater, soil and sewage his original solution, new preparations slurries. It is estimated that there are of bacteriophages were prepared to 1030–1032 phages in the biosphere and target other bacteria, including Vibrio every 48 h, phages infect and destroy cholera and Yersinia pestis. Soon, reports about half the bacteria in the world. The began to emerge that these new phage Coloured transmission electron micrograph of bacteriophage particles (turquoise) attacking a bacterial cell (red). AMI Images / Science Photo Library remarkable thing about phages is their solutions were successfully treating

Microbiology Today Aug 16 | www.microbiologysociety.org 123 thousands of people in China, Laos, India, Vietnam and Africa who were suffering from cholera and bubonic plague. Pharmaceutical companies began to sell phage solutions commercially. D’Herelle’s laboratory in Paris produced phage preparations against various bacterial infections that were sold by what became the large French company L’Oréal. In the 1940s, Eli Lilly and Company (Indianapolis, Indiana, USA) also produced several phage products for human use that targeted pathogens such as staphylococci, streptococci and E. coli. These pathogens are still causing concern today as antibiotic-resistant species continue to increase. D’Herelle’s collaborations with scientists in Tbilisi, Georgia, ensured that Colour-enhanced transmission electron micrograph of enterobacteria phage T4, a phage that infects Escherichia coli phage therapy was adopted as a clinical bacteria. Omikron / Science Photo Library treatment for bacterial infection in the former Soviet Union and Eastern Europe. infections in humans, as well as food the cellular membrane to attack and Unfortunately, the results of these studies additives designed to reduce bacterial degrade the bacterial cell wall. Phage did not use the rigorous, double-blinded contamination of ready-to-eat meat, fruit, endolysins differ depending on whether clinical trials that are the current gold vegetables and dairy products. they target the cell wall of Gram-positive standard used to test new therapeutic Scientists are exploiting phages or Gram-negative cells. What makes drugs and interventions today. These in other ways. After phages enter the endolysins so interesting is that they can studies were extensively published bacterial cell they force the bacteria be applied exogenously as recombinant in non-English (primarily Russian, to synthesise new phage particles proteins to destroy bacterial cells. In Georgian and Polish) journals. As a that accumulate inside the cell. At addition, compared with many antibiotics result, they were not readily accessible the end of their replication cycle the still in clinical use, the endolysins have to the western scientific and medical phages use suicidal enzymes, called an added advantage; they exhibit a community, and phage therapy rapidly endolysins (phage lysins) to degrade genus or species specificity. These fell out of favour in the west, especially the peptidoglycan found in the bacterial characteristics offer exciting therapeutic after antibiotics became commercially cell walls and the progeny phages are possibilities. Lysins encoded by phages available. released into the environment. The lysis that infect Gram-positive hosts usually of the bacterial cell is also enabled by contain two distinct domains. They have Phage therapy in the future another group of phage enzymes, the an N-terminal catalytic domain (CD) and However, as there is a dearth of new, holins, which are produced during the a C-terminal cell-wall binding domain clinically relevant antibiotics emerging late stages of phage infection. Holins (CBD). Engineering chimeric lysins, or through the antibiotic discovery pipelines, and endolysins are encoded by the ‘chimeolysins’, by mixing and matching there is renewed interest in revisiting bacteriophage genomes and synthesised the two domains from various natural phage therapy. Currently, there are by the bacterial cellular machinery. As lysins offers opportunities for finding clinical trials, which are examining the the holins oligomerise, they punch holes novel enzymes with improved properties safety and efficacy of phages as topical through the inner bacterial membrane. such as increased solubility and an therapies for treating skin and ear This allows the lysins to move through extended lytic spectrum. Currently,

124 Microbiology Today Aug 16 | www.microbiologysociety.org methods for developing chimeolysins, Lysins offer enormous potential such as domain shuffling, depend largely as effective antibacterials in the fight on a trial-and-error approach, and this against infectious disease caused by limits the efficiency for discovering pathogens with multidrug resistance. effective chimeolysins. Antibiotics such as penicillin and Scientific esearchr is currently cephalosporin inhibit peptidoglycan underway to examine how lysins can synthesis, and only dividing cells are be used to treat and destroy pathogens lysed. In contrast, lysins destroy the associated with food production such as peptidoglycan directly, killing both Clostridium perfringens, which causes growing and non-growing cells. And, necrotic enteritis in poultry and is one unlike antibiotics that often kill bacteria of the leading causes of food poisoning indiscriminately, lysins display a in humans. In addition, Staphefekt is the high specificity that ensures that the first endolysin to be registered for human normal commensal microflora is left

use against common skin conditions undisturbed. As bacteriophages are such as eczema, acne and rosacea, considered the most abundant biological which are caused or exacerbated by entities on Earth, they are a rich natural Staphylococcus aureus. source of these enzymes. It is hoped Colour-enhanced transmission electron micrograph of enterobacteria phage T4, a phage that infects Escherichia coli Despite the promise of viable that bioinformatic and proteomic studies bacteria. Omikron / Science Photo Library antibiotic alternatives and a previous will lead to new opportunities for

medical history, phage therapy and phage domain swapping, and the production derivatives still provoke serious concerns of specifically engineered designer among the scientific establishment. chimeolysins with diverse applications Issues include the fact that bacteria are that will be part of the solution to the Lysins destroy the just as capable of developing resistance growing problem of antibiotic resistance. to phages and phage lysins as they are peptidoglycan directly, to develop resistance to antibiotics. But Laura Bowater killing both growing and phages“ can also mutate their endolysins The Norwich Medical School, University and holins, enabling them to re-infect of East Anglia, Norwich NR4 7TJ, UK non-growing cells. And, phage-resistant bacteria, and this [email protected] process happens rapidly and naturally; unlike antibiotics that often it does not rely on human intervention. Further reading Although humans and animals are Chanishvili, N. (2012). Phage Therapy – kill bacteria indiscriminately, constantly exposed to phages, much History from Twort and d’Herelle Through Soviet more research is required to ensure Experience to Current Approaches. Advances in lysins display a high that phages and phage-derived therapy Virus Research, pp. 3–40. Elsevier. do not cause adverse reactions. As Fenton, M. & others (2010). Recombinant specificity that ensures lysins are proteins, they are capable of bacteriophage lysins as antibacterials. Bioeng stimulating an immune response when Bugs 1, 9–16. “that the normal commensal administered mucosally or systemically. Stern, A. & Sorek, R. (2011). The phage–host This immune response can potentially arms race: shaping the evolution of microbes. microflora is left decrease the lysis activity. It is absolutely BioEssays News Rev Mol Cell Dev Biol 33, undisturbed. essential that the phages or phage 43–51. derivatives, such as lysins, are screened Twort, F. W. (1925). The Discovery of the to make sure that they are safe to use for “Bacteriophage.” The Lancet 205, 845. human and animal consumption.

Microbiology Today Aug 16 | www.microbiologysociety.org 125 Annual Conference 2017 #Microbio17

Work continues with the preparations for our Annual Conference 2017, which takes place Impact of Annual at the Edinburgh International Conference Centre (EICC) between 3 and 6 April. Abstracts submission will be opening soon and closing just before Christmas, so start working on your Conference attendance submissions today to be in with a chance of presenting your work through an offered paper As part of our evaluation following or poster! You can now register online and take advantage of our early bird rate, and don’t this year’s event we discovered forget to apply for your grants in time to ensure you meet the deadlines. that the impact of attending our Conference is far greater than we Visit the Society website to view all of the information about the Annual Conference and could have imagined. Here are some follow the Twitter hashtag #Microbio17 for regular updates. of the outcomes delegates shared with us, relating to professional development and developing

Thinkstock connections: Professional development • I gained experience of presenting my work Destination Edinburgh some of the must-see attractions as • I updated my knowledge Scotland’s capital city is globally recommended by Convention Edinburgh: • I have been invited to present at recognised as a world-leading • Make some time to explore the other meetings authority in the sciences, technology delights of Edinburgh Castle, which • I identified funding opportunities and education. Home to more than is visible from almost every main • I identified the latest research to 3,000 researchers and 100 market- street in the city centre. It’s a support with my dissertation topics leading companies, it is a major part medieval fortress built upon the site • I arranged an external examiner for my PhD student of Scotland’s science and technology of an extinct volcano and provides • I met the expert in my field sector – a sector that continues to lead breath-taking panoramic views of • I found the opportunity to recruit the world. Edinburgh. young talent Did you know that it was in • Scotland’s Parliament Building is • I was able to identify job Edinburgh that chloroform was first located at the bottom of the Royal opportunities and new job leads used as an anaesthetic? Dolly the sheep was Mile and is open to visitors all year Developing connections cloned at the University of Edinburgh’s round, except when Parliament is • I was able to meet face-to-face with The Roslin Institute, and the first ‘bionic’ in recess. Guided tours are free of others hand – a powered prosthetic with charge. • I developed contacts and research articulating fingers – was developed • The Royal Yacht Britannia is one groups • I met with collaborators by a spin-out company from the city’s of the UK’s top tourist attractions. • I developed joint projects Princess Margaret Rose Hospital. Berthed at Leith Docks, it’s available • I developed long-term friendships So along with Edinburgh’s status as for guided group tours. and networks a thriving scientific hub; plus plenty of • The National Museum of Scotland • I networked with researchers at a attractions, lots of history, superb travel provides a diverse collection similar stage in their career to me systems and fantastic accommodation, it that will take you on a journey of • I met and discussed with people who were able to advise on current was an obvious choice as the location for discovery through the history of difficulties in my research our 2017 Annual Conference. Scotland, the wonders of nature and • Some of the companies at the world cultures – all under one roof stalls have agreed to sponsor our Must-sees in Edinburgh and with free admission. research/send us free samples. There’s so much to see and do in Search online for more must-sees • I discovered new products from the Edinburgh, why not plan some time and further information on tickets and commercial stands I found a PhD student working on the before or after the conference to visit opening times. • same topic as me

126 Microbiology Today Aug 16 | www.microbiologysociety.org Keep up-to-date with events, follow the Society Thinkstock on Twitter: @MicrobioSoc Accommodation and Travel

Because Edinburgh provides so much our booking service via Reservation together? Perhaps you can share a twin to see, do and explore, the destination Highways. We have instructed them to room or even hire an apartment; plus,- attracts many visitors all year round secure negotiated rates at hotels to suit when travelling together you may be and therefore hotels are continuously a range of budgets and you can make able to save on fares with some train in use. So if you’re planning on joining your reservation today, or you may wish companies who offer discounts for us for Conference next year we would to make your own enquiries by searching multiple bookings. And why not share highly recommend you secure your online travel agents or visiting the local taxi fees when you arrive? accommodation and make your travel tourist board. Either way, avoid delay so Getting to Edinburgh is easy and plans as early as possible. you can get the best value for money! below is a guide to assist your plans: To aid you with securing your One last thought – if you are Air Edinburgh Airport is served by accommodation you can visit our planning on travelling as a group, why more than 40 airlines, connecting 130 website where we provide a link to not try and make your travel plans worldwide destinations. Only 12 km from the city centre, it’s served by excellent Conference Programme 2017 bus, tram and taxi services that link directly to the town. More information You can view the current 2017 programme online, including a list of over 150 available from www.edinburghairport. invited speakers and their talk titles and abstracts. Here is a list of the scientific com sessions for 2017: Rail Edinburgh station is linked to all the • Regulation of RNA expression Main symposia: UK’s major cities and airports. A high- during virus infection • Anaerobes in infection speed link to London takes just over four • Synthetic and systems biology • Aquatic microbiology hours. The Eurostar service connects to approaches to microbiology • Cell biology of pathogen entry Paris in around eight hours. There are into host cells Prokaryotic forums: trains to major cities across the UK, as • Circadian rhythms well as to Scotland’s favourite tourist • Prokaryotic Genetics and Genomics • EndCritical health challenges in medical destinations. More information available Forum mycology? from www.nationalrail.co.uk • Prokaryotic Microbial Infection Forum • Epigenetic and non-coding RNAs • Environmental and Applied Microbiology Road Edinburgh is easily accessed by a in eukaryotes Forum network of motorways and trunk roads, • Geomicrobiology • Microbial Physiology, Metabolism and chiefly the M74, A1 and A68 from the • Heterogeneity and polymicrobial Molecular Mechanisms Forum south and the M8 from the west. The M9 interactions in biofilms and M90 head north. Edinburgh’s bus • Just passing through – virus infections of Virus workshops: station on St Andrew Square connects the gastrointestinal tract • Antivirals and vaccines to all the major cities in Britain. More • Macromolecular machines • Clinical virology information on bus services available • Microbial cell surfaces • Evolution and virus populations from www.nationalexpress.com • Microbial genomics: whole population to • Gene expression and replication single cell • Innate immunity Visit the website to book your • Microbial mechanisms of plant pathology • Pathogenesis place at the Annual Conference: • Annual General Meeting of Protistology- • Plant virology www.microbiologysociety.org/ UK Society: Intracellular infection and • Viral assembly events endosymbiosis within protists *Titles are subject to change We look forward to seeing you there.

Microbiology Today Aug 16 | www.microbiologysociety.org 127 Focused Meetings

Each year the Society runs a series of Focused Meetings in the UK and Ireland that have been submitted by our members. These meetings are a fantastic way to learn from, network with and present to the leaders working in each specific field of microbiology.

Irish Division Host–Pathogen Interactions

The first in our series of Focused Meetings took place at the end of June in Trinity College, Dublin. Invited speakers from local and international institutes presented their research on host–pathogen interactions.

The meeting took place over two half- days and included plenty of networking opportunities and poster presentations, along with offered papers during the programmed talks. Gerald Barry / Massimo Palmarini Gerald

Molecular Biology of Archaea 5

The second in our series has just taken place at the London School of Hygiene and Tropical Medicine. It attracted a large number of international delegates and over 65 abstract submissions were added to the programme as posters and offered papers. The event also included an evening at the Charles Darwin Centre in the Natural History Museum, where the delegates enjoyed a supper, to round off an excellent meeting. Thorsten Allers Thorsten

Thanks to all of those who helped make the meetings a success, and please continue to submit ideas for future meetings.

128 Microbiology Today Aug 16 | www.microbiologysociety.org Keep up-to-date with events, follow the Society on Twitter: @MicrobioSoc Still to come in the series… Irish Division #MISinterface Exploring the Microbe–Immune System Interface 1–2 September 2016 – Rochestown Park Hotel, Cork, Ireland In this meeting, we will explore our current understanding of how commensal and pathogenic bacteria interact with the host immune system during infection, and the consequences of these interactions in health and disease. Topics will include: Speakers will include: • Host colonisation Jose Bengoechea (Queen’s University Belfast, UK) • Host–microbiome interactions Lydia Lynch (Harvard Medical School, USA) • Infection and immunity John O’Shea (National Institutes of Health, USA) • Immune regulation Vincent Young (University of Michigan, USA) Juergen Berger / Science Photo Library / Science Photo Berger Juergen The Dynamic Fungus #DynamicFungus 5–7 September 2016 – Mercure Exeter Rougemont Hotel, Exeter, UK This meeting aims to celebrate the dynamic nature of fungi and provide an overview of some of the most cutting-edge current research in fungal biology.

Topics will include: Speakers will include: • Dynamics of the fungal cell Geoff Gadd (University of Dundee, UK) • Mathematical modelling in fungal science Jeff Gore (Massachusetts Institute of • Dynamics of cellular differentiation Technology, USA) • Dynamics in fungal pathogenicity Balázs Papp (Biological Research Centre, Hungary) • Dynamic evolution and adaptation of fungi Han Woesten (University of Utrecht, Netherlands) Gero Steinberg Gero Molecular Biology and #Avian16 Pathogenesis of Avian Viruses 27–29 September 2016 – Charles Darwin House, London, UK This timely meeting focusing on avian viruses will bring together the international scientific community to assess the extent of the problem and help find solutions. Topics will include: Speakers will include: • Molecular biology and genetics of avian virus Wendy Barclay (Imperial College London, replication UK) • Tropism and host range restriction Paul Britton (The Pirbright Institute, UK) • Pathogenesis of avian viruses Laura Delgui (National University of Cuyo, • Host antiviral responses and virus Argentina) immunomodulation Roy Duncan (Dalhousie University, • New and improved approaches to the control Canada) of avian viruses Thinkstock

You can view each of the programmes, along with a list of invited speakers, on our website www.microbiologysociety.org/events where registration remains open for each meeting. Visit the website today to secure your spot.

Microbiology Today Aug 16 | www.microbiologysociety.org 129 Legionella turns 40

PhD student (Imperial College London/ PHE), who presented the application of metagenomics for improved detection of Legionella in low-abundance specimens. In May–July 2012, a large LD outbreak occurred in Edinburgh, Scotland. Professor J. Ross Fitzgerald presented data on WGS analysis of the clinical isolates, which revealed subtypes within a single sequence type, raising the possibilities of diversification within a single environmental source or multiple point sources. The title of Professor Julian Parkhill’s talk was Delegates attending the Legionella symposium at Colindale. Mark Greenley ‘Recent emergence of L. pneumophila disease-associated clones: what 2016 marks the 40th year since the now infamous outbreak at the 58th American does it mean?’, with the somewhat Legion’s convention, Bellevue-Stratford Hotel, Philadelphia, USA, in 1976 controversial hypothesis that the only resulting in many unexplained deaths. It was Joseph McDade, a specialist in possible explanation is that human infection is involved in the transmission rickettsia at the Centers for Disease Control and Prevention (CDC), Atlanta, USA, cycle. Finally, the day also marked the who along with his team finally discovered the causative agent, the Legionnaires’ retirement of Dr Tim Harrison who disease (LD) bacterium, by inoculating guinea pigs with clinical material from gave his insightful ‘Reflections from the Legionella Reference Laboratory’. patients. This bacterium was later formally named as Legionella pneumophila. Acknowledgments o mark this milestone, Public experience’ and described the link Special thanks to Professor Maria Health England (PHE) arranged a between increased incidence of LD and Zambon, PHE Directorate and Tone-day symposium at Colindale, climate conditions. Dr Sophie Jarraud Microbiology Society for financial support; London, on 31 March 2016 called reported on an investigation into one of Professor Nick Phin and Dr Tim Harrison ‘Legionella pneumophila (1976 to 2016) – the largest LD outbreaks in France in (Chairs), Mark Greenley (audio visual from whole guinea pigs to whole genome 2004, illustrating new insights gained support), Sheila Culkin (administrative sequencing’. The aim was to bring from the application of WGS analyses. support) and Kastro Doda (catering). together Legionella experts and those Dr Natalia Kozak-Muiznieks, from the with an interest in Legionella to gain CDC, illustrated how both the current Norman Fry new insights, particularly concerning consensus typing method, sequence- Deputy Head, Respiratory and Vaccine the application of the new disruptive based typing (SBT), and WGS analysis Preventable Bacteria Reference Unit, technology of whole genome sequencing of a US healthcare-associated outbreak, PHE Colindale, London NW9 5EQ, UK (WGS) to public health. led to a revisiting of the concept of L. [email protected] The opening presentation was pneumophila subspecies. Sophia David, a by Professor Jacob Moran-Gilad on final-year PhD student (Sanger Institute/ This event was supported by the Society via a ‘Legionellosis in Israel - it’s not the PHE), described the evaluation of WGS Society-Supported Conference Grant. Find out heat, it’s the humidity’, which covered for the epidemiological typing of L. about other Society-Supported conferences an infant death due to LD caused by a pneumophila and the rationale for a new taking place this year and how you could domestic humidifier. Next, Dr Valeria core genome WGS scheme. The next apply for a grant for your event at www. microbiologysociety.org/ssconferences Gaia presented ‘Legionella: the Swiss speaker was Sharon Carney, a first-year

130 Microbiology Today Aug 16 | www.microbiologysociety.org

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h e n SubmitSubmit your your next next biotech biotech articlearticle to to Microbiology Microbiology

Microbiology invites submissions of research articles and reviewsMicrobiology that focus invites on established submissions and of emergingresearch articlestopics in and biotechnology.reviews that focus on established and emerging topics in biotechnology. There are a wide range of applications for microbiology in the Therefield areof biotechnology, a wide range fromof applications industrial forand microbiology in environmentalthe field of processesbiotechnology, to developments from industrial in and biopharmaceuticals.environmental processes Articles areto developments welcome that in utilise microbialbiopharmaceuticals. systems to develop Articles technologies are welcome and that products utilise

Thinkstock Luchschen Thinkstock microbial systems to develop technologies and products

Thinkstock Luchschen Thinkstock that could improve our lives or our environment. that could improve our lives or our environment. Articles are invited on the following topics: Articles are invited on the following topics: Editor-in-Chief • Metabolic engineering • Bioremediation Dr TanyaEditor-in-Chief Parish • Metabolomics• Metabolic engineering • Bioengineering• Bioremediation InfectiousDr Tanya Disease Parish • Genetic• Metabolomics engineering • Protein• Bioengineering engineering ResearchInfectious Institute, Disease USA • Synthetic• Genetic biology engineering • Microbes• Protein as engineering cell factories Research Institute, USA • Directed• Synthetic evolution biology • Bioenergy• Microbes as cell factories The biotechnology section of • Directed evolution • Bioenergy the journalThe biotechnology is overseen section by: of the journal is overseen by: Senior Editor Dr GavinSenior Thomas Editor UniversityDr Gavin of ThomasYork, UK Microbiology combines editorial expertise from around the University of York, UK worldMicrobiology with exceptional combines breadth editorial of coverage. expertise The from journal’s around the Editor world with exceptional breadth of coverage. The journal’s Editor Impact Factor is 2.268. There are no submission or page Dr Louise Horsfall chargesImpact and Factor the average is 2.268. time There to firstare no decision submission is four or weeks.page UniversityDr Louise of Edinburgh, Horsfall charges and the average time to first decision is four weeks. UK University of Edinburgh, Authors can also choose to publish with our gold open access UK option,Authors OpenMicrobiology. can also choose to publish with our gold open access option, OpenMicrobiology. Editor ProfEditoressor Saul Purton UniversityProfessor College Saul Purton London,University UK College Submit your article at London, UK Submit your article at mic.microbiologyresearch.orgmic.microbiologyresearch.org

Find out more at mic.microbiologyresearch.org Find out more at mic.microbiologyresearch.org Connect with us @MicrobioSoc #MicrobioJ Microbiology Today Aug 16 | www.microbiologysociety.org 131 Connect with us @MicrobioSoc #MicrobioJ Microbial Genomics: One-Year Anniversary!

July marked the one-year anniversary of the Society’s newest open Meet the Senior Editors Find out about their subject access journal, Microbial Genomics (MGen). We recap on the journal’s category, what they are most extraordinary first year and meet the journal’s Senior Editors to find proud of achieving so far and out what achievement they are most proud of and what papers they what subjects they would like to see more of in MGen in the next hope to see published in the journal in the future. 12 months.

he past year has seen a wave and lead author Timothy Dallman Jennifer Gardy of change across the Society’s acknowledged, “I think this shows BCCDC Canada journal publishing department, the impact that social media, and T Microbial and paving the way in innovation is the particularly Twitter, can have in rapidly Evolution and Society’s open access and open data disseminating scientific knowledge to Epidemiology journal Microbial Genomics (MGen), which our peers, the press and the public.” is the place for aims to champion the discoverability At six months, Senior Editor Kat papers that shine a and accessibility of open research data. Holt reviewed the variety of papers light on microbial population In support of this mission, four MGen published, from cutting-edge genomic genomics, and we’re proud to say Editors joined a panel discussion on Open epidemiology, antibiotic resistance, to it’s becoming a go-to spot for papers Data (https://microbepost.org/tag/ several articles focusing on functional in the emerging field of genomic exploring-open-data) at the Society’s genomics to investigate microbial gene epidemiology. Annual Conference to discuss why it’s regulation. Also key to developing the I am so proud of the remarkable important to share datasets in real-time area of microbial genomics are methods calibre of authors we’ve attracted and connect on a wider scale to target papers, which are critical to driving for MGen! Looking through the list of outbreaks; the differences in open data research forward. authors who have published in the needs for academics and public health Six months on and submissions journal it really is a who’s who of the clinicians; and being open about science are on the increase, with the launch of microbial genomics community – and the importance of metadata. the Microbial Genomics poster prizes, there are so many people doing truly Another exciting achievement for focused interviews with the Senior leading-edge work! the journal is its highest Altmetric score Editors of each subject category and a I’d love to see more genomic in the Society. The journal’s published wealth of inspiring scientists drawing epidemiology outbreak reports in the paper ‘Applying phylogenomics to interest for the journal’s feature journal – we can really carve out a understand the emergence of Shiga- ‘Standing on the Shoulders of Giants’ niche for ourselves here. I’d also love toxin-producing Escherichia coli O157:H7 (http://microb.io/1OcuHOl). to see our community start taking a strains causing severe human disease As we continue to build a strong look at combining genomic datasets in the UK’ (http://microb.io/1TPZYtf) community of Microbial Genomics published by multiple groups in order peaked at an Altmetric score of 84. This researchers, we interviewed each of our to do some really large-scale is an incredible achievement for Senior Editors to get a better insight into comparative genomics. a journal that is barely a year old, their subject categories.

132 Microbiology Today Aug 16 | www.microbiologysociety.org Thinkstock

Alan Walker University of Aberdeen, UK Carmen Buchrieser Microbial Communities publishes research Pasteur Institute, France employing marker gene, full metagenomic or Microbe-Niche Interactions metatranscriptomic sequencing to characterise should improve our understanding and understand the structure, function and of the interactions that pathogenic, dynamics of microbial communities present in commensal or environmental a range of host-associated and environmental microbes have with their niches. niches. One of the key strengths of the journal is the commitment to The journal welcomes articles that use new genomic, open data and open access, ensuring that both results and raw data proteomic, transcriptomic, metabolomic or imaging are freely available to the wider scientific community immediately techniques to analyse these interactions. Having an upon publication. The hope is that this will benefit submitting authors excellent, gender-balanced, motivated and innovative by increasing audience exposure to their research, and also benefit Editorial Board that loves working together and the scientific process by allowing others to validate or further utilise promotes the journal is a great achievement. We data. We would like to encourage further submissions in the area of would like more high-throughput imaging and host–microbiota interactions, with a particular emphasis on studies metabolomics analysis of the interactions and where the sequence data provides real biological insight into the role influences of the microbe on the host and the host that microbial communities play in host health. on the microbe in future papers.

Jukka Corander University of Oslo, Norway Kathryn Holt Genomic Methodologies highlights the new University of Melbourne, Australia depths of microbial sequencing that necessitates Responses to Human new tools to make the most out of the data and Interventions covers genomic the Genomic Methodologies focus of MGen aims studies of microbial responses to promote methods which make a difference to human interventions including for turning bases into biological insights. This clinical interventions such as new journal has very rapidly attracted the attention of both method antibiotic therapy, vaccines or infection control developers and practitioners, publishing biological highlights with a measures; as well as agriculture and environmental steady pace from the start. Novel methods for pangenomic analysis changes such as climate change or pollution. I am are particularly welcome as subjects as the need for such tools is proud of the variety and quality of articles published increasing as the data sets get more and more complex. so far in MGen and also the journal’s innovative feature ‘Standing on the Shoulders on Giants’ which Christos Ouzounis CPERI, CERTH, Greece highlights pioneers in the field of genomics. In future Systems Microbiology covers a combination we would like to engage the experimental evolution of systems biology and microbial genomics, community to publish their ground-breaking studies addressing big-data issues, omics approaches, on evolutionary responses to antimicrobial stresses, perturbation experiments, networks and as well as those with longitudinal genomic data sets pathways, microbial diversity and population examining evolution in response to treatment and dynamics under a systems perspective. MGen host-associated stresses. has already published some very interesting studies on the subject, including the comparative genomics of bacterial strains, genome- View the journal online: wide analyses of environmental responses, clinical proteomics, http://mgen.microbiologyresearch.org and others. I would love to see more of those efforts flourish, and invite work on single-cell genomics, comparative-omics, including Harriet Pope transciptomics, pangenome analyses and perhaps emerging Product Executive application domains, such as metabolic engineering work for biofuels [email protected] and bioenergy.

Microbiology Today Aug 16 | www.microbiologysociety.org 133 Diagnosing and sequencing Ebola in Sierra Leone

In November 2014, I was deployed with Public Health England (PHE) to establish one of the first diagnostic laboratories in Sierra Leone. Our team of 10 scientists was composed primarily of biomedical or clinical scientists from various UK hospitals or diagnostic laboratories. We were

sent to Makeni, to work in an Ebola treatment centre (ETC) operated by Professor Ian Goodfellow in the Public Health International Medical Corps (IMC). England diagnostic laboratory in Makeni, Sierra Leone. I. Goodfellow

hen we arrived the ETC was available from days to as short as four evolving in real-time rather than being still under construction so the hours. reliant on the release of data following Wfirst weeks were spent moving During my first deployment to publication, which often occurred many boxes and equipment around a bustling Sierra Leone, and in discussions with months after samples had been isolated. building site. The early days were rather Dr Tim Brookes (PHE) and Professor The ability to sequence viruses in Sierra chaotic, logistics were unpredictable Jeremy Farrar (Wellcome Trust), we had Leone rapidly has been critical in the so the establishment of the laboratory identified an urgent need to be able to recent emergence of new cases from required a lot of creative approaches sequence viruses in real-time. The ability persistently infected survivors. including the repurposing of household to sequence patient samples within a I returned home on 23 December items purchased from the local market 24–48 h time period would make the data 2015 and what followed was a frantic for use in the laboratory. of use for epidemiological tracing and the period of contacting suppliers, obtaining The diagnostic lab opened on identification of transmission networks. quotations and putting together a grant 13 December 2014 and this significantly By making the data freely available, it to perform next-generation sequencing reduced the time taken between samples would also enable scientists to gain a in a tent next to the diagnostic lab. In being collected to the results being better insight into how the virus was April 2015, my third deployment to

Makeni Team #1 within the PHE diagnostic laboratory. The team were sent to establish the diagnostic laboratory Professor Ian Goodfellow discussing the at the Mateneh Ebola treatment centre in Makeni. From left to right: Dr Stan Ko, University of Central Lancashire; interpretation of Ebola virus sequencing data with Prof. Ian Goodfellow, University of Cambridge; Cristina Leggio, PHE Porton; Stephanie Leung, PHE Porton; Lisa Berry, a member of the World Health Organization Ebola University Hospitals Coventry and Warwickshire (UHCW) NHS Trust; Steve Diggle, PHE Manchester; Laura Grice, PHE response team. I. Goodfellow Manchester; Catherine Moore, PHW Cardiff; Kate Baldwin, North Bristol NHS; Angela Short, PHE Bristol.I. Goodfellow

134 Microbiology Today Aug 16 | www.microbiologysociety.org Makeni Team #1 preparing to establish the diagnostic laboratory at the Mateneh Ebola treatment centre run by International Medical Corps. From left to right: Kate Baldwin, North Bristol NHS; Lisa Berry, University Hospitals Coventry and Warwickshire (UHCW) NHS Trust; Catherine Moore, PHW Cardiff; Steve Diggle, PHE Manchester; Dr Stan Ko, University of Central Lancashire; Stephanie Leung, PHE Porton; Cristina Leggio, PHE Porton; Angela Short, PHE Bristol; Laura Grice, PHE Manchester; Prof. Ian Goodfellow, University of Cambridge. I. Goodfellow

Sierra Leone, I returned to Makeni to scientists with access to in-country diseases in secondary school students install an Ion Torrent sequencer in the sequencing technology. The laboratory within Sierra Leone and will run until ETC using support from the Wellcome has provided support to a number of September 2017. Trust. Dr Armando Arias, a postdoctoral ongoing clinical trials and is providing fellow in my lab, and I set off to install longer-term capacity building of Ian Goodfellow all the equipment and establish the scientists in state-of-the-art research University of Cambridge, Addenbrooke’s sequencing workflow. Neither of us had methods. The sequencing facility Hospital, Hills Road, Cambridge CB2 2QQ operated a sequencer before but we continues to provide essential support to [email protected] had 5 days of training beforehand to the outbreak, including the sequencing enable us to install, run and, if necessary, of the last cases in Sierra Leone and the repair the equipment. The sequencer new cases from Guinea. and the other equipment arrived on 13 I would encourage all members of April 2015 and within a period of 3 days the Society to get involved in work in we were sending our first Ebola virus low- and middle-income countries, as sequences back to our collaborators it has provided me with an invaluable at the Wellcome Sanger Centre and insight into the impact of infectious University of Edinburgh for analysis. We diseases on vulnerable populations. have since sequenced >1,200 samples, Although challenging, the six months I providing >600 viral genomes, ~1/3 of all spent in Sierra Leone working as part of the viral sequences from the outbreak. the response was without a doubt one of By sharing our data with other scientists, the most rewarding experiences of my including a team run by Dr Nick Loman, scientific career. also a Microbiology Society member who I have just recently returned was performing real-time sequencing from a two-week trip to Makeni to in Guinea, we were able to provide run microbiology practicals for the invaluable information on the movement undergraduate students at the local of cases across the borders in real-time. university (UniMak) and to begin a public In September 2015, we subsequently engagement project in collaboration with relocated to the University of Makeni EducAid, a UK-based charity involved in (UniMak) and established the UniMak education in Sierra Leone. The Wellcome Professor Ian Goodfellow and Sunday Kilara (International Infectious Disease Research Laboratory Trust-funded engagement project aims Medical Corps) preparing to unpack the equipment required to provide both local and international to improve the awareness of infectious to establish the Ebola virus sequencing facility. I. Goodfellow

Microbiology Today Aug 16 | www.microbiologysociety.org 135 Developing microbiology around the world

The Society’s International Development Fund supports members’ The first grant supported a workshop to exchange knowledge development activities in low-income economy countries. We spoke to of detection methods for resistant two previous recipients to find out more about their projects. bacteria in Romanian diagnostic laboratories. This was the first time such a meeting of human and veterinary Professor Iruka Okeke, University of microbiologists was held in Romania, Ibadan, Nigeria and participants received protocols “It is important to me to teach at for detection of resistant bacteria to the highest level – the International support implementation of routine Development Fund allowed us to make a testing. Dorina continued this work in high-impact curricular change and start a 2014 by implementing a pilot scheme collaborative research project that engages for detection of carbapenamase- students as well as faculty and staff producing Gram-negative bacteria members.” (CPGNB) in Romanian hospitals. She In 2015, Iruka received Society established wide collaborations and support to transform the Masters course generated evidence of the need for a in Pharmaceutical Microbiology at the national surveillance system to rapidly University of Ibadan, Nigeria. Iruka identify CPGNB-infected or colonised relocated to Ibadan from Haverford patients to allow implementation of College, USA, after receiving an African appropriate infection control measures. Research Leader’s Award under the Discovery-based learning course: student Pelumi Participating laboratories continue to Medical Research Council/Department Adewole loading his agarose gel. Ayandiran Tunmise, use these protocols with the aim of them for International Development concordat PHM712 class being implemented as part of a wider agreement. She noticed that, due to screening policy throughout Romania. resource limitations, graduates joining Dr Dorina Timofte, University of the Pharmaceutical Microbiology Liverpool, United Kingdom Advice for prospective applicants programme often had little microbiology “Having worked in the UK for several years, Iruka: “Apply! The awards have wet-lab experience. I see major gaps that occur in antimicrobial truly transformative potential. The Society funding helped develop resistance detection, surveillance and opportunities are far greater than we a discovery-based laboratory course, antimicrobial stewardship in countries had predicted and we’re working on where students with limited previous like Romania. The grants were the perfect sustaining our discovery-based course.” experience of bacteriology learned vehicles to enable significant impact in this Dorina: “The Fund is a great way to practical skills in microbiology, area.” help microbiology in less economically molecular biology and bioinformatics Dorina received Society support fortunate parts of the world. If you have via investigation of leaf microbiomes of in 2012 and 2014 for antimicrobial an idea that fits with the remit of the plants used in Nigerian ethnomedicine. resistance (AMR)-focused projects in scheme, do not hesitate to apply – you The project has brought the expertise Romania. During previous collaborations never know what opportunities it may of individuals in the department – in Romania, she noticed that one lead to.” bacteriologists, molecular biologists and contributing factor for AMR was the lack The next closing date for the natural product researchers – together of national guidance regarding routine International Development Fund is around a new research endeavour. testing and reporting methodology for 1 October 2016. Contact grants@ According to Iruka, the wet-lab and detection of antimicrobial-resistant microbiologysociety.org for details. research training has been invaluable bacteria. As an Antibiotic Action champion, both for the development of the Dorina addressed this by implementing Maria Fernandes individuals, and future generations of rapid diagnostic methods in order to Professional Development Manager microbiologists that they will go on to improve patient care and prevent the [email protected] train. spread of resistance in hospitals.

136 Microbiology Today Aug 16 | www.microbiologysociety.org Membership

home in one organisation and not in another. And this is why we need your “I had a brilliant day!” help. We need you to tell us what you think I mentioned in the last issue that we are undertaking some research about your membership of the Society – what we do well, what we don’t do to help us identify what we may need to do differently in future, to well and what we could do differently ensure Society membership remains relevant and attractive. in future. You don’t have to be a current member. You can be a former member or not even have been a member at all. talked to members and delegates at observations, opportunities to meet If you have thoughts, ideas and opinions our Annual Conference in Liverpool other members, talk to them, discuss around the subject, please let us have in March, to find out just what it is ideas and compare notes all seem to be I them. We’d love to hear from you. that continues to draw participants in high up our members’ wish list. So too There are a number of ways to join the ever increasing numbers to our annual does having the opportunity to listen to conversation. You can: showcase event. After all, it’s a serious some great science and get closer to Take part in our Member Survey. commitment both in terms of time and the originators of it. Wrap this all up in • It’s a short online questionnaire for money. The quote above is not from a supportive and friendly environment current, former and ‘never been’ me (although I did have a brilliant day (along with regular opportunities to members. You should have already too, getting to meet and talk to many socialise) and we seem to be on the received an email invitation to take from across the organisation). The right track in delivering, at least in part, part, but if you haven’t, please quote is actually from Mark Jones, an what many members are looking for. contact me and I will send one to you. undergraduate student at John Moores In a world increasingly dominated by Take part in a focus group University in Liverpool. Mark had come online interactions, these opportunities • meeting. There will be face-to- along to the Annual Conference for the to engage face-to-face are ones that face group meetings taking place first time and summed up his experience seem to be striking a real chord with across September 2016 to share in these words. He’d arrived not really members. insight and discuss membership- knowing what to expect, listened to a But these are just my observations related issues in more detail. If variety of presentations, met new people, from Conference. What about the you would like to register your discussed his work, and went home very members who don’t go to Conference? interest, please email me. Final happy. What about those who live overseas? selections, timings and locations It was a sentiment that was echoed What about those members who are will be advised closer to the time. by many. Increasingly, members seem less than satisfied and feel we fall Email me with your thoughts and to be enjoying the opportunity to meet short? What about members who want • suggestions on how we can make first-hand, hear world-class presentations a more enhanced online experience? membership even more attractive on a very wide range of topics and enjoy And what about those who have made in the future. the interactions that naturally follow both a conscious decision not to join us? With your help, we want to create within and outside the formal programme. We have members – and prospective a membership offering that is attractive So maybe we shouldn’t try to members – with a wide range of and relevant, valued and recognised. In over-complicate the member research views and opinions and we need to short we want many more of you to be that’s currently underway, looking for ensure these are captured too. So able to say ‘I had a brilliant day’. all the answers in an in-depth analysis inevitably there has to be some form of questionnaires and spreadsheets. of information collection and analysis Paul Easton Maybe some of the answers are closer to get a representative cross-section of Head of Membership Services and more obvious than we think. From views that will help us better understand [email protected] my (admittedly not very scientific) why a member feels a sense of place or

Microbiology Today Aug 16 | www.microbiologysociety.org 137 Schoolzone

Bioinformatics is the method of using computer programming to interpret biological data. The teaching of bioinformatics in schools is becoming an integral Old School, part of the curriculum and understanding the evolutionary relationships between species at the genomic level is an important skill. However, the basic process of how these relationships are determined is fundamental in understanding all of New School: these relationships. The activities below demonstrate how the evolutionary history of different determining phylogenetic relationships and evolutionary strains of a virus or other micro-organism can be determined by using both history of micro-organisms using bioinformatics modern (computer) and historical (observation) methods.

Phylogenetic comparisons of genetic sequence data to to be selected (it is the only option with the basic software). The alignment can determine the origins of HIV take a few minutes to process. HIV (human immunodeficiency virus) is a type of virus that attacks and weakens the immune system and 6. A new alignment file will appear in the most notably can lead to AIDS (acquired immune deficiency syndrome). Since the discovery of HIV in 1981, top window, select this and click on the the origin of the virus has been widely debated. The most widely accepted hypothesis, which is backed ‘Tree’ button to create a phylogenetic up by the scientific evidence, is that HIV came from cross-species transmission of related viruses. Closely tree. Another box will appear and the related non-human primates (monkeys and apes) carry viruses that are very similar to HIV, called SIVs following options need to be selected; (simian immunodeficiency viruses). Genetic Distance Model: Jukes-Cantor, By comparing the genetic sequences of these closely related viruses it is possible to make Tree build method: Neighbour-Joining, and Outgroup: No Outgroup. Make sure assumptions about evolutionary relationships and origins of different strains. The following procedure is the ‘Resample Tree’ box is unchecked taken from a case study on the DNA to Darwin website (www.dnadarwin.org). The activity makes use of and select ‘OK’. Geneious software, which aligns protein sequences following nucleotide translation and builds phylogenetic 7. Your phylogenetic tree should appear! trees that allow the user to visualise how closely related sequences are to one another. You can zoom in and out and re-size Student procedure selected by clicking and highlighting the tree to properly study it. The 1. Download the free software, Geneious, the file name in the top window of the tree should show you the different from www.geneious.com and the program. Click on the ‘Translate’ button subgroups of the HIV-1 family and Geneious document from www. to convert the nucleotide sequence to how these relate to the SIV sequences dnadarwin.org/casestudies/7 a protein sequence. (A box will appear, found in gorillas and chimpanzees. containing the ‘DNA-HIV1andSIV. make sure the genetic code selected is An easy way to visualise the tree is geneious’ file. This data file contains 16 ‘Standard’ and the translation frame is to print it out and highlight different nucleotide sequences of the pol gene ‘1’). relationships you can see. from the HIV and SIV viruses from A new file will be created and as you 4. Questions to consider chimpanzees, gorillas and humans. did in Step 2, you can zoom in on this • Can you visualise the branches of the 2. Open the Geneious program and the sequence and view the sequences tree that are from human, chimpanzee DNA-HIV1andSIV.geneious file. The at the amino acid level. Each letter or gorilla origin? 16 sequences will be visible on the represents a different amino acid. How closely related are the different central window. Use the zoom function Can you guess which amino acid is • HIV subgroups to each other and to the (magnifying glass button) to view the represented by the letters? SIV sequences. What can you conclude sequences at the nucleotide level. 5. Now to ensure the sequences are from this about the origins of HIV? You should be able to see differences aligned (they should be already, between the sequences at the base however). Make sure the protein For further information, see our level. How can you tell the difference sequence file is selected in the top factfile on HIV and AIDS on our education between an RNA and DNA virus at the window and select the ‘Alignment’ website, Microbiology Online: www. nucleotide level? button. A box will appear and the microbiologyonline.org.uk/teachers/ 3. Make sure all 16 sequences are ‘Geneious Alignment’ method needs resources

138 Microbiology Today Aug 16 | www.microbiologysociety.org Building a phylogenetic tree based on morphological 5. To find the pairs of organisms that have a common ancestor that lived characteristics before the most recent pair, look for A simple way to make assumptions about the evolutionary history of a group of organisms is to observe the micro-organisms that have two morphological similarities between them and build a picture of how recently they shared a common differences. Draw a dot below these ancestor based on the number of different features. The activity below is a basic introduction to how organisms and link these too. phylogenetic trees can be created using morphological characteristics of different micro-organisms. The 6. Do the same for both three differences trees will show common origins and how species can be related to one another based on morphology. and when the organisms differ by Student procedure four: this means they share the oldest common ancestor and the tree is 1. Below are five illustrations (A–E) of well-known bacteria. They each have complete. differences and similarities to one another. An example of a smaller phylogenetic A B C tree developed using this technique is shown below: Maurizio De Angelis/SPL Maurizio De Angelis/SPL Spencer Sutton/SPL

D E

Things to consider: • Are there other ways to classify the morphology of these micro-

Science Artwork/SPL Science Artwork/SPL organisms? Think about possible lab 2. Using the table below, record the features for each of the micro-organisms. The techniques that you could use. features listed are purely suggestions, include your own. • Is it possible any of the morphological features have evolved independently Shape Flagella Texture Colour (rod or cocci) present? (smooth or spiked) of the other species (convergent A evolution); are there ways you could B determine this? C • Look at the world around you; think D of other everyday items you could E determine relatedness of with this technique. A tasty suggestion is a 3. In the following table, record the 4. To draw the phylogenetic tree, the five biscuit sampler tray; you could look at number of differences between each micro-organism letters will be placed features such as chocolate coating and micro-organism. The numbers in each at the top of the tree as these are the shape. of the boxes indicate how the different currently living micro-organisms. micro-organisms relate to one another Firstly, you need to identify which of For further information about based on their morphology. the micro-organisms are the closest bioinformatics, see our open access relatives; these will have the least journal, Microbial Genomics: http://mgen. number of differences between them. BCDE microbiologyresearch.org Place this pair next to each other at the A top of the tree. These two will share a B recent common ancestor; place a dot Hannah Forrest Public Affairs Administrator C below the two letters and draw lines leading to the letters showing their [email protected] D evolutionary lineages.

Microbiology Today Aug 16 | www.microbiologysociety.org 139 Outreach Light at the bottom of the sea

Recipient of the Education and Outreach Grant, Society member Dr Jenny Search shares her work on engaging primary school children using bacterial luminescence.

Large anglerfish at the school’s Lumiere festival.J. Search

have worked in the field of science communication for over 10 years now Iand spend most of my time working with school children, promoting all the science, technology, engineering and research (STEM) subjects and encouraging youngsters to think of themselves as potential scientists and engineers of the future. It is always exciting when I get to do something linked to immunology and microbiology – the topics of my own undergraduate and postgraduate studies. Sadly, there is little microbiology in the current English national curriculum at primary school level. I was approached by Denise Armstrong from Vane Road Primary School in County Durham, as she wanted to run a microbiology event in school but, like many primary schools, they couldn’t afford the time to run activities not directly linked to the national curriculum. Denise also wanted the children to develop something that could be used Primary school children making anglerfish.J. Search

140 Microbiology Today Aug 16 | www.microbiologysociety.org as part of the school’s activities for their producing symbiotic bacteria. They bacteria being the same, and bacteria Lumiere festival. We came up with the were quite taken with the wide range being only known as causing disease. topic of ‘Light at the bottom of the sea’ of strange, glowing creatures that The children found making the light- where we linked bacterial luminescence live under the sea. They came up with up anglerfish tricky and they needed with the circuit-making requirements of their own ideas for creating light-up perseverance to get it to work; however, the national curriculum. creatures that could be used as their most of the children stated this was their I developed a workshop to part of the school’s annual Lumiere favourite part of the workshop. They also introduce the children (aged 10–11) to festival. They worked with an artist to enjoyed finding out about marine life and bacteria, symbiosis and light production, create larger model anglerfish and other building circuits. particularly bioluminescence under bioluminescent sea creatures and then I the sea. The children then used craft helped them add circuits and glow-sticks Jenny Search materials and electrical components to their models so they lit up. http://drresearch.co.uk to create a model anglerfish with a As well as producing a fantastic @DocReSearch light that can be turned on and off display of light at the bottom of the with a basic switch. As the light in sea, the children found out a lot The next deadline for the Education the anglerfish lure is provided by more about general microbiology, and Outreach Grant is 1 October. bioluminescent bacteria, this provides the scale of micro-organisms and For more information, please see a good link to exploring the marine the wide range of environments in www.microbiologysociety.org/ microbial environment. which they live. Evaluation showed the grants or contact The children investigated several workshop countered some common [email protected] marine creatures that have light- misconceptions, such as viruses and

Jenny Search demonsrating luminol (above) and jellyfish at the school’s Lumiere festival (below). J. Search

Primary school children making anglerfish.J. Search Making anglerfish.J. Search

Microbiology Today Aug 16 | www.microbiologysociety.org 141 Policy

Professor Mike Skinner and Professor Wendy Barclay attended a Parliamentary Science policy update and Scientific Committee meeting on the safety of pathogen research. Society staff and members also The Society’s policy team work to ensure appropriate scientific information and attended Parliamentary Links Day at expert opinion is made available to policy-makers. We also engage in science the UK Parliament and Science and the policy to ensure that microbiology is supported as a discipline. Here is an Assembly in Wales. The Society is also represented on overview of just some of our activities over a busy first half of 2016. several external policy committees and groups. Professor Dawn Arnold was Antimicrobial resistance In May, the Society endorsed elected to the advisory committee of the Antimicrobial resistance (AMR) remains recommendations made by Lord O’Neill’s UK Plant Sciences Federation, while Dr a key priority for our policy work. As a independent review on AMR, including John McGrath replaced Dr Nigel Ternan founding member of the Learned Society building AMR research capacity and as the Society’s representative on the Partnership on AMR (LeSPAR) we aim to raising global awareness. Northern Ireland Learned Societies and support research and present a unified Professional Bodies Forum. scientific voice on AMR http://microb.( Policy consultation responses io/1Ontlzi). Meeting with research Our consultation response to the Fungal diseases funders and policy-makers, the Societies House of Commons Environmental and microbiomes have been highlighting issues and ideas Audit Committee UK Soil Health We continue raised by our collective memberships inquiry in January highlighted to proactively at a series of interdisciplinary LeSPAR environmental microbiology, while highlight important workshops held last year. our joint response with the Society microbiology In February, the Microbiology Society for Applied Microbiology to the House issues and was invited to a stakeholder workshop of Commons Science and Technology research to policy- in Brussels about the European Committee inquiry on Science in makers through Union’s AMR Action Plan; there was emergencies: UK Lessons from Ebola our series of policy interest in the report from our LeSPAR saw several issues our members briefings, including our one on Human workshops and we were able to highlight raised, such as better drawing on and Fungal Diseases, which was sent to UK the importance of research and the supporting microbiology volunteers and Irish policy-makers. environmental dimension of AMR. in disease emergencies, cited in the All briefings are accessible on our Committee’s report. We also highlighted website: www.microbiologysociety.org/ Get involved our forward-thinking Antibiotics briefings Our policy work depends on the Unearthed outreach project in our The Society has also established expertise and input of our members. response to the Committee’s Science a Microbiome Expert Working Group, It is a great way to promote Communication inquiry. chaired by Professor Julian Marchesi, microbiology and increase its View our consultation responses on to produce a major evidence-based impact. Contact the team at policy@ our website: www.microbiologysociety. document for policy-makers, which will microbiologysociety.org to find org/consultationresponses explore the research on human, animal out how to get involved or flag an and environmental microbiomes. issue. Keep an eye on the monthly Linking microbiologists and newsletter and our website (www. policy-makers Paul Richards microbiologysociety.org/policy) Early-career members questioned Policy Officer for policy news, opportunities and leading UK politicians on science policy [email protected] resources. at Voice of the Future 2016. Members

142 Microbiology Today Aug 16 | www.microbiologysociety.org www.microbepost.org Timber rattlesnake (Crotalus horridus). Frupus / Flickr

t’s been a busy few months for us whether a pipette works upside down! here on the blog, and we’ve covered Parasitic worms affect millions Iresearch from across the world (and of people and represent a significant out of it). health burden worldwide. However, Back in April, we learnt about Best might there be an upside to these Ophidiomyces ophidiicola, the causative infections? On the April podcast, Anand agent of Snake Fungal Disease, an interviewed Dr Ken Cadwell from emerging pathogen that is spreading of the New York University, who has been rapidly across North America. Anand investigating whether worms can cure Jagatia interviewed Dr Jeffrey Lorch inflammatory conditions of the digestive from the National Wildlife Health Center tract (http://microb.io/1VEBgfp). in America about his work on the fungus. blog Finally in this round up, we looked First reported on the East Coast in 2006, at the history of vaccines, from their this often-fatal disease has now been beginnings in the 15th century through detected in 16 American states and in Environment at the Library, explain the to the modern methods used to create Canada (http://microb.io/1rffeTQ). service (http://microb.io/1TiqViJ). them today (http://microb.io/1VYrZgQ). Members of the UK Parliament For the podcast in June (http:// I’ll be back again in the next often have to debate issues outside of microb.io/28IFjar), I was lucky enough issue to tell about some more of the their area of expertise. How do they get to interview Dr Kate Rubins, a virologist content you might have missed. In the the background information they need who at this moment is orbiting the Earth meantime, keep an eye on the blog at to stay up-to-date on the latest at 17,000 miles an hour aboard the www.microbepost.org and search for research? The answer is the Commons International Space Station. At the time the podcast on iTunes or on SoundCloud. Library, staffed by 70 specialist staff of our interview, Kate was in the final few working across eight subject areas. weeks of training ahead of the mission’s Benjamin Thompson Our Policy Officer, Paul Richards, launch. We talked about her transition Head of Communications reviewed a ‘Policy Lunchbox’ event that from one career to another, having [email protected] saw Ed Potton, Head of Science and to learn chemistry and physics, and

Microbiology Today Aug 16 | www.microbiologysociety.org 143 Membership Q&A This is a regular column to introduce our members. In this issue, we’re pleased to introduce Dr Martin Cole. M. Cole M.

What is your current position? a novel substance made by the soil further and higher education in Epsom. I I retired some years ago after a microbe Streptomyces clavuligerus, which still enjoy reading Microbiology Today, The career at Beecham Pharmaceuticals we named clavulanic acid. Developing Biologist, The Biochemist and Chemistry and SmithKline Beecham. I joined George its use for treating amoxicillin-resistant & Industry. Rolinson’s Department of Microbiology at infections was a bit of an uphill struggle Brockham Park in 1958, later becoming as there was no precedent for such What might your colleagues head of the Department of Biochemistry an approach. A product containing not know about you? and a Project Manager. When I retired I amoxicillin with potassium clavulanate That I have flown over the City of London was Director of Research Programmes was eventually launched under the trade and up the Thames in the helium-filled at Brockham Park. name Augmentin (co-amoxiclav) and is Skyship 500, taking photographs over widely used around the world. my old college – Imperial College. Also, What was your area of I successfully took a course on driving specialisation? What was the worst moment the famous steam locomotive Flying Microbial biochemistry, particularly of your career? Scotsman but did not exceed 25 mph. searching for potentially useful Having my briefcase stolen from my substances. Initially I worked on car while I was in a local pub with What do you do to relax? 6-aminopenicillanic acid (6-APA), the colleagues after work. It contained Together with my wife, Maureen, son nucleus of all penicillins, which is a draft of a multi-author paper and and daughter we restored our 2.5 made in small amounts by Penicillium my draft for the patent application on acre Victorian garden and now enjoy chrysogenum and from which a great clavulanic acid. With the help of our looking after it. With the help of our local variety of semi-synthetic penicillins local police chief and two young boys museum and history group, and also the could be made. One of my early from the nearby village, all the pages Surrey Gardens Trust, we researched achievements was to find an enzyme, were retrieved, sodden, from roadside the history of where we live and have made by a strain of Escherichia ditches, having been chucked away by just published a book, The Capel Leyse coli, which would readily deacylate the criminals when they found no Estate, South Holmwood, Surrey. I also benzylpenicillin to give 6-APA and money or drugs. I was very relieved! enjoy visiting other peoples’ gardens, hence allow bulk production of the supporting the Royal Botanic Gardens, new penicillins such as amoxicillin. What happened when you retired? Kew and going on fungal forays. Oh, I Initially, I did consultancy work for my nearly forgot, I collect things but there is You worked in the field of previous employer. I also helped the not much space left in my study or my antibiotic resistance, what did United Nations Industrial Development Victorian summer house! this involve? Organisation in China and the Royal The very useful broad-spectrum Botanic Gardens at Kew. As more If you would like to be featured in penicillin, amoxicillin, is destroyed time became available I joined local this section or know someone who by beta-lactamases made by many organisations, becoming Chairman may, contact Paul Easton, Head of clinically important bacteria. A big of Dorking Museum, Convenor of the Membership Services, at p.easton@ effort was mounted to see if we could Local History Group and President of microbiologysociety.org find an inhibitor of these enzymes. My the Rotary Club in Dorking. I was also colleagues and I found such an agent, Vice-Chairman of NESCOT, a college of

144 Microbiology Today Aug 16 | www.microbiologysociety.org Microbiology Today Aug 16 | www.microbiologysociety.org 145 Reviews

Biofilms in Bioremediation: Ch. 2 is a comprehensive account aggregations (flocs) and rhizosphere Current Research and of biofilm biology and its advantages for communities in Ch. 10. bioremediation. However, 19th century Ch. 11 provides an excellent Emerging Technologies trickling filter technology, one of the introduction to biofilm permeable Edited by G. Lear earliest uses of biofilms for used water reactive barriers, with many useful Caister Academic Press (2016) bioremediation, is not mentioned. I’m illustrations. Ch. 12 is not particularly £159.00 ISBN 978-1910190296 unconvinced that shear forces act on convincing about biofilm cells having microbial cells at surfaces other than in higher rates of biodegradation than The book has three sections, with the lab, but I’m glad this boundary layer planktonic ones. each chapter written by one or more is mentioned in relation to substrate Ch. 13a presents interesting growth- specialists in the area. This makes for transport. promoting effects of phenol-degrading easy navigation and chapters of specific In ‘Methods and Monitoring’, Fig. bacteria on Lemna roots, while Ch. 13b interest can be read as stand-alone items. 4.11 shows a more comprehensive presents convincing data for the use of However, there is considerable repetition range of biofilm reactors than in Ch. 1 specific strains of Sphingobium. of basic biofilm information if read as a and I wonder why it was not included A useful book for those new to the whole. there. Ch. 4 also considers the role of area, but probably as a library copy. Ch. 1 is a useful introduction, if only chemotaxis – anomalous in a book about A second edition would benefit from for microbiologists to understand the biofilms. closer collaboration between the chapter importance of mass transfer! This should The brief Ch. 6, on microscopy, lacks authors, to avoid unnecessary repetition. help to inform their research so that it is images illustrating the applications of of more direct use to engineers. From my each type. In Ch. 8, immobilisation of Mike Dempsey point of view, Table 1.2 is missing ‘landfill cells in gels or foam cubes should not Manchester Metropolitan University leachate’. be considered as ‘biofilm’, nor ‘floating’

Tuberculosis deserved a section of its own. The second section starts with a focus on the biology Edited by S. H. E. Kaufmann, E. J. Rubin of the tubercle bacillus. I particularly & A. Zumla liked the interlinking of this fundamental Cold Spring Harbor Laboratory Press (2015) knowledge to subsequent chapters US$135.00 ISBN 978-1621820734 on drug development, discovery and use. The final section is dedicated to Tuberculosis is a comprehensive clinical aspects of TB, including updated and updated amalgamation of information on epidemiology, diagnostics, contemporary knowledge of fundamental TB in the context of HIV co-infection, and clinical research on tuberculosis (TB) management of latent TB, the worrying and its aetiological agent, Mycobacterium trend of drug resistance, and advanced tuberculosis. The first section, the most imaging methods for diagnostics. wide range of readers including, but not diverse, covers topics including basic Tuberculosis also offers limited to, PhD students, early career research on TB-related cell biology alternatives or challenges to conventional postdoctoral fellows and clinicians and immunology, advances in vaccine views on TB, such as a newer look at pursuing research on TB. development, and the development of the granuloma as a structure that is laboratory models of infection, ranging beneficial to mycobacterial expansion, Apoorva Bhatt from zebrafish to primates. Diverse and a critical analysis of comparative University of Birmingham animal models have accelerated our genomics in defining ‘virulence’ factors, understanding of host–pathogen and of the protective role of humoral More reviews can be found at: interactions and vaccine design and I felt immune response. The book offers www.microbiologysociety.org/ that this topic (four chapters) perhaps updated information and views to a MicrobiologyToday

146 Microbiology Today Aug 16 | www.microbiologysociety.org Comment Moving microbial diagnostics into the 21st century Justin O’Grady Rapid and accurate diagnosis is critical for the effective treatment of life-threatening infections, such as bloodstream, respiratory tract and complicated urinary tract infections. These clinical syndromes have complex aetiology and require the recognition of pathogens within a variety of Thinkstock challenging sample types. he ‘gold standard’ culture and, at best, one to identify pathogens therapy is often unnecessarily potent techniques are labour intensive, and test their antimicrobial susceptibility. and, conversely, sometimes ineffective Thave long turnaround times and Meanwhile, in the absence of pathogen due to inherent or acquired resistance. often offer poor clinical sensitivity. Even or resistance information, the patient is Nucleic acid amplification-based with the addition of mass spectrometry treated empirically with broad-spectrum diagnostic tests are a step in the right for rapid identification of bacterial therapy, which has evolved over time direction, providing results within isolates, and automated culture systems to include previously reserved potent hours, but are limited by the range of for isolation, the turnaround time is at antibiotics as a result of increasing pathogens and resistances they can least two days: one to grow the bacteria antimicrobial resistance. This empirical detect.

Microbiology Today Aug 16 | www.microbiologysociety.org 147 investigate the utility of metagenomics pathogen numbers in blood vs large sequencing for rapid diagnosis and numbers of leukocytes and pathogens antimicrobial susceptibility testing in in urine; therefore, we modify our clinical microbiology. enrichment/depletion strategies So why are researchers and clinical depending on the sample type being microbiologists across the world not tested. This ensures that the cost–benefit implementing this technology in their is appropriate for the various sample laboratories as we speak? You’ve types. Our current metagenomics MinION. Oxford Nanopore Technologies guessed it – sample preparation is the sequencing pipeline (including host remaining bottleneck, a commonly DNA depletion) for UTI and hospital- A paradigm shift in diagnostics neglected research area as it produces acquired pneumonia samples takes technology is required, to allow the few ‘glamour journal’ articles. Working approximately 4 hours from sample development of a universal diagnostic directly with clinical samples, without to answer (pathogen identification that can detect any pathogen or any culture enrichment steps, is and antimicrobial resistance gene resistance. Next-generation sequencing challenging as competing nucleic acid, identification) using MinION nanopore (NGS) technology, particularly rapid particularly that of the human host, sequencing. The sepsis pipeline takes nanopore sequencing using Oxford make metagenomics sequencing- approximately 7 hours. Nanopore Technologies’ MinION based pathogen diagnosis insensitive By providing comprehensive, rapid device, has the potential to drive and costly (spending time and money pathogen and antimicrobial resistance this shift by combining rapidity with sequencing unwanted host nucleic acid identification, metagenomics sequencing comprehensiveness beyond that of and struggling to find all or any of the will reduce the time that patients remain culture or PCR. All pathogens contain pathogen genome with the sequencing on empirical broad-spectrum antibiotics, nucleic acids and so identification in depth available). Some sample types enabling clinicians to move away from a clinical samples by NGS, for example, are particularly challenging, e.g. blood, ‘one-size-fits-all’ approach to antibiotic metagenomics sequencing, is potentially where the ratio of human:bacterial DNA treatment to a stratified medicine universal (i.e. capable of detecting in a septic blood sample can be as high approach. This will lead to a reduction bacteria, viruses and fungi). Nothing as 10 billion:1. Enrichment is necessary, in the use of inappropriate antibiotics, has to be known about the pathogen in but, for metagenomics sequencing, reduced risk of antibiotic resistance and advance of testing, unlike PCR which rather than time-consuming culture reduced patient morbidity and mortality. only targets predetermined pathogens enrichment we need pathogen nucleic In the final O’Neill report, Jim O’Neill or culture, which requires different acid enrichment and/or host nucleic called for governments to “mandate now approaches for bacteria, fungi and acid depletion. For metagenomics that by 2020, all antibiotic prescriptions viruses. Recent advances in nanopore sequencing-based clinical microbiology will need to be informed by up-to-date sequencing technology include: to succeed, effective enrichment is vital. surveillance information and a rapid reduced capital and running costs We are currently developing diagnostic test wherever one exists”. (US$1,000 returnable deposit for the novel pathogen DNA enrichment and MinION metagenomics sequencing will MinION nanopore sequencer and as little human DNA depletion strategies and ensure that a rapid diagnostic test exists as US$45/sample when barcoding 12 applying them to blood, sputum and for every disease. samples per flow cell); portability (MinION urine for the metagenomics sequencing- devices were recently used to track Ebola based diagnosis of sepsis, hospital- Justin O’Grady in rural West African communities) and acquired pneumonia and complicated Norwich Medical School, University rapid real-time analysis (pathogen ID urinary tract infections (UTIs). The of East Anglia, James Watson Road, within 10 mins of starting the device) different sample types come with Norwich, NR4 7UQ using freely available analysis tools in different challenges, such as [email protected] Metrichor. It has now become realistic to large numbers of leukocytes and low

148 Microbiology Today Aug 16 | www.microbiologysociety.org

43:3 August 2016 43:3 August Nanoscale imaging infrared spectroscopyTwo-dimensional microscopy electron Cryogenic CRISPR-Cas Phage therapy Future Tech Microbiology TODAY

Microbiology Today 43:3 August 2016 Future Tech