OFFICIALOFFICIAL JOURNALJOURNAL OFOF THETHE AUSTRALIAN SOCIETY FOR MICROBIOLOGY INC.INC.

VolumeVolume 3636 NumberNumber 33 SeptemberSeptember 20152015

Anaerobic Confirmed Plenary speakers

Professor Peter Professor Dan Assoc Prof Susan Lynch Dr Brian Conlon Professor Anna Hawkey Andersson University of California Northeastern Durbin University of Upsalla University San Francisco University, Boston Johns Hopkins Birmingham Environmental pollution Colitis, Crohn's Disease Drug discovery in Dengue and vaccines Nosocomial by and its and Microbiome soil bacteria infection control and role in the evolution of Research resistance resistance

As with previous years, ASM 2016 will be co-run with NOW CONFIRMED! EduCon 2016: Microbiology Educators’ Conference 2016 Rubbo Oration Watch this space for more details on the scientific and Professor Anne Kelso social program, speakers, ASM Public Lecture, workshops, CEO NHMRC ASM awards, student events, travel awards, abstract deadlines and much more..

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www.theasm.org.au www.westernaustralia.theasm.org.au Annual Scientific Meeting and Trade Exhibition The Australian Society for Microbiology Inc. OFFICIAL JOURNAL OF THE AUSTRALIAN SOCIETY FOR MICROBIOLOGY INC. 9/397 Smith Street Fitzroy, Vic. 3065 Tel: 1300 656 423 Volume 36 Number 3 September 2015 Fax: 03 9329 1777 Email: [email protected] www.theasm.org.au Contents ABN 24 065 463 274 Vertical For Microbiology Australia Transmission 102 correspondence, see address below. Jonathan Iredell Editorial team Guest Prof. Ian Macreadie, Mrs Jo Macreadie Editorial 103 and Mrs Hayley Macreadie Anaerobic bacteria 103 Editorial Board Dena Lyras and Julian I Rood Dr Chris Burke (Chair) Dr Gary Lum Under the Prof. Mary Barton Dr John Merlino Microscope 104 Prof. Linda Blackall Prof. Wieland Meyer The complex factors that contribute to Clostridium difficile infection 104 Prof. Sharon Chen Prof. William Rawlinson Kate E Mackin and Dena Lyras Prof. Peter Coloe Dr Paul Selleck Dr Narelle Fegan Dr David Smith Clostridium difficile infection: an Australian clinical perspective 106 Dr Geoff Hogg Ms Helen Smith Grant A Jenkin Prof.. Jonathan Iredell Dr Jack Wang Dr Ipek Kurtböke Dr Paul Young Predicting genome variations between passages of Clostridium difficle Subscription rates by ribotypes 109 Current subscription rates are available Volker Gürtler from the ASM Melbourne offi ce. Community-acquired Clostridium difficile infection and Australian Editorial correspondence food animals 111 Prof. Ian Macreadie/Mrs Jo Macreadie Michele M Squire, Daniel R Knight and Thomas V Riley Tel: 0402 564 308 (Ian) Email: [email protected] Clostridium perfringens extracellular toxins and : 20 and counting 114 Published four times a year Sarah A Revitt-Mills, Julian I Rood and Vicki Adams in print and open access online by Necrotic enteritis in chickens: an important disease caused by Clostridium perfringens 118 Robert J Moore The manufacture of veterinary clostridial vaccines 120 Unipark, Building 1, Level 1 Robert Dempster 195 Wellington Road, Clayton, Vic. 3168 http://microbiology.publish.csiro.au Anaerobic and animals 122 David J Hampson, Nyree D Phillips and Tom La Publishing enquiries Jenny Bennett Anaerobic microorganisms and bioremediation of organohalide pollution 125 Email: [email protected] Matthew Lee, Chris Marquis, Bat-Erdene Judger and Mike Manefield Production enquiries Helen Pavlatos Coupling anaerobic bacteria and microbial fuel cells as whole-cell Email: [email protected] environmental biosensors 129 Advertising enquiries Lara T Bereza-Malcolm and Ashley E Franks Doug Walters Interesting anaerobes in the environment 133 Tel: 03 9545 8505 Mobile: 0419 357 779 Linda L Blackall Email: [email protected] ASM © 2015 The Australian Society for Microbiology Inc. Affairs 136 The ASM, through CSIRO Publishing, reserve all rights to the content, artwork and photographs in Microbiology Report from ASM 2015: One Microbiology 136 Australia. Permission to reproduce text, photos and artwork must be sought from CSIRO Publishing. Science meets Parliament 2015 140 The Australian Copyright Act 1968 and subsequent Visit from Turkish Society of Microbiology 142 amendments permit downloading and use of an article by an individual or educational institution for non- Vale Stephen Davis 143 commercial personal use or study. Multiple reproduction of any Microbiology Australia article in a study block is governed by rights agreement managed by Copyright Hot Topic 144 Agency Limited and fees may apply. Out of Africa: response to Ebola in the developed world; Authors published in Microbiology Australia have the moral right under Australian law to be acknowledged as lessons for the future 144 the creator. Lyn Gilbert and Peter Collignon ISSN 1324-4272 eISSN 2201-9189 While reasonable effort has been made to ensure the accuracy of the content, the Australian Society for Microbiology, CSIRO, and CSIRO Publishing accept no responsibility for any loss or damage from the direct or indirect use of or reliance on the content. The opinions expressed in articles, letters, and advertisements in Cover image: Scanning electron microscope image of Clostridium diffi cile spores. (SEM image provided Microbiology Australia are not necessarily those of the by the laboratory of Dena Lyras). Australian Society for Microbiology, the Editorial Board, CSIRO, and CSIRO Publishing.

MICROBIOLOGY AUSTRALIA • SEPTEMBER 2015 101 Vertical Transmission

positions will be finalised and the agenda for the next AGM dis- cussed. This is likely to include significant constitutional change and we will be inviting the entire membership to engage in discussions about the future of Microbiology and the ASM in this country.

Data from ASN and our VP Corporate Affairs, Cheryl Power, tell us that membership continues to rise with >2100 financial members as Jonathan Iredell of mid-2015, the distribution by state roughly equating to that of the President of ASM general population. Approximately 5% of our members are Fellows and as the Society undergoes the natural evolution that is occurring in all general societies, we will be looking to our senior members Constitutional reformwas onthe agenda again atthe Annual General including the Divisional and State Chairs and the Fellowship as a Meeting in Canberra, as it will be for the next several AGMs I think, whole for guidance. Among the key issues on which advice will be as there is clear recognition from the membership that the Society sought before being put to Council and ultimately the entire is facing change. The roles of the Divisional chairs and State chairs membership at the AGMs are the review of Society structure and and the nature of the general meeting were discussed in the light of governance and the ongoing review of meetings. overall structure. The Council is the principal decision-making body but because the Council only meets twice-yearly, interactions inside Fellowship is achieved by examination or by submitting relevant the Executive form the operational and strategic engine of ASM. The evidence of a members seniority and excellence within their disci- current Executive includes the incumbent President, with either the pline and we would encourage all those who would be eligible to incoming (President-Elect) or outgoing (Immediate Past President), apply. While we will be looking to increased consultation of the the Vice-President Scientific Affairs, Vice-President Corporate Affairs Fellowship and the Divisions in advising Council, the State branches and Vice-President Communications. The Chair roles have long will always be the main drivers of society business outside the been tasked with national leadership but without a role in Executive national meetings, delivering content for members by members, they have been preoccupied almost exclusively with the national hosting VSP participants from overseas and interstate, and mentor- meeting and speak only at the National Scientific Advisory Com- ing the Microbiologists of tomorrow. Recent rises in capitation and mittee, which has an enormous operational role managing the bulk regular contact between State branch representatives and national of the scientific awards and meetings. The recent move of the office by teleconference have been successful in increasing cohe- Division Chairs into Council as invitees over the last year or two sion and communication, and the reform of the VSP process that has has worked well and the increased involvement of State Chairs just been completed by Peter Traynor is expected to increase the similarly. We will be now ratifying these arrangements by seeking utility of this mechanism. formal endorsement at an extraordinary general meeting of I would urge society members to suggest new meetings, new the membership to bring the Constitution into line with these themes, even new Divisions. Ask yourselves if the current structure arrangements. with multiple special interest groupings is the right one for you? We will also be seeking formal ratification at the EGM (probably If you’re thinking about new meetings, how can the Society help you at the coming BacPath meeting) of the shift in the membership do it? The ASM is an easy way for a good idea to become a regular renewal data to better synchronise with the auditing and end conference, by providing infrastructure and corporate protection. of financial year, as discussed at previous AGM. Formal notice of Your state branch or Division chairs are easy points of contact and these minor adjustments will come out to members soon. The are listed on the website. The Society is here to serve us all and so next Council meeting will be In February at which next executive must we all aim to constantly improve its capacity to do so.

102 10.1071/MA15034 MICROBIOLOGY AUSTRALIA * SEPTEMBER 2015 Guest Editorial

Anaerobic bacteria

a ‘One-Health’ dimension to our understanding of C. difficile epidemiology in Australia. Other disease-causing clostridial are also discussed, starting with Clostridium perfringens and its plethora of toxins and extending into an examination of C. perfringens-mediated necrotic enteritis in chickens. Given the current, and growing, veterinary importance of clostridial diseases, the article on the manufacture of veterinary clostridial vaccines is timely. A discussion of anaerobic spirochaetes and their importance in animals provides another dimension to the theme of disease pathogenesis. Rounding out the issue are articles with a focus on Dena Lyras and Julian I Rood interesting anaerobes in the environment, including a discourse on Department of Microbiology, Monash University, Vic. 3800, Australia bioremediation of organohalide pollution. Finally, the cutting-edge approach of coupling anaerobic bacteria and microbial fuel cells as whole-cell environmental biosensors is described, providing food-for-thought as we move into a new age of environmental In the beginning there was no oxygen. The anaerobes ruled the awareness. earth at that time and they continue to play an important role in our oxygenated world, in food microbiology, microbial ecology and The Australian Society for Microbiology is pleased to showcase bacterial pathogenesis. Welcome to this special issue of Microbio- anaerobic microbes in this special issue. Australia has been a driving logy Australia, which is dedicated to anaerobic microbes. force in the study of anaerobes and is leading the way in many aspects, including understanding disease pathogenesis mechan- Anaerobes represent an extraordinarily diverse group of microbes isms, developing preventative measures and vaccines and applying that thrive in many environments, including the gastrointestinal knowledge of anaerobes and anaerobic processes to environmental tracts of humans and many animals, food, soil, sediments, subsur- problems. We hope you find this special issue relevant and valuable. face aquifers, and other environmentally extreme habitats. Their We would like to thank Ian Macreadie and the Microbiology activities have attracted wide attention, beginning with the demon- Australia editorial team for guiding us through the production of stration by Louis Pasteur in 1861 that yeast cells produce ethanol by this issue and all of the authors for their carefully considered fermentation. Many important anaerobic processes have been contributions. revealed in subsequent studies. Metabolically, anaerobes catalyse reactions that are critical to the global cycling of carbon and other elements, environmental detoxification, biocorrosion, biofuel pro- Biographies duction, methanogenesis and waste treatment. Anaerobes also A/Prof Dena Lyras is an ARC Future Fellow located at Monash encompass formidable of humans and animals, exem- University. She heads Clostridium difficile research in the Depart- plified by Clostridium species among many others. This group of ment of Microbiology, which is aimed at defining disease patho- organisms therefore has exceptional economic, medical, veterinary genesis mechanisms by combining molecular microbiology and academic importance in our world. techniques with host- interaction studies. The outcomes of these research efforts form the basis of vaccine and therapeutic This issue presents 11 articles that offer insights into anaerobic approaches to the prevention and treatment of C. difficile microbiology with an Australian focus, highlighting some of the infections. important areas of current relevance to our part of the world. Clostridium difficile is discussed from a number of different Julian I Rood is a Professor of Microbiology at Monash University perspectives, beginning with a clinical view and a discussion of and has led the field in clostridial conjugation mechanisms for factors that contribute to infection and disease and moving on many years. He also studies bacterial pathogenesis, predominantly to genomic variation. A thought-provoking article on community- mechanisms utilised by the human and animal pathogen, Clostrid- acquired and food animal C. difficile infection is included, bringing ium perfringens, and the sheep pathogen, Dichelobacter nodosus.

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The complex factors that contribute to Clostridium difficile infection

Kate E Mackin Department of Microbiology Dena Lyras Monash University, Clayton Vic. 3800, Australia Department of Microbiology Tel: +61 3 9902 9182 Monash University, Clayton Email: [email protected] Vic. 3800, Australia

Over the past decade Clostridium difficile has emerged as a Disease is caused by the production of two major toxins, toxin A and serious public health issue, causing both hospital-based toxin B, which are produced by strains that encode the Pathogenicity epidemics and community-associated disease. The most Locus (PaLoc); strains that do not contain PaLoc are non-toxigenic commonly recognised cause of antibiotic-associated diar- and considered unable to cause disease. All toxigenic strains pro- rhoea in the human population, C. difficile was initially seen duce toxin B; however, not all of them produce toxin A9. Both toxins as a nuisance pathogen causing limited disease in the hos- are monoglucosyltransferases that inactivate host cell Rho-family pital setting. However, the emergence of ‘hypervirulent’ GTPases, resulting in disruption of the host cell actin cytoskeleton strain types, associated with an increase in both morbidity and cell death10,11. This, in turn, leads to the loss of tight junction and mortality, has made it a pathogen of great concern integrity in the gut, and movement of fluid into the intestinal worldwide. Infection with C. difficile is also being increas- lumen, causing diarrhoea10. Toxins A and B also induce inflamma- ingly documented in animals, with suggestions that animals tory responses, which contribute to host tissue damage during destined for human consumption may provide a reservoir infection12,13. for disease. The use of antibiotics is considered the main risk Aside from the two major toxins, the virulence factors of C. difficile factor for the development of human infection; however, are still poorly characterised. Sporulation and germination are many other factors such as strain type, patient age, and important in the disease process, as the infectious particle ingested host immune response all contribute to disease caused by by patients is the spore14,15. Once in the gut, germination occurs and C. difficile. the vegetative cell expresses factors required for survival and path- ogenesis in the host14. C. difficile may produce a number of these A Gram-positive, spore-forming, anaerobic rod, C. difficile was first factors, which include fibronectin-binding ,S-layer, capsule, identified as causing disease in 19781,2. Disease arising from type IV pili, and flagella12,13. Some strains also produce an additional C. difficile infection (CDI) may range from mild and self-limiting toxin termed binary toxin, or CDT (C. difficile ). An ADP- diarrhoea throughto pseudomembranous colitis; insome cases CDI ribosylating toxin, CDT does not appear essential for disease16,17; may progress to toxic megacolon, perforation of the bowel, sepsis however, it has been suggested that this toxin may promote colo- and death3,4. Extra-intestinal cases of CDI are rare5. While nearly half nisation in the host18. of all infections are in those younger than 65, more than 90% of deaths occur in people aged 65 or older6. C. difficile can be isolated Antibiotic use is the most important risk factor for developing CDI19, from about 3% of healthy adults, while up to 35% of hospital patients as exposure to antibiotics disrupts the normal gut microbiota, thus can be carriers7. Children under the age of two years are commonly reducing the normal ‘colonisation resistance’ ofthe large intestine20 asymptomatic carriers of C. difficile; however, disease can occur in and allowing C. difficile to colonise14. Antibiotic use is associated the paediatric population as well8. with both carriage of C. difficile and with diarrhoea, and the duration

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of an antibiotic course is also a risk factor for CDI21. Most antibiotics factors drive the disease process, including strain type, patient age, have been implicated in the development ofCDI in patients22,which and host immune response. While the use of antibiotics and the is often associated with acute care hospitals19 and with the elderly21. development of resistance have selected for particular strain types Decreased host defences, common in the elderly population, may associated with outbreaks of severe disease, other strain types are also play a role in the development of infection. A predisposition to also emerging. For this reason, C. difficile infection is of significant recurrent disease is associated with an inadequate immune re- clinical and veterinary concern, and will continue to be so into the sponse23. Relapses of CDI are common, and may lead to a cascade future. of interrelated complications, which can ultimately be fatal24. Relapses may follow an initial infection in about 20% of cases25, and rates of subsequent relapses can rise to 40–60%26. While these References 1. Bartlett, J.G. et al. (1978) Role of Clostridium difficile in antibiotic-associated recurrences may be caused by relapsing infection with the same pseudomembranous colitis. Gastroenterology 75,778–782. – strain, it has been suggested that between 33 75% of cases may be 2. George, R.H. et al. (1978) Identification of Clostridium difficile as a cause of due to infection with a new strain25. pseudomembranous colitis. BMJ 1, 695. doi:10.1136/bmj.1.6114.695 3. Borriello, S.P. (1998) Pathogenesis of Clostridium difficile infection. J. Antimi- The epidemiology of CDI has changed over the last decade. The rise crob. Chemother. 41,13–19. doi:10.1093/jac/41.suppl_3.13 fi of ‘hypervirulent’ ribotype 027 strains has seen an increase in the 4. Heinlen, L. and Ballard, J.D. (2010) Clostridium dif cile infection. Am. J. Med. Sci. 340,247–252. doi:10.1097/MAJ.0b013e3181e939d8 fi 27,28 morbidity and mortality associated with C. dif cile . Prior to the 5. García-Lechuz, J.M. et al. (2001) Extra-intestinal infections caused by outbreaks in 2002 and onwards, ribotype 027 strains were not Clostridium difficile. Clin. Microbiol. Infect. 7,453–457. doi:10.1046/j.1469- 0691.2001.00313.x commonly isolated29, but are now considered to be one of the 6. Korman, T.M. (2015) Diagnosis and management of Clostridium difficile infec- most clinically important strain types of C. difficile. This strain type is tion. Semin. Respir. Crit. Care Med. 36,31–43. doi:10.1055/s-0034-1398741 30 now well established worldwide, including in Australia . However, 7. Kuijper, E.J. et al. (2006) Emergence of Clostridium difficile-associated disease in North America and Europe. Clin. Microbiol. Infect. 12(Suppl 6), 2–18. other strain types are also becoming a cause for concern, with doi:10.1111/j.1469-0691.2006.01580.x ribotype 017 and ribotype 078 strains associated with more severe 8. Enoch, D.A. et al. (2011) Clostridium difficile in children: colonisation and disease in human patients31,32. These strain types have been iden- disease. J. Infect. 63, 105–113. doi:10.1016/j.jinf.2011.05.016 tified locally33, highlighting the need for epidemiological vigilance 9. King, A.M. et al. (2015) Emergence of toxin A-negative, toxin B-positive Clostrid- ium difficile strains: epidemiological and clinical considerations. Future Micro- when it comes to C. difficile. New variants will also continue to biol. 10,1–4. doi:10.2217/fmb.14.115 emerge, as recently seen in Australia with ribotype 244 strains34. The 10. Voth, D.E. and Ballard, J.D. (2005) Clostridium difficile toxins: mechanism of – fl action and role in disease. Clin. Microbiol. Rev. 18,247263. doi:10.1128/ development of uoroquinolone resistance has been linked to the CMR.18.2.247-263.2005 35 global spread of ribotype 027 strains , but why other strain types 11. Aktories, K. (2011) Bacterial toxins that modify host regulatory GTPases. become more or less prevalent is not always clear34. Without Nat. Rev. Microbiol. 9, 487–498. doi:10.1038/nrmicro2592 12. Awad, M.M. et al. (2015) Clostridium difficile virulence factors: insights into an ongoing monitoring, it will be difficult to ascertain changes in strain anaerobic spore-forming pathogen. Gut Microbes 5,579–593. doi:10.4161/ type within the local population. 19490976.2014.969632 13. Vedantam, G. et al. (2012) Clostridium difficile infection: toxins and non-toxin Community-associated CDI is becoming increasingly important, virulence factors, and their contributions to disease establishment and host response. 3, 121–134. doi:10.4161/gmic.19399 with a greater awareness of cases occurring outside of the traditional Gut Microbes 14. Sarker, M.R. and Paredes-Sabja, D. (2012) Molecular basis of early stages of hospital setting. The spectrum of disease seen in these patients is Clostridium difficile infection: germination and colonization. Future Microbiol. similar to that seen in healthcare-associated CDI. Mild and self- 7, 933–943. doi:10.2217/fmb.12.64 limiting disease is common, and many community-based patients 15. Sorg, J.A. and Sonenshein, A.L. (2008) Bile salts and glycine as cogerminants for Clostridium difficile spores. J. Bacteriol. 190,2505–2512. doi:10.1128/ 36 may not seek medical attention . The infection source for com- JB.01765-07 munity-acquired CDI is not known, and these cases cannot always 16. Carter, G.P. et al. (2015) Defining the roles of TcdA and TcdB in localized gastrointestinal disease, systemic organ damage, and the host response be linked to the major risk factors associated with CDI37. Animals during Clostridium difficile infections. MBio 6, e00551-e15. doi:10.1128/ and animal-derived food products have both been suggested as mBio.00551-15 potential sources of C. difficile38, but this has not yet been clearly 17. Kuehne, S.A. et al. (2014) Importance of toxin A, toxin B, and CDT in virulence of an epidemic Clostridium difficile strain. J. Infect. Dis. 209,83–86. doi:10.1093/ demonstrated. infdis/jit426 18. Schwan, C. et al. (2014) Clostridium difficile toxin CDT hijacks microtubule fi Undoubtedly, C. dif cile has emerged as an important public health organization and reroutes vesicle traffic to increase pathogen adherence. Proc. problem, no longer just confined to hospitals as a nuisance path- Natl. Acad. Sci. USA 111, 2313–2318. doi:10.1073/pnas.1311589111 19. McDonald, L.C. . (2006) fi infection in patients discharged ogen. As discussed elsewhere in this issue, C. difficile is present in et al Clostridium dif cile from US short-stay hospitals, 1996-2003. Emerg. Infect. Dis. 12,409–415. Australia and is found in human and animal populations. Multiple doi:10.3201/eid1205.051064

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20. Reeves, A.E. et al. (2011) The interplay between microbiome dynamics and 33. Mackin, K.E. et al. (2015) Molecular characterization and antimicrobial suscepti- pathogen dynamics in a murine model of Clostridium difficile Infection. Gut bilities of Clostridium difficile clinical isolates from Victoria, Australia. Anaerobe Microbes 2,145–158. doi:10.4161/gmic.2.3.16333 34,80–83. doi:10.1016/j.anaerobe.2015.05.001 21. Bignardi, G.E. (1998) Risk factors for Clostridium difficile infection. J. Hosp. Infect. 34. Lim, S.K. et al. (2014) Emergence of a ribotype 244 strain of Clostridium difficile 40,1–15. doi:10.1016/S0195-6701(98)90019-6 associated with severe disease and related to the epidemic ribotype 027 strain. – 22. Owens, R.C., Jr. et al. (2008) Antimicrobial-associated risk factors for Clostridium Clin. Infect. Dis. 58, 1723 1730. doi:10.1093/cid/ciu203 difficile infection. Clin. Infect. Dis. 46(Suppl 1), S19–S31. doi:10.1086/521859 35. He, M. et al. (2013) Emergence and global spread of epidemic healthcare- fi – 23. Drudy, D. et al. (2004) Human antibody response to surface layer proteins in associated Clostridium dif cile. Nat. Genet. 45,109 113. doi:10.1038/ng.2478 Clostridium difficile infection. FEMS Immunol. Med. Microbiol. 41, 237–242. 36. Hensgens, M.P. et al. (2012) Clostridium difficile infection in the community: doi:10.1016/j.femsim.2004.03.007 a zoonotic disease? Clin. Microbiol. Infect. 18,635–645. doi:10.1111/j.1469- 24. Pepin, J. et al. (2005) Increasing risk of relapse after treatment of Clostridium 0691.2012.03853.x difficile colitis in Quebec, Canada. Clin. Infect. Dis. 40, 1591–1597. doi:10.1086/ 37. Kuntz, J.L. et al. (2011) Incidence of and risk factors for community-associated 430315 Clostridium difficile infection: a nested case-control study. BMC Infect. Dis. 11, 25. Johnson, S. (2009) Recurrent Clostridium difficile infection: a review of risk 194. doi:10.1186/1471-2334-11-194 factors, treatments, and outcomes. J. Infect. 58, 403–410. doi:10.1016/j.jinf. 38. Rupnik, M. (2007) Is Clostridium difficile-associated infection a potentially 2009.03.010 zoonotic and foodborne disease? Clin. Microbiol. Infect. 13,457–459. 26. Surawicz, C.M. and Alexander, J. (2011) Treatment of refractory and recurrent doi:10.1111/j.1469-0691.2007.01687.x Clostridium difficile infection. Nat. Rev. Gastroenterol. Hepatol. 8, 330–339. doi:10.1038/nrgastro.2011.59 27. Warny, M. et al. (2005) Toxin production by an emerging strain of Clostridium Biographies difficile associated with outbreaks of severe disease in North America and Europe. Lancet 366, 1079–1084. doi:10.1016/S0140-6736(05)67420-X Dr Kate Mackin is a Research Fellow in the Department of fi 28. Pépin, J. et al. (2005) Mortality attributable to nosocomial Clostridium dif cile- Microbiology at Monash University. She completed her PhD in associated disease during an epidemic caused by a hypervirulent strain in Quebec. CMAJ 173, 1037–1042. doi:10.1503/cmaj.050978 2014, exploring the diversity of Clostridium difficile clinical isolates, 29. McDonald, L.C. et al. (2005) An epidemic, toxin gene-variant strain of Clostridium identifying important strain types in local hospitals. She also difficile. N. Engl. J. Med. 353, 2433–2441. doi:10.1056/NEJMoa051590 demonstrated that variant strain types regulate virulence factors 30. Richards, M. et al. (2011) Severe infection with Clostridium difficile PCR ribotype 027 acquired in Melbourne, Australia. Med. J. Aust. 194,369–371. differentially, through the master regulator Spo0A. Her current 31. Goorhuis, A. et al. (2008) Emergence of Clostridium difficile infection due to a work extends on these themes, to identify other factors important new hypervirulent strain, polymerase chain reaction ribotype 078. Clin. Infect. Dis. in the pathogenesis of C. difficile. 47, 1162–1170. doi:10.1086/592257 32. Drudy, D. et al. (2007) Toxin A-negative, toxin B-positive Clostridium difficile. Int. J. Infect. Dis. 11,5–10. doi:10.1016/j.ijid.2006.04.003 The biography for A/Prof Dena Lyras is on page 103.

Clostridium difficile infection: an Australian clinical perspective

Kingdom and Europe, associated with the emergence of fl Grant A Jenkin a uoroquinolone-resistant clone known as restriction en- donuclease type BI, pulsed field type NAP1 or PCR ribotype Monash Infectious Diseases – Monash Medical Centre 027 (RT027) Clostridium difficile (CD)1 3. CD is now the 246 Clayton Road fi Clayton, Vic. 3168, Australia most commonly identi ed nosocomial pathogen in the Department of Microbiology USA4–6 and in 2011 there were approximately 450 000 Monash University Clayton, Vic. 3800, Australia incident cases of CDI in the USA and 29 300 deaths at Tel: +61 3 9594 4564 day 30 post diagnosis6. Using an estimated attributable Fax: +61 3 9594 4533 Email: [email protected] mortality rate of 50%, approximately 15 000 deaths due to CDI occurred in the USA in 2011.

Australian data have been slow to emerge, but a coordinated survey The scale of the problem now posed by Clostridium difficile of lab diagnosis of CDI in 450 Australian public hospitals in the 2-year infection (CDI) is becoming frighteningly clear. Since 2001, period 2011–2012 has provided the first comprehensive nationwide a dramatic increase in the incidence and severity of CDI profile of CDI. The study identified 12 683 cases of CDI and the has occurred, particularly, in North America, the United incidence of disease increased by 26% over the period of the study7,

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confirming that CDI is responsible for substantial morbidity and Genomic analysis of CD RT027 isolates from around the world presumably mortality in Australia. has identified two distinct epidemic lineages with evidence of intercontinental transmission, including repeated introductions There is clear evidence that CD RT027 causes more severe disease8 from North America to the United Kingdom21. Australian CD and also has a reduced cure rate and an increased risk of relapse9. RT027 isolates were shown to have been imported from North Other emergent strains of CD have contributed to the increasing America and the transmission of the Australasian RT244 clone burden of disease and also the changing the pattern of CDI10,11. found in a patient who developed CDI on return to the UK20 CD RT078 for example, shares genetic virulence characteristics with emphasises the potential for rapid international dissemination of CD RT027, has been shown to cause severe disease and is associated virulent clones. with community-acquired infection10. To further emphasise the potential for CDI to pose new challenges, Australia was forewarned of the possibility for arrival of CD RT02712, we found that RT244 produces a variant Toxin B that has an altered although the fact that routine diagnostics are based on toxin N-catalytic domain more similar to C. sordellii lethal toxin and detection, and isolates are not routinely cultured, hampered produces atypical cytotoxicity on Vero cells in vitro18. This was the surveillance efforts. The first CD RT027 strain identified in first report of this variant toxin being associated with a severe CDI Australia occurred in a patient returned from hospitalisation cluster, but the role this variant toxin may play in the virulence of in North America13 but did not apparently result in secondary RT244 CDI has not yet been established. cases. Locally acquired infection was then reported in Melbourne in early 201014, although subsequent surveys of Australian CD A particular challenge for clinicians is the identification and treat- isolates indicate that RT027 has not become endemic within ment of relapses that occur in 20–25% of patients following the initial Australia15,16. episode, but increase following each relapse and exceeds 50% following multiple recurrences. In the USA, 83 000 (21%) of CDI In this setting of heightened awareness and understanding of the patients experienced first recurrences in 2011 and the high associ- potential for outbreaks of severe CDI, two patients at Monash Health ated morbidity and costs associated with relapses have lent strong developed clinically severe CDI in mid-2011. While not unusual, we impetus to the development of new therapeutics that may prevent were actively surveying for the appearance of RT027 using the Xpert their occurrence6. Fidaxomicin is a non-absorbable macrolide an- C. difficile PCR (Cepheid, Sunnyvale, CA, USA) as the confirmatory tibacterial recently licensed for CDI. Although substantially more diagnostic test for CDI. This assay includes a detection probe for the expensive and no more effective than oral , fidaxomicin tcdC nucleotide 117 deletion (tcdCD117) characteristic of CD RT027 reduces relapse rates in non-RT027 CDI22 and has therefore found strains, although this deletion is present in some other CD a role in some CDI treatment guidelines23,24. A number of new nonRT-027 strains17. Faecal samples from both patients signaled antibacterials are currently in Phase III trial including the quinolo- as presumptive CD RT027 but subsequent analysis found the strain nyloxazolidinone cedazolid, and the lipopeptide analogue of to be PCR ribotype 244 (RT244) and fluoroquinolone susceptible. Daptomycin, CB-183,315 as well as repurposed agents such as the We eventually identified 12 patients at our institution over a period non-absorbable rifamycin, rifaximin and nitazoxanide25. of 9-months with RT244 CDI and a retrospective case-control study found that RT244 CDI was more severe, and death was 13-times CDI is associated with antibacterial induced alteration in the gut more likely in patients infected with this strain compared to non- microbiome and recurrent disease is associated with persistent gut RT244 CDI controls18. One-third of infections were community dysbiosis. Restoration of the gut microbiome by administration of acquired. In October 2011 CDI due to RT244 was reported also donor-derived faecal suspension is highly effective in preventing in New Zealand and was also found to cause more severe disease recurrence and restoration of gut microbial diversity26. Theoretical and was also frequently community acquired19. Whole genome concerns as to the lack of knowledge of long-term effects, trans- sequencing of Australian outbreak strains found that RT244 CD was mission of pathogens and lack of standardisation of donor bacterial from the same clade as RT027 but significantly divergent from it. flora as well as the logistic difficulties have hampered access to this A whole-genome sequence comparison of 15 Australian CD RT244 treatment. Efforts to define a minimal set of bacterial strains that strains from Victoria, New South Wales and Western Australia could provide standardised on-demand treatment seem likely to be confirmed the CD RT244 strains formed a highly related clone with the way forward27. A recent Phase II study offers the prospect of 14 isolates having 8 or fewer single nucleotide polymorphisms20. highly targeted bacteriotherapy using non-toxigenic CD for preven- The reasons for the sudden appearance of this virulent strain, and its tion of recurrent infection28 and another current study in a mouse subsequent disappearance are unknown. model found that Clostridium scindens offered protection from

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fi CDI by altering bile acid metabolism in the gut so as to inhibit CD 13. Riley, T.V. et al. (2009) First Australian isolation of epidemic Clostridium dif cile PCR ribotype 027. Med. J. Aust. 190, 706–708. spore germination29. 14. Richards, M. et al. (2011) Severe infection with Clostridium difficile PCR ribotype 027 acquired in Melbourne, Australia. Med. J. Aust. 194,369–371. Further hope is offered by immune mediated protection to 15. Australian Commission on Safety and Quality in Health Care (2015) Consultation CDI. Absence of anti-toxin antibody is associated with disease and on surveillance and monitoring of Clostridium difficile infection in Australia: discussion paper. ACSQHC, Sydney. http://www.safetyandquality.gov.au/our-work/ recurrence and passive immunisation by administration of bivalent healthcare-associated-infection/consultation-on-clostridium-difficile/ (accessed 9 monoclonal anti-Toxin A and anti-Toxin B antibodies reduced CDI June 2015). fi recurrence from 25% to 7%30. A Phase III study examining the 16. Foster, N.F. et al. (2014) Epidemiology of Clostridium dif cile infection in two tertiary-care hospitals in Perth, Western Australia: a cross-sectional study. New relative protective effect of anti-Toxin A, anti-Toxin B or the bivalent Microbes New Infect. 2,64–71. doi:10.1002/nmi2.43 combination has been completed and results are anticipated 17. Carter, G.P. et al. (2011) The anti-sigma factor TcdC modulates hypervirulence in an epidemic BI/NAP1/027 clinical isolate of Clostridium difficile. PLoS Pathog. 7, shortly31. e1002317. doi:10.1371/journal.ppat.1002317 fi Intensively applied control measures including anti-microbial stew- 18. Lim, S.K. et al. (2014) Emergence of a ribotype 244 strain of Clostridium dif cile associated with severe disease and related to the epidemic ribotype 027 strain. ardship, enhanced identification and reporting of cases and imple- Clin. Infect. Dis. 58, 1723–1730. doi:10.1093/cid/ciu203 mentation of mandated infection control measures can reduce 19. De Almeida, M.N. et al. (2013) Severe Clostridium difficile infection in 11 New Zealand associated with an emerging strain, PCR-ribotype 244. N. Z. Med. CDI ; however, the huge burden of CDI, and the emergence of J. 126,9–14. community acquired disease, challenges how further CDI control 20. Eyre, D.W. et al. (2015) Emergence and spread of predominantly community- fi is to be achieved. An international Phase III placebo-controlled onset Clostridium dif cile PCR ribotype 244 infection in Australia, 2010 to 2012. Euro Surveill. 20, 21059. vaccine trial with a bivalent Toxin-A and Toxin-B toxoid vaccine is 21. He, M. et al. (2013) Emergence and global spread of epidemic healthcare- now underway in immunocompetent at-risk patients over 50 years associated Clostridium difficile. Nat. Genet. 45,109–113. doi:10.1038/ng.2478 of age, including at a number of Australian sites and if successful, 22. Louie, T.J. et al. (2011) Fidaxomicin versus vancomycin for Clostridium difficile infection. N. Engl. J. Med. 364, 422–431. doi:10.1056/NEJMoa0910812 offers an alternative and complementary strategy to reduce the 23. Wilcox, M. et al. (2013) Updated guidance on the management and treatment of 32 global problem presented by CDI . Clostridium difficile infection. https://www.gov.uk/government/publications/ clostridium-difficile-infection-guidance-on-management-and-treatment (accessed 9June2015) 24. Debast, S.B. et al. (2014) European Society of Clinical Microbiology and Infectious References Diseases: update of the treatment guidance document for Clostridium difficile infection. Clin. Microbiol. Infect. 20(Suppl 2), 1–26. doi:10.1111/1469-0691.12418 1. McDonald, L.C. et al. (2005) An epidemic, toxin gene-variant strain of Clostridium fi difficile. N. Engl. J. Med. 353,2433–2441. doi:10.1056/NEJMoa051590 25. Jarrad, A.M. et al. (2015) Clostridium dif cile drug pipeline: challenges in discovery and development of new agents. J. Med. Chem. 58, 5164–5185. 2. Loo, V.G. et al. (2005) A predominantly clonal multi-institutional outbreak of doi:10.1021/jm5016846 Clostridium difficile-associated with high morbidity and mortality. N. Engl. J. Med. 353, 2442–2449. doi:10.1056/NEJMoa051639 26. van Nood, E. et al. (2013) Duodenal infusion of donor feces for recurrent Clostridium difficile. N. Engl. J. Med. 368, 407–415. doi:10.1056/NEJMoa1205037 3. Kuijper, E.J. et al. (2006) Emergence of Clostridium difficile-associated disease in North America and Europe. Clin. Microbiol. Infect. 12(Suppl 6), 2–18. 27. Lawley, T.D. et al. (2012) Targeted restoration of the intestinal microbiota with a fi fi doi:10.1111/j.1469-0691.2006.01580.x simple, de ned bacteriotherapy resolves relapsing Clostridium dif cile disease in mice. PLoS Pathog. 8, e1002995. doi:10.1371/journal.ppat.1002995 4. Magill, S.S. et al. (2014) Multistate point-prevalence survey of health care- associated infections. N. Engl. J. Med. 370, 1198–1208. doi:10.1056/NEJMoa 28. Gerding, D.N. et al. (2015) Administration of spores of nontoxigenic Clostridium fi fi 1306801 dif cile strain M3 for prevention of recurrent C. dif cile infection: a randomized clinical trial. JAMA 313, 1719–1727. doi:10.1001/jama.2015.3725 5. Leffler, D.A. and Lamont, J.T. (2015) Clostridium difficile infection. N. Engl. J. fi Med. 372, 1539–1548. doi:10.1056/NEJMra1403772 29. Buf e, C.G. et al. (2015) Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile. Nature 517,205–208. doi:10.1038/ 6. Lessa, F.C. et al. (2015) Burden of Clostridium difficile infection in the United nature13828 States. N. Engl. J. Med. 372, 825–834. doi:10.1056/NEJMoa1408913 30. Lowy, I. et al. (2010) Treatment with monoclonal antibodies against Clostridium 7. Slimings, C. et al. (2014) Increasing incidence of Clostridium difficile infection, difficile toxins. N. Engl. J. Med. 362, 197–205. doi:10.1056/NEJMoa0907635 Australia, 2011–2012. Med. J. Aust. 200, 272–276. doi:10.5694/mja13.11153 31. A study of MK-3415 MK-6072, and MK-3415A in participants receiving antibiotic 8. See, I. et al. (2014) NAP1 strain type predicts outcomes from Clostridium difficile therapy for Clostridium difficile infection (MK-3415A-001) (MODIFY I). https:// infection. Clin. Infect. Dis. 58,1394–1400. doi:10.1093/cid/ciu125 clinicaltrials.gov/ct2/show/NCT01241552 (accessed 9 June 2015). 9. Petrella, L.A. et al. (2012) Decreased cure and increased recurrence rates for 32. Study of a candidate Clostridium difficile toxoid vaccine in subjects at risk for Clostridium difficile infection caused by the epidemic C. difficile BI strain. Clin. C. difficile infection. https://clinicaltrials.gov/ct2/show/NCT01887912 (accessed Infect. Dis. 55, 351–357. doi:10.1093/cid/cis430 9 June 2015). 10. Goorhuis, A. et al. (2008) Emergence of Clostridium difficile infection due to a new hypervirulent strain, polymerase chain reaction ribotype 078. Clin. Infect. Dis. Biography 47, 1162–1170. doi:10.1086/592257 11. Wilcox, M.H. et al. (2012) Changing epidemiology of Clostridium difficile Grant Jenkin is a physician and Director of the Mycobacterial infection following the introduction of a national ribotyping-based Infection Service at Monash Infectious Diseases. His research inter- surveillance scheme in England. Clin. Infect. Dis. 55, 1056–1063. doi:10.1093/ cid/cis614 ests include the genetic virulence determinants and immunology 12. Riley, T.V. (2006) Epidemic Clostridium difficile. Med. J. Aust. 185, 133–134. of mycobacterial and clostridial infections.

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Predicting genome variations between passages of Clostridium difficle by ribotypes

not address the fundamental underlying issue of how the prediction of the systematic similarities and differences in operons can be treated in this way for typing bacterial strains6. The purpose of this short note is to demonstrate how the analysis of rrn Types can be Volker Gürtler standardised, with simplicity, to provide more reliable typing infor- School of Applied Science mation, just by including the flanking genes of each rrn operon. By RMIT University Bundoora, Vic. 3152, Australia including this information a surprising amount of detail can be Email: [email protected] obtained regarding the insertion of whole operons between strains7.

First, the genomic position of all the rrn operons can be generalised between strains according to their proximity to Type-specific flank- Ribotyping is the most widely used method for differentiat- ing genes (Figure 1a). This makes it possible to identify specifically ing strains of Clostridium difficile for epidemiological stud- deletions, insertions and double rrn operons in some strains of ies and infection control. Recently there have been calls for affected Types. The insertions and deletions affect whole genes standardisation of the technique to which sophisticated (16S, 23S and 5S) as well as extragenic regions (ITS1, pre-16S and technical solutions have been offered. The present note post-5S). However, all specific Types have identical genes flanking offers a solution for standardisation based on conserved their rrn operons (e.g. Type 1 has sigB and smb in all strains except rrn operon Type-specific flanking genes. Furthermore, this CF5 and M120). The only exception is when the flanking genes are technique can be used to detect Type-specific rrn operon not present and in this case the rrn operons are absent too. deletions in passages from a single strain of C. difficile. Therefore in these strains the whole Type is missing or rearranged. As can be seen, the genes directly flanking the rrn operons are good The ribosomal RNA operon is present in up to 12 copies per genome for identifying rrn Types and their associated deletions, insertions in Clostridium difficile1. Since the early 1990s it has been used for and rearrangements. ‘ribotyping’ strains of C. difficile by exploiting sequence variations between operons on the same genome and between operons from Second, the only Type that has been duplicated in the 630DERM different strains2. Detailed analysis of the main ribotypes for which strain can be reliably identified and tracked in the related strains as whole genome sequencing (WGS) are available has been per- ‘Type 1’ (Figure 1b). Even though the deletion of rrnA (16S and 23S formed1 and will not be presented here. Rather, it will be shown genes) was useful for differentiation of the three CD630 passages how the rrn operons are predictably related to each other between it was not specific to CD630, also occurring in CD196 (Figure 1a). strains by their flanking genes according to Type position on the But of particular note was the observation that Single Nucleotide genome. I will draw on two recent studies3,4 of the well characterised Polymorphisms (SNPs) in the two flanking genes (Figure 1b, Open 630 strain that show that according to ribotype, the relatedness of Reading Frames (ORFs) ‘sigB’ and ‘smb’ labelled yellow) could be flanking genes (Figure 1) makes it possible to reliably show that rrn clearly used to differentiate the three 630 passages from the other operons have been lost and gained between passages of the same five strains (G46R2, BI1, CD196, 2007855 and R20291) with smb strain (e.g. Figure 1a, rrnA is absent in 630AM but present in 630CP having up to 80 times more SNPs than the rrn genes. and 630DERM). This method of analysis has determined that in the rrn operon of Many ribotyping schemes have been used and reported for ‘Type 1’ of C. difficile strains, the duplication of rrnA has occurred C. difficile1 and there is increasing interest in standardisation in multiple strains of different lineages (i.e. in strains 630AM and of methodology with a recent Internationally-Standardised, High- CD196). There is potential for the method outlined here to be used Resolution Capillary Gel-Based Electrophoresis PCR-Ribotyping to differentiate between passages of other C. difficile strains and Protocol providing the technical basis5. However, this protocol does strains of different bacterial species.

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(a)

(b)

Figure 1. (a) Identification of ‘Types 1-9’ by genes (yellow) immediately flanking the Type-specific rrn operons marked in respective colours. ‘Types 1,3,4’ have double rrn operons in some strains. (b) Alignment for ‘Type 1’ showing two lineages: (i) three 630 (ribotype 12) passages and (ii) ribotype 027 strains G46R2, BI1, CD196, 2007855 and R20291. The % SNPs is stated under identity and the number of deletions for each gene region in parentheses. Abbreviations: sigB, RNA polymerase sigma-37 B factor; smb, putative small-molecule-binding protein; tnf, putative glycosyl transferase; pycA, pyruvate carboxylase; cwlD, germination-specific N-acetylmuramoyl-L-alanine amidase, Autolysin; aspC, aspartate aminotransferase; nrdG, anaerobic ribonucleoside-triphosphate reductase-activating protein; CHP00159, conserved hypothetical protein; ATPase, putativeK/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporting P-type ATPase; DedA, putativemembraneprotein;mgeN, putativemannosyl-glycoprotein endo- beta-N-acetylglucosamidase; aaa_cwp16, putative N-acetylmuramoyl-L-alanine amidase,autolysin; C60B, putative peptidase C60B, B; PSD, putative polysaccharide deacetylase; ABC, ABC-type transport system, sugar-family ATP-binding protein; sirC, precorrin-2 dehydrogenase; speA, putative arginine decarboxylase; lysS, lysyl-tRNA synthetase (Lysine–tRNA ).

References 6. Gürtler, V. et al. (2014) Bacterial typing and identification by genomic analysis of 16S–23S rRNA intergenic transcribed spacer (ITS) sequences. In Methods in 1. Gürtler, V. and Grando, D. (2013) New opportunities for improved ribotyping of Microbiology. (Goodfellow, M., Sutcliffe, I., Chun, J., eds). 41; 253–274. C. difficile clinical isolates by exploring their genomes. J. Microbiol. Methods 93, 257–272. doi:10.1016/j.mimet.2013.02.013 7. Gürtler, V. et al. (2015) Typing of Vibrio parahaemolyticus environmental and clinical strains by ribosomal rna operon allele-specific flanking genes. Front. 2. Gürtler, V. (1993) Typing of Clostridium difficile strains by PCR-amplification of Microbiol. (in press). variable length 16S-23S rDNA spacer regions. J. Gen. Microbiol. 139, 3089–3097. doi:10.1099/00221287-139-12-3089 3. Riedel, T. et al. (2015) Genome resequencing of the virulent and multidrug- resistant reference strain Clostridium difficile 630. Genome Announc. 3, Biography e00276-15. doi:10.1128/genomeA.00276-15 ’ 4. van Eijk, E. et al. (2015) Complete genome sequence of the Clostridium difficile Dr Gürtler s research interest has been in developing diagnostic laboratory strain 630Derm reveals differences from strain 630, including translo- tests primarly using the ribosomal RNA operon for the last 25 years. cation of the mobile element CTn5. BMC Genomics 16, 31. doi:10.1186/s12864- 015-1252-7 He has recently developed a web site where more information on 5. Fawley, W.N. et al. (2015) Development and validation of an internationally- the techinques discussed in this note can be obtained (www. standardized, high-resolution capillary gel-based electrophoresis PCR-ribotyping ribotyping.net). He is also Editor of the Journal of Microbiological protocol for Clostridium difficile. PLoS One 10, e0118150. doi:10.1371/journal. pone.0118150 Methods (Elsevier).

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Community-acquired Clostridium difficile infection and Australian food animals

Michele M Squire A, Daniel R Knight A and Thomas V Riley A,B AMicrobiology and Immunology, School of Pathology and Laboratory Medicine, The University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands, WA 6009, Australia BTel: +61 8 6383 4355, Fax: +61 8 9346 2912, Email: [email protected]

Clostridium difficile is an anaerobic Gram positive spore- from food production animals outside Australia10, and this strain is forming bacterium, the leading cause of infectious diarrhoea now commonly isolated from human infections11,12. (C. difficile infection; CDI) in hospitalised humans. The Increasing CA-CDI and genetic diversity of circulating C. difficile assumption that CDI is primarily a hospital-acquired infec- strains suggest a reservoir of C. difficile outside healthcare facilities. tion is being questioned. Community-acquired CDI (CA-CDI) Similarity between community and animal strains has focussed is increasing1 particularly in groups previously considered at attention on animals, or environmental sources common to animals low risk2,3. In Australia, CA-CDI rates doubled during 2011 and humans, as potential infection reservoirs. and increased by 24% between 2011 and 20124. Two poten- tially high-risk practices in Australian food animal husband- C. difficile in animals and food ry may present a risk for CA-CDI: slaughtering of neonatal C. difficile is an enteric pathogen of companion animals (cats, dogs, animals for food, and effluent recycling to agriculture. horses) and food animals (cattle, sheep, goats, pigs)13,14. Neonates are typically colonised with C. difficile due to the lack of colonisation CA-CDI strains are genetically diverse, dominated by previously resistance afforded by mature intestinal microflora; hence preva- unidentified PCR ribotypes5. These strains often cause hospital lence decreases with age15,16. outbreaks when patients are admitted with CDI from the commu- nity. A whole genome sequencing (WGS) study of isolates from 1250 C. difficile spores contaminate retail meat and meat products patients with CDI at hospitals and in the community around Oxford, outside Australia10,17–23, ostensibly via gut contamination of car- UK, found that 45% were genetically diverse and distinct from all cases at slaughter. Food-borne transmission is possible as spores previous human cases6. Recent local studies showed a range of survive the recommended cooking temperature for ground meat unique PCR ribotypes (RTs) in humans not previously described in (718C)24. Salads and vegetables are also contaminated with – Australia or elsewhere7,8. Transmission of C. difficile has been linked C. difficile spores14,25 27. A plausible explanation for this is that to non-healthcare sources by molecular typing. In The Netherlands, C. difficile spores resist pond-based effluent treatment, the by- WGS demonstrated RT 078 (toxinotype V, NAP 7/8, REA group BK) products of which are applied to agricultural land and used in strains isolated from pigs and pig farmers were identical9. However, compost manufacture; there is evidence for this in Australian this is not surprising; RT 078 is the predominant genotype isolated livestock operations28.

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Potential sources of CDI in Australian food Conclusion production animals C. difficile is commonly isolated from food production animals in C. difficile is commonly found in Australian piglets, with 67% period Australia, although prevalence is species- and age-dependent. Cir- prevalence in a study of neonatal herds29. These rates are higher cumstantial evidence based on similarity of RT isolated from food than that reported in major pork-producing countries30–32. RT 078 animals, their effluent, and humans in the community suggests that has not been isolated from Australian piglets. Instead there is a spillover of C. difficile strains is occurring in Australia. Plausible heterogeneous mix of RTs, the majority of which (61%) have not avenues of transmission include effluent recycling and consumption been previously described in animals or humans. Piglet strains are of neonatal animals. Targeted research using highly-discriminatory overwhelmingly toxigenic (87%). Human and piglet RTs overlap but WGS is required to confirm this. One stumbling block to learning epidemiological links have not been determined. more about CDI in animals is that most diagnostic tests used for laboratory diagnosis of CDI in humans do not perform well in Suckling piglets are not slaughtered for meat on a large scale, so the animals42. Further work is required to address this problem. risk of carcass contamination is low. Contamination of the piggery environment with C. difficile spores poses a risk for spore dissem- ination however. Spore contamination in an affected farrowing unit References is high (average: 4.08 Â 105 spores/ pen in 82% of pens) (M. Squire, 1. Khanna, S. et al. (2012) The epidemiology of community-acquired Clostridium difficile infection: a population-based study. Am. J. Gastroenterol. 107,89–95. in prep.), likely a result of high-pressure hosing of sheds using doi:10.1038/ajg.2011.398 treated liquid effluent. This is presumably true for other intensively 2. Naggie, S. et al. (2010) Community-associated Clostridium difficile infection: farmed animal settings where C. difficile is endemic and effluent experience of a veteran affairs medical center in southeastern USA. Infection 38, 297–300. doi:10.1007/s15010-010-0025-0 reuse occurs. Airborne spore dispersal and exposure of workers 3. Wilcox, M.H. et al. (2008) A case-control study of community-associated Clos- to bioaerosols could occur via pumping of raw effluent in open tridium difficile infection. J. Antimicrob. Chemother. 62,388–396. doi:10.1093/ jac/dkn163 channels, use of treated liquid effluent for flushing under-pen 4. Slimings, C. et al. (2014) Increasing incidence of Clostridium difficile infection, gutters and irrigating crops/pasture, and tunnel ventilation of Australia, 2011-2012. Med. J. Aust. 200, 272–276. doi:10.5694/mja13.11153 sheds. Manure storage facilities, compost bunds or treatment 5. Bauer, M.P. et al. (2009) Clinical and microbiological characteristics of community- onset Clostridium difficile infection in The Netherlands. Clin. Microbiol. Infect. lagoons also provide the potential for bioaerosols to disseminate 15, 1087–1092. doi:10.1111/j.1469-0691.2009.02853.x in high winds. Runoff from treatment ponds to local water courses 6. Eyre, D.W. et al. (2013) Diverse sources of C. difficile infection identified on and application of pond sludge to land are direct mechanisms of whole-genome sequencing. N. Engl. J. Med. 369, 1195–1205. doi:10.1056/ NEJMoa1216064 dispersal. 7. Foster, N.F. et al. (2014) Epidemiology of Clostridium difficile infection in two tertiary-care hospitals in Perth, Western Australia: a cross-sectional study. New fi C. dif cile prevalence in Australian cattle at slaughter ranges from Microbes New Infect. 2,64–71. doi:10.1002/nmi2.43 33 56% in veal calves <7 days of age to 1.8% in adult cattle . This is 8. Huber, C.A. et al. (2014) Surveillance snapshot of Clostridium difficile infection – higher than other cattle producing countries34 38, possibly because in hospitals across Queensland detects binary toxin producing ribotype UK 244. Commun. Dis. Intell. Q. Rep. 38, E279–E284. of differences in slaughter age. Some Australian veal is slaughtered at 9. Knetsch, C.W. et al. (2014) Whole genome sequencing reveals potential spread <7 days compared with 21 weeks of age in North America, increasing of Clostridium difficile between humans and farm animals in the Netherlands, 2002 to 2011. Euro Surveill. 19, 20954. the risk of carcass contamination with C. difficile. Recycled effluent 10. Songer, J.G. et al. (2009) Clostridium difficile in retail meat products, USA, 2007. from abattoirs processing veal calves and dairy feedlots also presents Emerg. Infect. Dis. 15, 819–821. doi:10.3201/eid1505.081071 a risk. Three toxigenic RTs predominate (77%) in veal calves in 11. Bauer, M.P. et al. (2011) Clostridium difficile infection in Europe: a hospital-based survey. Lancet 377,63–73. doi:10.1016/S0140-6736(10)61266-4 Australia: RT127, RT033 and RT126. Along with RT 078, these fi 39 12. Jhung, M.A. et al. (2008) Toxinotype V Clostridium dif cile in humans and food genotypes form part of the genetically divergent clade 5 . These animals. Emerg. Infect. Dis. 14, 1039–1045. doi:10.3201/eid1407.071641 RTs have been isolated from humans with CDI in Australia although 13. Keel, M.K. et al. (2006) The comparative pathology of Clostridium difficile- – RT033 may be underreported as it is poorly detected by commonly associated disease. Vet. Pathol. 43, 225 240. doi:10.1354/vp.43-3-225 fi 40 14. Rupnik, M. et al. (2010) Clostridium dif cile: its potential as a source of foodborne used molecular tests . disease. Adv. Food Nutr. Res. 60C,53–66. doi:10.1016/S1043-4526(10)60003-4 15. Rodriguez-Palacios, A. et al. (2013) Clostridium difficile in foods and animals: Based on a small sample, sheep and lambs present a lower risk for history and measures to reduce exposure. Anim. Health Res. Rev. 14,11–29. CDI spillover with an overall prevalence rate of 4% (lambs 6.5% and doi:10.1017/S1466252312000229 sheep 0.6%)41; however, effluent treatment and reuse on intensive 16. Weese, J.S. et al. (2010) Longitudinal investigation of Clostridium difficile shed- ding in piglets. Anaerobe 16, 501–504. doi:10.1016/j.anaerobe.2010.08.001 lamb finishing lots may present an opportunity for expansion and 17. Bouttier, S. et al. (2010) Clostridium difficile in ground meat France. Emerg. dissemination of C. difficile. Infect. Dis. 16, 733–735. doi:10.3201/eid1604.091138

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18. Indra, A. et al. (2009) Clostridium difficile: a new zoonotic agent? Wien. Klin. 35. Houser, B.A. et al. (2012) Prevalence of Clostridium difficile toxin genes in the Wochenschr. 121,91–95. doi:10.1007/s00508-008-1127-x feces of veal calves and incidence of ground veal contamination. Foodborne – 19. Von Abercron, S.M. et al. (2009) Low occurrence of Clostridium difficile in retail Pathog. Dis. 9,32 36. doi:10.1089/fpd.2011.0955 ground meat in Sweden. J. Food Prot. 72, 1732–1734. 36. Avbersek, J. et al. (2009) Diversity of Clostridium difficile in pigs and other – 20. Jöbstl, M. et al. (2010) Clostridium difficile in raw products of animal origin. animals in Slovenia. Anaerobe 15, 252 255. doi:10.1016/j.anaerobe.2009.07.004 Int. J. Food Microbiol. 138,172–175. doi:10.1016/j.ijfoodmicro.2009.12.022 37. Hoffer, E. et al. (2010) Low occurrence of Clostridium difficile in fecal samples 21. Bouttier, S. et al. (2007) Screening for Clostridium difficile in meat from French of healthy calves and pigs at slaughter and in minced meat in Switzerland. – retailers. In European Congress of Clinical Microbiology and Infectious Diseases, J. Food Prot. 73, 973 975. Munchen. 38. Costa, M.C. et al. (2011) Epidemiology of Clostridium difficile on a veal farm: 22. Rodriguez-Palacios, A. et al. (2007) Clostridium difficile in retail ground meat, prevalence, molecular characterization and tetracycline resistance. Vet. Microbiol. – Canada. Emerg. Infect. Dis. 13, 485–487. doi:10.3201/eid1303.060988 152, 379 384. doi:10.1016/j.vetmic.2011.05.014 fi 23. Weese, J.S. et al. (2009) Detection and enumeration of Clostridium difficile spores 39. Stabler, R.A. et al. (2012) Macro and micro diversity of Clostridium dif cile isolates in retail beef and pork. Appl. Environ. Microbiol. 75, 5009–5011. doi:10.1128/ from diversesources and geographical locations.PLoS One 7, e31559. doi:10.1371/ AEM.00480-09 journal.pone.0031559 fi 24. Rodriguez-Palacios, A. et al. (2011) Moist-heat resistance, spore aging, and super- 40. Androga, G.O. et al. (2015) Evaluation of the Cepheid Xpert C. dif cile/Epi and fi dormancy in Clostridium difficile. Appl. Environ. Microbiol. 77, 3085–3091. meridian bioscience illumigene C. dif cile assays for detecting Clostridium fi – doi:10.1128/AEM.01589-10 dif cile ribotype 033 strains. J. Clin. Microbiol. 53,973975. doi:10.1128/ JCM.03297-14 25. Bakri, M.M. et al. (2009) Clostridium difficile in ready-to-eat salads, Scotland. fi Emerg. Infect. Dis. 15,817–818. doi:10.3201/eid1505.081186 41. Knight, D.R. et al. (2013)Prevalence ofgastrointestinal Clostridiumdif cile carriage in Australian sheep and lambs. Appl. Environ. Microbiol. 79,5689–5692. 26. Metcalf, D. et al. (2011) Clostridium difficile in seafood and fish. Anaerobe 17, doi:10.1128/AEM.01888-13 85–86. doi:10.1016/j.anaerobe.2011.02.008 42. Knight, D.R. et al. (2014) Laboratory detection of Clostridium difficile in piglets 27. Metcalf, D.S. et al. (2010) Clostridium difficile in vegetables, Canada. Lett. Appl. in Australia. J. Clin. Microbiol. 52, 3856–3862. doi:10.1128/JCM.01225-14 Microbiol. 51,600–602. doi:10.1111/j.1472-765X.2010.02933.x 28. Squire, M.M. et al. (2011) Detection of Clostridium difficile after treatment in a two-stage pond system. In Manipulating Pig Production XIII (van Barneveld, R.J., ed), p. 215, APSA Biennial Conference, Australasian Pig Science Association. Biographies 29. Knight, D.R. et al. (2015) Nationwide surveillance study of Clostridium difficile in Michele Squire has recently completed her PhD in microbiology Australian neonatal pigs shows high prevalence and heterogeneity of PCR ribo- types. Appl. Environ. Microbiol. 81,119–123. doi:10.1128/AEM.03032-14 at The University of Western Australia. Her research focused on 30. Koene, M.G. et al. (2012) Clostridium difficile in Dutch animals: their presence, Clostridium difficile infection in neonatal piglets. characteristics and similarities with human isolates. Clin. Microbiol. Infect. 18, 778–784. doi:10.1111/j.1469-0691.2011.03651.x Daniel Knight is completing his PhD in microbiology at The 31. Chan, G. et al. (2013) A retrospective study on the etiological diagnoses of diarrhea in neonatal piglets in Ontario, Canada, between 2001 and 2010. Can. J. Vet. Res. 77, University of Western Australia. His research interests include the 254–260. molecular epidemiology of Clostridium difficile, antimicrobial 32. Susick, E.K. et al. (2012) Longitudinal study comparing the dynamics of Clostrid- resistance and One Health. ium difficile in conventional and antimicrobial free pigs at farm and slaughter. Vet. Microbiol. 157, 172–178. doi:10.1016/j.vetmic.2011.12.017 33. Knight, D.R. et al. (2013) Cross-sectional study reveals high prevalence of Tom Riley holds a Personal Chair at The University of Western Clostridium difficile non-PCR ribotype 078 strains in Australian veal calves at Australia. He has had a long standing interest in healthcare-related slaughter. Appl. Environ. Microbiol. 79, 2630–2635. doi:10.1128/AEM.03951-12 infections, particularly the diagnosis, pathogenesis and epidemiol- 34. Rodriguez-Palacios, A. et al. (2006) Clostridium difficile PCR ribotypes in calves, Canada. Emerg. Infect. Dis. 12, 1730–1736. doi:10.3201/eid1211.051581 ogy of Clostridium difficile infection.

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Clostridium perfringens extracellular toxins and enzymes: 20 and counting

Sarah A Revitt-Mills A, Julian I Rood A and Vicki Adams A,B AMonash University, 19 Innovation Drive, Clayton, Vic. 3800, Australia, Tel: +61 3 9902 9139, Fax: +61 3 9902 2222 BEmail: [email protected]

Clostridium perfringens is a Gram-positive, anaerobic bac- toxins (alpha, beta, epsilon and iota)4,9. This typing scheme is now terium that is widely distributed in the environment; it is very much outdated, but it has been useful for classification as the found in soil and commonly inhabits the gastrointestinal different toxinotypes are often associated with specific diseases4,10 tract of humans and animals1,2. The ubiquitous nature of (Table 1). For example, clostridial myonecrosis and human food this bacterium has resulted in it becoming a major cause of poisoning are associated with type A strains, whereas type B, C histotoxic and enteric diseases3. The success of C. perfrin- and D strains are most strongly associated with enteric diseases of gens as both a pathogen and a commensal bacterium lies in livestock4. its ability to produce a large number of potent toxins and extracellular enzymes4. This diverse toxin repertoire results Toxins and toxin gene location in a broad range of diseases including gas gangrene, various The number of characterised C. perfringens toxins is ever enterotoxaemias, food poisoning and necrotic enteritis4–6. increasing; with more than 20 different toxins and enzymes classi- Since 2007, six new toxins have been identified, adding to the fied to date, see Table 13,5,9,11. With a few important exceptions, – ever-increasing range of potential C. perfringens virulence these toxins are encoded on large conjugative plasmids4,10,12 18, determinants. This paper briefly reviews the plethora of which allows for potential toxin gene transfer between different toxins and extracellular enzymes produced by C. perfrin- C. perfringens strains in the gastrointestinal tract and may prolong gens, highlighting their importance in disease and strain disease10. C. perfringens utilises chromosomally encoded toxins, classification as well as introducing the latest additions to such as alpha-toxin and perfringolysin O, during human histotoxic the ever increasing C. perfringens toxin family. infections or human food poisoning (C. perfringens enterotoxin, CPE)3. However, for reasons that are probably related to disease Toxinotype strain classification epidemiology, plasmid-encoded toxins are critical for non-food- Like many clostridial species, the virulence of C. perfringens is borne human gastrointestinal diseases, human enteritis necroticans dependent on the production of toxins7. Not all toxins are produced and gastrointestinal diseases of animals3,10. by any one strain, instead individual isolates vary in toxin carriage and production8. This toxin expression profile forms the basis of a The toxin categories of C. perfringens toxinotype classification scheme; in which strains are classified into The toxins of C. perfringens can be functionally classified into four five toxinotypes (A–E) based on the production of four different broad categories: membrane damaging enzymes, pore-forming

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Table 1. Properties of Clostridium perfringens toxins. Toxin/ Gene Activity Associated disease Gene location Alpha-toxin plc or cpa Phospholipase C and CM: humans and animals Chm Sphingomyelinase

Beta-toxin cpb Pore-forming toxin NE in humans and animals Plasmid

Epsilon-toxin etx Pore-forming toxin ET in sheep and goats Plasmid

Iota-toxin iap/ibp Actin-specific ADP E in sheep and cattle, ET in Plasmid ribosyltransferase rabbits

Enterotoxin cpe Pore-forming toxin C and E in domestic ungulates, Chm or Plasmid FP and GI in humans

Theta-toxin or perfringolysin O pfoA Pore-forming toxin, cholesterol- CM: humans and animals Chm dependent cytolysin

Beta2-toxin cpb2 Putative pore-forming toxin No confirmed association with Plasmid disease

TpeL tpeL Ras-specific mono- No confirmed association with Plasmid glucosyltransferase disease

NetB netB Pore-forming toxin NE in poultry Plasmid

BecA, BecB becA/B Actin-specific ADP- GE in humans Plasmid ribosyltransferase

NetE netE Putative pore-forming toxin No confirmed association with Plasmid disease

NetF netF Pore-forming toxin HE and NEC of dogs and foals Plasmid

NetG netG Putative pore-forming toxin No confirmed association with Plasmid disease

NanI nanI Sialidase Accessory role Chm

Kappa-toxin colA Collagenase No confirmed association with Chm disease

Mu-toxin nagH Hyaluronidase No confirmed association with Chm disease

Lambda-toxin lam No confirmed association with Plasmid disease

a- ccp No confirmed association with Chm disease

NanJ nanJ Sialidase No confirmed association with Chm disease

Delta-toxin cpd Pore-forming toxin No confirmed association with Plasmid disease

Chm, denotes a chromosomal gene location; CM, clostridial mynecrosis; C, colitis; E, enteritis; FP, food poisoning; GI, non-food borne gastrointestinal disease; NE, necrotising enteritis; ED, enteric disease; ET, enterotoxaemia; GE, gastroenteritis; HE, haemorrhagic enteritis; NEC, necrotising enterocolitis).

toxins, intracellular toxins, and hydrolytic enzymes4. Membrane comprise the largest toxin category and function to disrupt mem- damaging toxins such as alpha-toxin are enzymes that damage target brane permeability and ion transport by inserting into the mem- cell membranes through their ability to breakdown the constituents brane and forming a permeable channel or pore20. This category of the mammalian cell membrane19. The pore-forming toxins includes toxins such as perfringolysin O, beta-toxin, CPE, NetB and

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epsilon-toxin. Intracellular toxins, such as TpeL, BEC and iota-toxin, extracellular enzymes to cause a myriad of diseases. Note that the are internalised into target host cells where they act to disrupt the primary function of these toxins is most likely not to cause disease, cellular cytoskeleton21. Hydrolytic enzymes, such as sialidases and but to provide nutrients for the growth of C. perfringens cells, which hyaluronidases, are secreted by C. perfringens and degrade surface have limited capability to synthesise amino acids and essential associated glycans or glycoproteins4,22. These enzymes are not co-factors. Many of the toxins that are crucial contributors to disease, essential for disease; however, they may still contribute to the for example NetB, are encoded on conjugative plasmids10, and overall virulence of the bacterium23. therefore may be readily disseminated to other strains. Recent findings support the theory that C. perfringens strains have a tight Recent toxin discoveries association with the species in which they cause disease; for exam- In recent years, the number of characterised C. perfringens toxins ple, NetB-producing strains in birds and NetF-producers in foals and has increased significantly. The latest editions to the C. perfringens dogs. Just about everywhere we look, if there is an unclassified arsenal are the six novel toxins or putative toxins: NetB, BEC, TpeL, disease from which lots of C. perfringens cells can be isolated, the NetE, NetF and NetG. chances are good that another toxin is waiting to be discovered ... stay tuned! NetB is a beta-barrel pore-forming toxin and, like many other C. perfringens toxins, it is encoded on large conjugative plasmids24,25. Since its discovery, NetB-encoding plasmids have been identified in Acknowledgements many avian necrotic enteritis isolates; and netB deletion studies SAR-M is the recipient of an Australian Postgraduate Scholarship. have indicated that NetB toxin, is required for the development of necrotic enteritis in chickens26. References BEC is a novel binary toxin, composed of two distinct components, 1. McClane, B. et al. (2013) Clostridium perfringens.InFood microbiology: funda- BECa and BECb and it appears to function in a similar fashion to iota- mentals and frontiers, Fourth edn, pp. 465–489. ASM Press, Washington, DC. toxin15, with the BECa component having actin-specific ADP-ribo- 2. McClane, B.A. (2014) Clostridium perfringens.InEncyclopedia of Toxicology, Third edn (Wexler, P., ed.), pp. 987–988. Academic Press. syltranferase activity. BEC was discovered after two unrelated food 3. Uzal, F.A. et al. (2014) Towards an understanding of the role of Clostri- poisoning outbreaks that were caused by C. perfringens strains that dium perfringens toxins in human and animal disease. Future Microbiol. 9, did not encode CPE; the toxin typically associated with human food 361–377. doi:10.2217/fmb.13.168 poisoning15. Further studies showed that these strains produced a 4. Petit, L. et al. (1999) Clostridium perfringens: toxinotype and genotype. Trends Microbiol. 7, 104–110. doi:10.1016/S0966-842X(98)01430-9 novel binary toxin, designated as BEC, suggesting that this new toxin 5. Uzal, F.A. et al. (2010) Clostridium perfringens toxins involved in mammalian was responsible for the enteric symptoms observed during these veterinary diseases. Open Toxinology J. 2,24–42. doi:10.2174/1875414701003 020024 outbreaks15. 6. Brynestad, S. et al. (2001) Enterotoxin plasmid from Clostridium perfringens is TpeL is a member of the clostridial monoglycosyltransferase toxin conjugative. Infect. Immun. 69, 3483–3487. doi:10.1128/IAI.69.5.3483-3487.2001 7. Hatheway, C.L. (1990) Toxigenic clostridia. Clin. Microbiol. Rev. 3,66–98. family, which mediate cytotoxic effects through the glycosylation of 8. Robertson, S. et al. (2014) Bacteria: Clostridium perfringens.InEncyclopedia host cell proteins, and is related to the large toxins, TcdA and TcdB, of Food Safety (Motarjemi, Y., ed), pp. 395–402. Academic Press. 27 of Clostridium difficile . There is no definitive evidence that TpeL 9. Rood, J.I. and Cole, S.T. (1991) Molecular genetics and pathogenesis of Clostri- – is involved in disease; however, it is postulated that this toxin may dium perfringens. Microbiol. Rev. 55,621 648. 27 10. Li, J. et al. (2013) Toxin plasmids of Clostridium perfringens. Microbiol. Mol. Biol. make a contribution to virulence . Rev. 77, 208–233. doi:10.1128/MMBR.00062-12 The most recently characterised toxins are the cytotoxic pore- 11. Rood, J.I. (1998) Virulence genes of Clostridium perfingens. Annu. Rev. Micro- biol. 52, 333–360. doi:10.1146/annurev.micro.52.1.333 forming toxin NetF and the putative pore-forming toxins NetE and 12. Brynestad, S. and Granum, P.E. (2002) Clostridium perfringens and foodborne NetG, which were shown to be encoded on plasmids in isolates infections. Int. J. Food Microbiol. 74,195–202. doi:10.1016/S0168-1605(01) 00680-8 causing necrotizing gastroenteritis and necrotizing enterocolitis in 13. Bannam, T.L. et al. (2011) Necrotic enteritis-derived Clostridium perfringens 28 fi foals and dogs . There is a signi cant association between NetF- strain with three closely related independently conjugative toxin and antibiotic producing strains and enteric disease in these animals; however, the resistance plasmids. MBio 2, e00190-11. doi:10.1128/mBio.00190-11 function of NetE and NetG in disease remains to be investigated28. 14. Hughes, M.L. et al. (2007) Epsilon-toxin plasmids of Clostridium perfringens type D are conjugative. J. Bacteriol. 189,7531–7538. doi:10.1128/JB.00767-07 15. Yonogi, S. et al. (2014) BEC, a novel enterotoxin of Clostridium perfringens Conclusion found in human clinical isolates from acute gastroenteritis outbreaks. Infect. Immun. 82, 2390–2399. doi:10.1128/IAI.01759-14 The production of toxins is essential for C. perfringens-mediated 16. Nagahama, M. et al. (2015) Recent insights into Clostridium perfringens beta- disease and this bacterium utilises an arsenal of different toxins and toxin. Toxins (Basel) 7, 396–406. doi:10.3390/toxins7020396

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17. Katayama, S. et al. (1996) Genome mapping of Clostridium perfringens strains 27. Chen, J. and McClane, B.A. (2015) Characterization of Clostridium perfringens with I-CeuI shows many virulence genes to be plasmid-borne. Mol. Gen. Genet. TpeL toxin gene carriage, production, cytotoxic contributions, and trypsin sen- 251,720–726. sitivity. Infect. Immun. 83, 2369–2381. doi:10.1128/IAI.03136-14 18. Miyamoto, K. et al. (2006) Complete sequencing and diversity analysis of the 28. Gohari, I.M. et al. (2015) A novel pore-forming toxin in type A Clostridium perfrin- enterotoxin-encoding plasmids in Clostridium perfringens type A non-food- gens is associated with both fatal canine hemorrhagic gastroenteritis and fatal borne human gastrointestinal disease isolates. J. Bacteriol. 188, 1585–1598. foal necrotizing enterocolitis. PLoS One 10, e0122684. doi:10.1371/journal.pone. doi:10.1128/JB.188.4.1585-1598.2006 0122684 19. Sakurai, J. et al. (2004) Clostridium perfringens alpha-toxin: characterization and mode of action. J. Biochem. 136,569–574. doi:10.1093/jb/mvh161 Biographies 20. Popoff, M.R. (2014) Clostridial pore-forming toxins: powerful virulence factors. Anaerobe 30,220–238. doi:10.1016/j.anaerobe.2014.05.014 Sarah A Revitt-Mills is a PhD student in the Department of 21. Sakurai, J. et al. (2009) Clostridium perfringens iota-toxin: structure and function. Microbiology at Monash University and is studying conjugative toxin Toxins (Basel) 1,208–228. doi:10.3390/toxins1020208 plasmid biology as part of her PhD studies. 22. Zukaite,_ V. and Biziulevicius, G.A. (2000) Physico-chemical and catalytic properties of Clostridium perfringens hyaluronidase: an update. Anaerobe 6, 347–355. doi:10.1006/anae.2000.0356 Julian I Rood is a Professor of Microbiology at Monash University 23. Chiarezza, M. et al. (2009) The NanI and NanJ sialidases of Clostridium perfrin- and has led the field in clostridial conjugation mechanisms for gens are not essential for virulence. Infect. Immun. 77, 4421–4428. doi:10.1128/ IAI.00548-09 many years. He also studies bacterial pathogenesis, predominantly 24. Keyburn, A.L. et al. (2008) NetB, a new toxin that is associated with avian necrotic mechanisms utilised by the human and animal pathogen, Clostrid- fi enteritis caused by Clostridium per ngens. PLoS Pathog. 4, e26. doi:10.1371/ ium perfringens, and the sheep pathogen, Dichelobacter nodosus. journal.ppat.0040026 25. Parreira, V.R. et al. (2012) Sequence of two plasmids from Clostridium perfringens chicken necrotic enteritis isolates and comparison with C. perfringens conjugative Vicki Adams is a Research Fellow in the Department of Microbi- plasmids. PLoS One 7, e49753. doi:10.1371/journal.pone.0049753 ology at Monash University and has studied mobile genetic elements 26. Keyburn, A.L. et al. (2010) Association between avian necrotic enteritis and found in the clostridia, in addition to the biology of large conjugative Clostridium perfringens strains expressing NetB toxin. Vet. Res. 41, 21. doi:10.1051/vetres/2009069 toxin plasmids of Clostridium perfringens.

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Necrotic enteritis in chickens: an important disease caused by Clostridium perfringens

apicomplexan parasite), alter the gut microbiota (e.g. high protein levels in feed2), or compromise the (e.g. some viral Robert J Moore infections). It is only following the development of a better under- standing of these predisposing factors, and the application of Host-Microbe Interactions Laboratory some of them, that it has been possible to reliably induce disease School of Applied Sciences experimentally. The difficulty of consistently reproducing disease Health Innovations Research Institute held back research for a number of years but has now been largely RMIT University overcome. Bundoora, Vic. 3083, Australia Tel: +61 3 9925 7580 The generally held conceptual model for disease development Email: [email protected] hypothesised that the predisposing factors induced an expansion of the C. perfringens population in the gut resulting in higher toxin levels, gut damage, and frank disease. However, recent studies have Clostridium perfringens, a spore-forming, Gram-positive, indicated that the C. perfringens strains that are commonly present anaerobic bacterium, causes a variety of diseases throughout in low numbers within the healthy gut are generally distinct from the the animal kingdom. Each disease in each animal species strains that can go on to cause disease3,4. The origin of pathogenic tends to be caused by particular strains of C. perfringens and strains has not yet been clarified and the mechanisms driving is defined by the tissue tropism and toxin profile of the displacement of non-pathogenic strains by pathogenic strains are bacteria. In chickens toxinotype A strains cause necrotic not clear but may be partly driven by bacteriocin expression by the enteritis; a disease characterised by tissue damage to the pathogenic strains5. So, although C. perfringens population expan- proximal regions of the small intestine. In extreme cases the sion is a critical part of disease development, it appears that non- disease can be lethal but is more commonly seen as a sub- pathogenic native strains are displaced by virulent strains. Many of clinical disease that causes welfare issues and productivity the toxins that play key roles in disease development are encoded on losses within the poultry industry. The disease is currently large conjugative plasmids that have the potential to move from well controlled in Australia by good management practices strain to strain6; this may be an important factor in the emergence and, for some poultry producers, the use of antibiotics and epidemiology of disease causing strains. in the feed. However, the disease does cause significant issues in other regions including North America and Europe. (a) In Europe there was a spike of necrotic enteritis disease when antibiotics were withdrawn from animal feeds. It is probable that the disease will become more of an issue in the Australian poultry industry as in-feed antibiotic use is re- duced. Therefore, other methods of disease control are under investigation, including the development of vaccines.

Necrotic enteritis (Figure 1) has been estimated to cost the global poultry industry $2 billion per annum in control measures and 1 productivity losses . Although C. perfringens is clearly the causative (b) agent of necrotic enteritis a simple infection with the bacterium is not sufficient to induce disease. C. perfringens is a ubiquitous organism commonly found in many environments, in particular within the gastrointestinal tract (GIT) of healthy animals and humans. At low levels it causes no problems in the GIT; it is only in the face of predisposing factors that disease occurs. There are many interacting factors which can predispose birds to the devel- opment of necrotic enteritis. They can be broadly classified accord- Figure 1. Clostridium perfringens-induced necrotic enteritis lesions on ing to the effects that they have on the birds; they can directly the luminal surface of the chicken small intestine. Visible lesions can range from small focal lesion (a) to involvement of the whole intestinal damage the intestinal mucosa (e.g. infection with the Eimeria surface (b).

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A range of potential therapeutic and prophylactic treatments are References being actively developed to address the necrotic enteritis disease 1. Van der Sluis, W. (2000) Clostridial enteritis is an often underestimated problem. – burden within the global poultry industry. These include the appli- World Poult. 16,42 43. fi cation of prebiotics7, probiotics8, organic acids9, and plant extracts10 2. Wu, S.-B. et al. (2014) Two necrotic enteritis predisposing factors, dietary shmeal and Eimeria infection, induce large changes in the caecal microbiota of broiler as well as vaccines. Vaccines against other animal clostridial diseases chickens. Vet. Microbiol. 169,188–197. doi:10.1016/j.vetmic.2014.01.007 have been available for many years. Such vaccines are produced 3. Keyburn, A.L. et al. (2010) Association between avian necrotic enteritis and using toxoids; chemically inactivated culture supernatants contain- Clostridium perfringens strains expressing NetB toxin. Vet. Res. 41,21. doi:10.1051/vetres/2009069 ing the disease causing toxins and a range of other minor antigens. 4. Hibberd, M.C. et al. (2011) Multilocus sequence typing subtypes of poultry They are cheap to produce and very effective. However, satisfactory Clostridium perfringens isolates demonstrates disease niche partitioning. vaccines to protect poultry from necrotic enteritis have not been J. Clin. Microbiol. 49, 1556–1567. doi:10.1128/JCM.01884-10 commercially available. 5. Timbermont, L. et al. (2009) Intra-species growth-inhibition by Clostridium perfringens is a possible virulence trait in necrotic enteritis in broilers. Vet. The toxins produced by C. perfringens are excellent vaccine anti- Microbiol. 137, 388–391. doi:10.1016/j.vetmic.2009.01.017 gens because they are the major virulence factors responsible for 6. Bannam, T.L. et al. (2011) Necrotic enteritis-derived Clostridium perfringens with disease induction and, as secreted proteins, are readily accessible to three closely related three independently conjugative toxin and antibiotic resis- tance plasmids. MBio 2, e00190-e11. doi:10.1128/mBio.00190-11 the host immune system. For decades after the definitive descrip- 11 7. Vidanarachchi, J.K. et al. (2013) Natural plant extracts and prebiotic compounds as tion of necrotic enteritis by Parish it was thought that alpha-toxin, alternatives to antibiotics in broiler chicken diets in a necrotic enteritis challenge a toxin produced by all isolates of C. perfringens, was the main model. Anim. Prod. Sci. 53, 1247–1259. doi:10.1071/AN12374 virulence determinant. However, in 2006, we demonstrated that 8. Cao, L. et al. (2012) Reduced lesions in chickens with Clostridium perfringens- alpha-toxin was not essential for experimental disease induction12 induced necrotic enteritis by Lactobacillus fermentum 1.20291. Poult. Sci. 91, 3065–3071. doi:10.3382/ps.2012-02548 and then went on to discover and characterise the toxin, necrotic 9. Geier, M.S. et al. (2010) Comparison of alternatives to in-feed antimicrobials for enteritis toxin B-like (NetB), that does play an essential role in the prevention of clinical necrotic enteritis. J. Appl. Microbiol. 109, 1329–1338. virulence13,14. doi:10.1111/j.1365-2672.2010.04758.x 10. Lee, S.H. et al. (2013) Dietary supplementation of young broiler chickens with The belief that alpha-toxin was important in disease misdirected capsicum and turmeric oleoresins increases resistance to necrotic enteritis. vaccine efforts for many years but the recent advances made in our Br. J. Nutr. 110, 840–847. doi:10.1017/S0007114512006083 fundamental understanding of the basis of pathogenesis is now 11. Parish, W.E. (1961) Necrotic enteritis in the fowl Gallus gallus domesticus. 15–17 I. Histopathology of the disease and isolation of a strain of Clostridium welchii. enabling the development of effective vaccines . The key to J. Comp. Pathol. 71, 377–393. doi:10.1016/S0368-1742(61)80043-X fi successful vaccine design has been to ensure that there is a suf cient 12. Keyburn, A.L. et al. (2006) The alpha-toxin of Clostridium perfringens is not level of the key NetB protein to elicit a strong protective immune an essential virulence factor in necrotic enteritis in chickens. Infect. Immun. 74, – response. With efficacious experimental vaccines demonstrated 6496 6500. doi:10.1128/IAI.00806-06 the challenge for the industry now is to be able to formulate and 13. Keyburn, A.L. et al. (2008) NetB, a new toxin is associated with avian necrotic enteritis caused by Clostridium perfringens. PLoS Pathog. 4, e26. doi:10.1371/ deliver the vaccines in a cost effective and useful way. The broiler journal.ppat.0040026 (meat) chicken industry presents interesting challenges for vaccine 14. Yan, X.-X. et al. (2013) Structural and functional analysis of the pore-forming application as vaccines must be provided at very low cost and toxin NetB from Clostridium perfringens. MBio 4, e00019-e13. doi:10.1128/mBio. 00019-13 must be effective in very young birds – for instance the peak risk 15. Keyburn, A.L. et al. (2013) Vaccination with recombinant NetB toxin protects of developing necrotic enteritis is between 2 to 4 weeks of age. broiler chickens from necrotic enteritis. Vet. Res. 44, 54. doi:10.1186/1297-9716- We are continuing to address industry needs by investigating the 44-54 use of both conventional killed vaccines and live vector delivered 16. Keyburn, A.L. et al. (2013) Maternal immunization with vaccine containing vaccines for necrotic enteritis. recombinant NetB toxin partially protects progeny chickens from necrotic enter- itis. Vet. Res. 44, 108. doi:10.1186/1297-9716-44-108 17. Fernandes da Costa, S.P. et al. (2013) Protection against avian necrotic entertitis after immunisation with NetB genetic or formaldehyde toxoids. Vaccine 31, Acknowledgements 4003–4008. doi:10.1016/j.vaccine.2013.05.063 I thank my two key collaborators, Professor Julian Rood and Dr Anthony Keyburn, who have contributed so much to necrotic Biography enteritis research. The fundamental and applied research to under- Rob Moore has recently moved from CSIRO to establish a stand disease pathogenesis and apply that learning to vaccine new laboratory at RMIT University, where he is a Professor of development has been carried out within the Australian Animal Biotechnology. He is also an Adjunct Associate Professor in the Health Laboratory, CSIRO, Geelong, and the Department of Micro- Microbiology Department at Monash University and an Honorary biology, Monash University, with the support of the Poultry Coop- Fellow within the CSIRO Biosecurity Flagship at the Australian erative Research Centre, established and supported under the Animal Health Laboratory, Geelong. His research interests are in Australian Government’s Cooperative Research Centres Program, bacterial pathogenesis, vaccine design, the role of gut microbiota and the ARC Centre of Excellence in Structural and Functional in health and productivity, and the identification and development Microbial Genomics. of probiotic strains of bacteria.

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The manufacture of veterinary clostridial vaccines

toxin is a phospholipase. Its basic enzyme effect is to ‘make a hole’ in the membrane of acell by cleaving a cell membrane component.The cell contents leak out and the cell dies. The toxin of C. septicum acts in a similar way, forming a pore in the cell, again allowing the cell contents to leak. This ‘membrane-action’ seems to be a general trait of clostridial toxins. Although the specific signs of clostridial disease in animals depend on the causative species, gas formation and Robert Dempster haemolysis are common. The progression of disease is usually rapid Email: [email protected] making treatment with antibiotics impractical. This is why vaccines are so vital. A review of the important clostridial toxins is found in Hatheway3 and a good coverage of the diseases of animals caused by Clostridial vaccines are commonly used in most countries the clostridia can be found in Hungerford4. where farming of cattle, sheep, goats and horses occurs on a commercial scale. Vaccines to protect against clostridial All the clostridial vaccines sold in Australia are combination pro- fi diseases make up the second largest group of ruminant ducts. The combinations usually include up to ve clostridial spe- vaccines sold globally. In Australia the sales value of cies, however different products also include leptospiral or these vaccines makes up $46m of the $96m sheep and cattle Corynbacterium psuedotuberculosis antigens. Two products made vaccine market (Baron market data). This group of vaccines by Virbac combine a six-way vaccine with an anthelmintic (deworm- has become so ubiquitous, and competition between com- ing chemical) to give sheep producers the added convenience of petitors so fierce, that they have been reduced to the status vaccinating and deworming in one shot. In South America some of commodities where they can sell for less than 20c/dose. cattle vaccines contain up to 11 different antigens. Traditionally fi However, this definition does not do justice to the enormous sheep and cattle are vaccinated twice in the rst year of life and then value they have generated for many decades ‘behind the receive annual boosters. farm gate’. Production of clostridial vaccines involves growing the organism in The clostridials grown for veterinary vaccines include some species fermenters, configured for anaerobes, to produce large amounts of important in human health viz: tetanus (C. tetani) and botulism toxin. Scale-up starts witha ‘seed’vial taken fromliquid nitrogen and (C. botulinum). C. perfringens D is of veterinary importance but grown up through several stages to the terminal fermenter, which it has a ‘close cousin’, i.e. C. perfringens type A, which was for Virbac has a capacity of 6000 L. Each species usually has a specific responsible for the deaths of so many soldiers in the trenches media and set of fermentation conditions. This ‘know-how’ has during WW1. However, added to those species of human impor- been developed in-house over many years to give good toxin yields. tance are other species of mainly veterinary importance that can However, despite high levels of quality compliance and good be found in vaccines sold in Australia, i.e. C. septicum, C. noyvi B and manufacturing practice (GMP) yields of toxin from fermentation C. chauvoei. Other species such as C. sordellii and C. haemolyticum batches still vary. This yield variability is common to our industry but and other members of the C. perfringens group complete the list is a frustration especially to the planners and the accountants. After of clostridials of global veterinary significance. toxin production has peaked, formalin is added to start the detox- ification and inactivation process, turning toxins into toxoids. The Members of the genus Clostridium are ubiquitous and are found in formalin also kills the live bacteria. Following centrifugation to soil and in the gut of animals. Infection can be triggered by a variety remove whole cells, and diafiltration across an ultrafilter, the con- of conditions ranging from a change in diet through to shearing cuts centrated ‘antigen’ is then stored, waiting to be blended into and dog bites1. Toxins produced by the clostridia are responsible vaccines. For some antigens a whole cell culture is used in the final for much of the typical pathology. C. perfringens alpha-toxin is ‘blend’. an enzyme, but this is the exception not the rule. Most of the other clostridial pore-forming toxins, such as C. septicum alpha-toxin and Antigens can be stored in a sterile concentrated stage for C. perfringens epsilon-toxin are not enzymes2. C. perfringens alpha several years. Samples are taken at various stages throughout the

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processing to measure toxoid yield and to confirm non-viability and the Therapeutic Goods Administration (TGA). Compliance, audits, non-toxicity. standard operating procedures, specifications, change controls, validation and qualification are a necessary part of our industry that Vaccines are blended based on registered inputs of toxoids which we live with day to day. This brings a high degree of rigour and form part of the vaccine’s registration dossier. Adjuvants are added control to those who work in this industry. to non-specifically stimulate the immune response. At Virbac the adjuvants we use are aluminium salts or oil, depending on the References duration of immunity required. Following blending, vaccines 1. Songer, J.G. (1996) Clostridial enteric diseases of domestic animals. Clin. Microbiol. are aseptically filled, labelled and packed. Each batch of vaccine is Rev. 9, 216–234. – tested for potency via antibody responses in rabbits and a battery of 2. Popoff, M.R. (2009) Bouvet P. Clostridial toxins. Future Microbiol. 4, 1021 1064. doi:10.2217/fmb.09.72 chemical tests is also conducted. 3. Hatheway, C.L. (1990) Toxigenic clostridia. Clin. Microbiol. Rev. 3,66–98. 4. Hungerford, T.G. (1990) Diseases of Livestock. Ninth edition. McGraw-Hill Book The basic technology used to make clostridial vaccines has not Company, Sydney. changed in many years. In fact the concept of using formalin to 5. Ebisawa, I. (1987) The encounter of Gaston Ramon (1886–1963) with formalin: a biographical study of a great scientist. Kitasato Arch. Exp. Med. 60,55–70. detoxify toxins produced by bacteria and the use of aluminium and oil as vaccine adjuvants, date back to the work of Ramon in Biography the 1920s5. Dr Robert Dempster is the R&D and Licensing manager at Virbac The veterinary vaccine world is highly regulated and products must Australia. He holds a MSc in microbiology and PhD in pathology. be made and tested in compliance with the respective registration Virbac is a global animal health company. Robert has worked for dossier and the rules and regulations of Good Manufacturing eight local and international animal health companies in the past Practice. In Australia the veterinary industry is regulated by the 28 years. He has held a number of technical and management roles Agricultural Pesticides and Veterinary Medicines Authority (APVMA) but has spent most of his time working with vaccines for sheep and much in the same way the human medicine industry is regulated by cattle.

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Anaerobic spirochaetes and animals

David J Hampson A,B, Nyree D Phillips A,C and Tom LaA,D ASchool of Veterinary and Life Sciences, Murdoch University, Murdoch, WA 6150, Australia, Tel: +61 8 9360 2287, Fax: +61 8 9360 3130 BEmail: [email protected] CEmail: [email protected] DEmail: [email protected]

Anaerobic spirochaetes colonise the large intestine of many various species are classified as being either strongly or weakly avian and mammalian host species. The most well known haemolytic (Figure 1a). Under a phase contrast microscope indi- pathogenic species is the strongly haemolytic vidual spirochaetes are seen as slender rods, usually with two or hyodysenteriae that was first isolated from pigs with swine three undulating curves (Figure 1b). The cells stain weakly Gram (SD) in the early 1970s. Classical SD is a severe negative, and are easier to see using silver stains. There are relatively mucohaemorrhagic colitis that occurs in growing pigs and is few defining and consistent phenotypic differences between the endemic in most pig-rearing areas of the world. The spiro- seven officially named and other proposed Brachyspira species. chaete acts in concert with other components of the colonic Identification to the species level is important as some species are microbiota to disrupt the integrity of the colonic epithelium pathogenic and some are not. Identity is usually achieved using and induce inflammation. In recent years two new strongly species-specific PCRs directed at pathogenic species, or by sequenc- haemolytic species, the proposed ‘Brachyspira suanatina’ ing genes such as the NADH oxidase gene and constructing phy- and ‘Brachyspira hampsonii’, both with reservoirs in logenetic trees1,2. PCRs can be used on primary growth off the migratory water birds, have been described as new and isolation plates or in DNA extracted from faeces. Multilocus emerging agents of SD in the northern hemisphere. Weakly sequence typing has been developed to assist with species identi- haemolytic species also have been described, some of which fication as well as for sub-specific differentiation of isolates and have pathogenic potential. In particular Brachyspira pilo- recognition of clonal groups3. sicoli causes a mild colitis and diarrhoea in many species, including human beings, whilst Brachyspira intermedia is Swine dysentery a common pathogen in adult poultry. Infection with Swine dysentery (SD) manifests as a severe mucohaemorrhagic B. intermedia and/or B. pilosicoli can cause wet litter, faecal colitis of grower and finisher pigs (Figure 2). The disease is endemic staining of eggshells and delays in reaching peak egg in many countries around the world, including Australia. Although production. Options for control of these widespread and clinical manifestations can be controlled by the use of a small economically significant anaerobic infections are limited. number of antimicrobial agents that are licensed for use in pigs, resistance to these drugs by strains of the causative agents as Isolation and characterisation observed both in vitro and in vivo is a growing problem worldwide. Brachyspira species take at least 3–5 days to appear as a thin film of Detecting the condition and limiting the spread of infection surface growth on selective blood agar that is incubated at 37–418C between farms remains a major fundamental for control. Subclinical in an anaerobic environment. Lack of colony formation can cause a carriage of the pathogen can present challenges for controlling number of technical difficulties when manipulating cultures. The transmission. The classical agent of SD is the strongly haemolytic

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(a)(b) Figure 1. Brachyspira. (a) Growth on blood agar showing weak haemolysis on the top (Brachyspira innocens) and strong haemolysis on the bottom (Brachyspira hyodysenteriae). (b) Phase contrast microscopic appearance of Brachyspira species strains.

Figure 3. End-on attachment of Brachyspira pilosicoli to colonic Figure 2. Mucohaemorrhagic colitis in a pig with swine dysentery. enterocytes (arrow).

Brachyspira hyodysenteriae, but in the last decade two newly of colonic enterocytes (Figure 3). Brachyspira aalborgi can cause described strongly haemolytic species, proposed as ‘Brachyspira this manifestation in humans, but in birds and mammals (including suanatina’4 and ‘Brachyspira hampsonii’5, have been identified human beings) the agent of IS is the weakly haemolytic Brachyspira as emerging causes of SD. Both species appear to have reservoirs pilosicoli7. This species is widely distributed, and is a particular in migratory water birds6. To date description of ‘B. suanatina’ clinical problem where individuals live in close proximity, such as has been limited to Scandinavia, whilst ‘B. hampsonii’ has been with intensively reared pigs, caged adult chickens, or villagers in described as an emerging clinical problem in pigs in North America developing communities where basic hygiene is compromised. and Europe. Problems also arise where production animals or birds that are raised outside are exposed to water or soil that has been contam- Intestinal spirochaetosis inated by faeces (for example from wild water birds, which fre- Adefining but inconsistent feature of intestinal spirochaetosis (IS) is quently carry B. pilosicoli). In pigs the infection can cause colitis and the presence of spirochaetes attached by one cell end to the surface diarrhoea in groups of recently weaned or growing animals,

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Control of infection within a geographical area (by State authorities or private/company veterinarians) still largely rests on provision of accessible, reliable diagnostic services and routine surveillance. When coupled with implementation of strong biosecurity measures the transmission of infection between farms can be reduced or prevented.

References 1. Atyeo, R.F. et al. (1999) Differentiation of Serpulina species by NADH oxidase (nox) gene comparisons and nox-based polymerase chain reaction tests. Vet. Microbiol. 67,47–60. doi:10.1016/S0378-1135(99)00030-9 2. Song, Y. and Hampson, D.J. (2009) Development of a multiplex qPCR for detection Figure 4. Faecal staining of eggshells from a chicken with avian intestinal and quantitation of pathogenic intestinal spirochaetes in pigs and chickens. Vet. spirochaetosis. Microbiol. 137, 129–136. doi:10.1016/j.vetmic.2008.12.020 3. La, T. et al. (2009) Multilocus sequence typing as a tool for studying the molecular resulting in poor and uneven growth rates that reduce the profit- epidemiology and population structure of Brachyspira hyodysenteriae. Vet. – ability of farming enterprises. Infections in dogs and horses also have Microbiol. 138, 330 338. doi:10.1016/j.vetmic.2009.03.025 been described. 4. Råsbäck, T. et al. (2007) A novel enteropathogenic, strongly haemolytic spiro- chaete isolated from pig and mallard, provisionally designated ‘Brachyspira suanatina’ sp. nov. Environ. Microbiol. 9,983–991. doi:10.1111/j.1462-2920. Avian intestinal spirochaetosis 2006.01220.x The outcome of infection with Brachyspira species in adult chick- 5. Chander, Y. et al. (2012) Phenotypic and molecular characterization of a novel strongly hemolytic Brachyspira species, provisionally designated ‘Brachyspira ens (laying and breeding hens) and other poultry species has been hampsonii’. J. Vet. Diagn. Invest. 4, 903–910. 8 called avian intestinal spirochaetosis (AIS) . The condition does not 6. Rubin, J.E. et al. (2013) Isolation and characterization of Brachyspira spp. seem to occur naturally in young meat producing chickens (broi- including ‘Brachyspira hampsonii’ from lesser snow geese (Chen caerulescens lers), perhaps because there is insufficient opportunity and time for caerulescens) in the Canadian Arctic. Microb. Ecol. 66,813–822. doi:10.1007/ s00248-013-0273-5 colonisation to occur. AIS has been associated with reductions and 7. Trott, D.J. et al. (1996) Serpulina pilosicoli sp. nov., the agent of porcine intestinal delays in egg production, foamy brown faeces, wet litter, and faecal spirochetosis. Int. J. Syst. Bacteriol. 46, 206–215. doi:10.1099/00207713-46-1-206 staining of eggshells resulting in downgrading of valuable table eggs 8. McLaren, A.J. et al. (1997) Genetic and phenotypic characterization of intestinal for use in manufacturing (Figure 4). The two main (weakly haemo- spirochetes colonizing chickens, and allocation of known pathogenic isolates to – lytic) species that have been implicated in AIS are Brachyspira three distinct genetic groups. J. Clin. Microbiol. 35,412 417. 9. Song, Y. et al. (2009) A reverse vaccinology approach to swine dysentery vaccine intermedia and B. pilosioli, although B. alvinipulli occasionally development. Vet. Microbiol. 137, 111–119. doi:10.1016/j.vetmic.2008.12.018 has been reported to be involved in the condition in the USA and 10. Pluske,J.R.et al. (1998) Confirmation of therole of non-starchpolysaccharides and Europe. AIS appears to be widespread throughout the world, resistant starch in the expression of swine dysentery in pigs following experimental including in Australia, but the condition is only infrequently diag- infection. J. Nutr. 128, 1737–1744. nosed. This is probably because of the relatively non-specific nature 11. Hampson, D.J. et al. (2000) Influences of diet and vaccination on colonization of fi pigs with the intestinal Brachyspira (Serpulina) pilosicoli. Vet. of the signs, and the dif culty in isolating and identifying the Microbiol. 73,75–84. doi:10.1016/S0378-1135(99)00200-X causative anaerobic Brachyspira species without the availability of 12. Phillips, N.D. et al. (2004) A wheat-based diet enhances colonisation with the specialised diagnostic media, facilities and expertise. intestinal spirochaete Brachyspira intermedia in experimentally-infected laying hens. Avian Pathol. 33,451–457. doi:10.1080/0307945042000260620 Control Without effective disease control the health and welfare of affected Biographies animals can be compromised. Infections with the anaerobic Bra- Dr David Hampson is a veterinarian who is Professor of chyspira species can be suppressed by antimicrobial agents such as Veterinary Microbiology and Dean of the School of Veterinary and metronidazole, but these drugs are no longer available for use in Life Sciences at Murdoch University. He has maintained a special production animals. Indeed, relatively few drugs are registered for interest in Brachyspira species for most of his academic career. production animal species, and resistance to the remaining drugs by Dr Nyree Phillips completed her PhD on avian intestinal spiro- strains of the various Brachyspira species is an increasing problem. chaetosis at Murdoch University in 2006. Nyree is a Postdoctoral Currently there are no effective commercially vaccines available, Fellow at Murdoch University and works mainly on the diagnosis and although new recombinant vaccines are being developed for control control of Brachyspira infections in pigs and poultry. of SD9. Modifications to the diet fed to both pigs and chickens have been shown to change the colonic microenvironment and influence Dr Tom La completed his PhD on swine dysentery vaccine devel- – and potentially reduce colonisation by Brachyspira species10 12, opment at Murdoch University in 2006. Tom is a Postdoctoral Fellow but these diets are expensive and too specialised to be commercially at Murdoch University and works on diagnostics and vaccine de- viable for routine use in animals. velopment for Brachyspira species.

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Anaerobic microorganisms and bioremediation of organohalide pollution

Matthew Lee A, Chris Marquis A, Bat-Erdene Judger A and Mike Manefield A,B ASchool of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia, Tel: +61 2 9385 1780 BEmail: manefi[email protected]

Organohalide pollution of subsurface environments is ubiq- manufacture of polyvinyl chloride (PVC), perchloroethene (PCE) as uitous across all industrialised countries. Fortunately, strict- dry cleaning solvent and trichloromethane (chloroform; CF) as ly anaerobic microorganisms exist that have evolved using precursor chemical in the production of chlorofluorocarbon refrig- naturally occurring organohalides as their terminal electron erant gases. Because of their utility, these compounds have been acceptor. These unusual organisms are now being utilised to synthesised in enormous quantities. For example PCE production clean anthropogenic organohalide pollution. in the USA peaked at 320 million kg per annum in the 1980s2. Industrial activity on such a large scale has resulted in the discharge Subsurface water systems are a precious resource particularly in of organohalides into the environment, where because of their high Australia where they are used for agricultural, industrial and do- density and limited water solubility, tend to reside as solvent pools mestic purposes including for drinking. However, a large range of within subsurface water systems. These subsurface solvent pools are organic and inorganic pollutants often compromise this precious known as dense non-aqueous phase liquid (DNAPL) source zones. resource, such as nitrates from overuse of fertilisers, heavy metals The DNAPL slowly dissolves into the surrounding water creating a from the mining industry and BTEX compounds from petroleum toxic solvent plume radiating out from the source zone in the spillages. direction of the groundwater flow. To give some perspective on Bioremediation of organohalides has garnered a significant amount the extent of organohalide pollution, of the 1319 priority contam- of research interest over the past 20 years1.Organohalides are a large inated sites listed by the USA-EPA, 819 (61%) of them are polluted family of compounds ranging from simple C1 and C2 aliphatics to with PCE3. complicated polyaromatic structures such as dioxins (Figure 1). The chemical and physical characteristics of these compounds have Fortunately, strictly anaerobic bacteria exist that have evolved to enabled them to be used in a large range of applications. Some utilise organohalides as terminal electron acceptors. These organ- of the more notable examples include: vinyl chloride (VC) in the isms predate anthropogenic organohalide production and owe

Figure 1. Examples of organohalides that pollute Australian waterways. From left to right: perchloroethene (PCE); trichloromethane (chloroform; CF); and 2,3,7,8-tetrachlorobenzodioxin (TCDD).

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their existence to a wide range of naturally occurring organohalides Desulfuromonas and Dehalobacter were shown to be capable of in the environment4. Their metabolism results in the elimination of a partial dechlorination of PCE and TCE to cis-DCE and the highly halogen atom, which ultimately yields a much less toxic hydrocar- toxic carcinogen VC9–13 (Figure 3). bon. Organohalide respiring bacteria (ORB) perform the dehalo- For bioremediation, the production of partially chlorinated toxic genation reaction by virtue of enzymes known as reductive daughter compounds is an undesirable outcome. Therefore, the dehalogenases that are constructed around a cobalt-containing discovery of mccartyii strain 19514, which could cobalamin at the . The cobalt atom in its reduced transform vinyl chloride to ethene, was extremely important, as form is a strong nucleophile that attacks the carbon atom in a this made bioremediation of chlorinated ethanes and ethenes a carbon-halogen bond. In most ORB, the electrons that are ultimately viable option. Dehalococcoides strains since that time have become transferred to the organohalide are acquired from the oxidation of synonymous with bioremediation because of their versatility. Deha- hydrogen. The electrons are transferred via a series of membrane lococcoides strains harbor up to 32 reductive dehalogenase homo- associated quinones and cytochrome-like proteins to the reductive logs that are capable of dehalogenating not only halogenated C2 dehalogenase (RdhA). The transfer of electrons generates a proton aliphatics, but also halogenated aromatic compounds such as hexa- 5 motive force and subsequently ATP (Figure 2). – chlorobenzene and chlorinated dioxins15 17.

The first ORB reported about 30 years ago transformed 3-chloro- Dehalococcoides resides in the Chloroflexi, to which the benzoic acid to benzoic acid6. However, the greatest advance class Dehalococcoidetes was added to accommodate organohalide in terms of organohalide bioremediation was the discovery of respiring members18. Dehalococcoides cells morphologically are PCE-dechlorinating mixed cultures in the mid 1980s. These unusual being disc shaped and only 0.3–1 mm in diameter and cultures produced various daughter compounds ranging from 0.2 mm thick. Another unusual feature of Dehalococcoides is the cis-dichloroethene and VC to ethane7,8. Isolation and characterisa- apparent lack of layer in the cell-wall a feature typical tion of organisms capable of utilising PCE and trichloroethene (TCE) of most bacteria18. Electron micrographs have revealed an unusual as terminal electron acceptors revealed the mechanism behind the cell-wall S-layer resembling that found in archaea19. Dehalococ- dechlorination reactions observed in the mixed cultures. Strains coides like most ORB and other strict anaerobes operate only at very from genera including Dehalospirillium, Desulfitobacterium, low redox potentials (<110 mV)18. Therefore, minute traces of

Figure 2. A typical membrane associated electron transport system in ORB involved in the oxidation of molecular hydrogen and subsequent reduction of an organohalide. The proton motive force generated across the cytoplasmic membrane is used to synthesise ATP. MBH, membrane bound hydrogenase; MQ, menaquinone; RdhB, membrane anchor protein; RdhA, reductive dehalogenase (catalytic subunit).

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Cl H

HCI H Cl H2 HCI

H2

Cl Cl H HCI Cl H H H H 2 H H2 HCI

Cl Cl HCI Cl Cl H2 HCI H Cl H H

H2 H H

Cl Cl Figure 3. The transformation of the dry cleaning solvent (PCE) to the innocuous hydrocarbon ethene. Several genera of bacteria can capable of transforming PCE to cis-DCE or vinyl chloride; however, only Dehalococcoides strains are capable of complete dechlorination to ethene. oxygen quickly curtail dehalogenating activity. This presents a these compounds as terminal electron acceptors in a process called difficulty in cultivating these types of organisms under laboratory organohalide respiration. These organisms have been exploited to conditions, where equipment and chemicals must be scrupulously remediate sub-surface environments that have been polluted with deoxygenated in anaerobic chambers or with chemical reducing large quantities of anthropogenic organohalides. Research over the agents before they can be used. past 20 years has revealed numerous genera that are capable of organohalide respiration, with a large array of reductive dehalo- Over the past 20 years numerous obligate and facultative ORB have genases that can tackle most organohalides found at polluted sites. been reported, and between them, these bacteria can use the vast However, knowledge gaps still exist around how ORB interact with majority of anthropogenic chlorinated ethanes and ethenes as sole other cohabiting microorganisms such as those supplying hydrogen electron acceptors (reviewed in Koenig et al.3). However, in reality, and cobalamin cofactors. many organohalide-polluted sites contain more than one haloge- nated compound, which poses a challenge for ORB. The presence of chlorinated methanes in the solvent plume has been problematic, as they are strong inhibitors of most anaerobic microbial processes References 20,21 including organohalide respiration . CF is particularly problem- 1. Lovley, D.R. (2003) Cleaning up with genomics: applying molecular biology to – atic as it is a widespread pollutant and highly recalcitrant, with a half- bioremediation. Nat. Rev. Microbiol. 1,35 44. doi:10.1038/nrmicro731 2. Doherty, R.E. (2000) A history of the production and use of carbon tetrachloride, life of 3500 years in groundwater in the absence of appropriate tetrachloroethylene, trichloroethylene and 1, 1, 1-trichloroethane in the United microbes22. Until recently, the presence of CF has required site States: part 2–trichloroethylene and 1, 1, 1-trichloroethane. Environ. Forensics 1, 83–93. doi:10.1006/enfo.2000.0011 pretreatment for its removal, or the employment of abiotic strate- 3. Koenig, J. et al. (2015) Aliphatic organochlorine degradation in subsurface “ ” gies such as pump and treat or the installation of permeable environments. Rev. Environ. Sci. Biotechnol. 14,49–71. doi:10.1007/s11157- reactive iron barriers23. However, recent discoveries of CF respiring 014-9345-3 4. Gribble, G.W. (1992) Naturally occurring organohalogen compounds–a survey. Dehalobacter and Desulfitobacterium strains that transform CF J. Nat. Prod. 55, 1353–1395. doi:10.1021/np50088a001 to dichloromethane (DCM) have opened many CF-impacted sites 5. Mohn, W.W. and Tiedje, J.M. (1992) Microbial reductive dehalogenation. Micro- – to bioremediation strategies24 26. Although DCM is not a favourable biol. Rev. 56, 482–507. intermediate, recently Dehalobacter strains have been identified 6. DeWeerd, K.A. et al. (1990) Desulfomonile tiedjei gen. nov. and sp. nov., a novel anaerobic, dehalogenating, sulfate-reducing bacterium. Arch. Microbiol. 154, that can use DCM as a sole source of organic carbon and electrons, 23–30. doi:10.1007/BF00249173 producing acetate, hydrogen and carbon dioxide. Both the CF 7. Vogel, T.M. and McCarty, P.L. (1985) Biotransformation of tetrachloroethylene to trichloroethylene, dichloroethylene, vinyl chloride, and carbon dioxide under respiring and DCM fermenting strains have been Dehalobacter methanogenic conditions. Appl. Environ. Microbiol. 49,1080–1083. shown to work together to effect complete dechlorination of 8. DiStefano, T.D. et al. (1992) Hydrogen as an electron donor for dechlorination of CF26,27. tetrachloroethene by an anaerobic mixed culture. Appl. Environ. Microbiol. 58, 3622–3629. In conclusion, in subsurface environments where oxygen is in 9. Scholz-Muramatsu, H. et al. (1995) Isolation and characterization of Dehalospir- illum multivorans gen. nov., sp. nov., a tetrachloroethene-utilizing, strictly anaer- limited supply, microorganisms have evolved to use a multitude obic bacterium. Arch. Microbiol. 163,48–56. doi:10.1007/BF00262203 of alternative electrons acceptors for energy conservation. Organo- 10. Gerritse, J. et al. (1996) Desulfitobacterium sp. strain PCE1, an anaerobic halides are naturally occurring molecules in which the carbon halide bacterium that can grow by reductive dechlorination of tetrachloroethene or ortho-chlorinated phenols. Arch. Microbiol. 165,132–140. doi:10.1007/s0020300 bond is in a high oxidation state, hence bacteria have evolved to use 50308

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11. Krumholz, L.R. (1997) Desulfuromonas chloroethenica sp. nov. uses tetrachlor- 23. Lee, M. et al. (2015) Relative Contributions of Dehalobacter and Zerovalent oethylene and trichloroethylene as electron acceptors. Int. J. Syst. Bacteriol. 47, Iron in the Degradation of Chlorinated Methanes. Environ. Sci. Technol. 49, 1262–1263. doi:10.1099/00207713-47-4-1262 4481–4489. doi:10.1021/es5052364 12. Miller, E. et al. (1997) Comparative studies on tetrachloroethene reductive 24. Ding, C. et al. (2014) A Desulfitobacterium sp. strain PR reductively dechlorinates dechlorination mediated by Desulfitobacterium sp. strain PCE-S. Arch. Microbiol. both 1,1,1-trichloroethane and chloroform. Environ. Microbiol. 16, 3387–3397. 168,513–519. doi:10.1007/s002030050529 doi:10.1111/1462-2920.12387 13. Holliger, C. et al. (1998) Dehalobacter restrictus gen. nov. and sp. nov., a strictly 25. Grostern, A. et al. (2010) Chloroform respiration to dichloromethane by a anaerobic bacterium that reductively dechlorinates tetra-and trichloroethene in Dehalobacter population. Environ. Microbiol. 12, 1053–1060. doi:10.1111/ an anaerobic respiration. Arch. Microbiol. 169, 313–321. doi:10.1007/s0020300 j.1462-2920.2009.02150.x 50577 26. Lee, M. et al. (2012) Complete chloroform dechlorination by organochlorine 14. Maymó-Gatell, X. et al. (2001) Reductive dechlorination of cis-1, 2-dichloroethene respiration and fermentation. Environ. Microbiol. 14,883–894. doi:10.1111/ and vinyl chloride by ‘Dehalococcoides ethenogenes’. Environ. Sci. Technol. 35, j.1462-2920.2011.02656.x – 516 521. doi:10.1021/es001285i 27. Justicia-Leon, S.D. et al. (2014) Bioaugmentation with distinct Dehalobacter 15. Adrian, L. et al. (2000) Bacterial dehalorespiration with chlorinated benzenes. strains achieves chloroform detoxification in microcosms. Environ. Sci. Technol. Nature 408,580–583. doi:10.1038/35046063 48, 1851–1858. doi:10.1021/es403582f 16. Bunge, M. et al. (2003) Reductive dehalogenation of chlorinated dioxins by an anaerobic bacterium. Nature 421,357–360. doi:10.1038/nature01237 17. Kube, M. et al. (2005) Genome sequence of the chlorinated compound–respiring Biographies bacterium Dehalococcoides species strain CBDB1. Nat. Biotechnol. 23, 1269–1273. doi:10.1038/nbt1131 Matthew Lee is a Senior Research Associate at UNSW. His research fl 18. Löf er, F.E. et al. (2013) Dehalococcoides mccartyi gen. nov., sp. nov., obligately interests include microbiology in subsurface environments, partic- organohalide-respiring anaerobic bacteria relevant to halogen cycling and biore- mediation, belong to a novel bacterial class, Dehalococcoidia classis nov., order ularly chlorinated methane metabolising bacteria. Dehalococcoidales ord. nov. and family Dehalococcoidaceae fam. nov., within the phylum Chloroflexi. Int. J. Syst. Evol. Microbiol. 63, 625–635. doi:10.1099/ Chris Marquis is a bioprocess engineer who runs the UNSW ijs.0.034926-0 Recombinant Products Facility at UNSW and has interests in applica- 19. Kandler, O. (1993) biochemistry and three-domain concept of life. Syst. Appl. Microbiol. 16,501–509. doi:10.1016/S0723-2020(11)80319-X tions of microbiology and protein. 20. Bagley, D.M. et al. (2000) Acclimation of anaerobic systems to biodegrade tetrachloroethene in the presence of carbon tetrachloride and chloroform. Water Bat-Erdene Judger is a PhD student at UNSW. His research project – Res. 34,171 178. doi:10.1016/S0043-1354(99)00121-9 is to heterologous production of functional reductive dehalo- 21. Futagami, T. et al. (2006) Effects of chloromethanes on growth of and deletion of genases from and species. the pce gene cluster in dehalorespiring Desulfitobacterium hafniense strain Y51. Dehalobacter Dehalococcoides Appl. Environ. Microbiol. 72,5998–6003. doi:10.1128/AEM.00979-06 Mike Manefield is an Associate Professor at UNSW. His interests are 22. Mabey, W. and Mill, T. (1978) Critical review of hydrolysis of organic compounds in water under environmental conditions. J. Phys. Chem. Ref. Data 7, 383–398. in environmental microbiology and biotechnology development.

Retraction notice to ‘Subversion of immunity by schistosomes’.[Microbiology Australia 2013, 34(3), 137–141. doi:10.1071/MA13046]

Bernd KalinnaA and Anna WalduckB ACentre for Animal Biotechnology, Faculty of Veterinary Science, The University of Melbourne, Melbourne, Vic., Australia BSchool of Applied Sciences, RMIT University, Melbourne, Vic., Australia

Refers to: Kalinna, B., Walduck, A. (2013) Subversion of immunity by schistosomes. Microbiology Australia 34, 137–141. After due consideration, the editors of the journal and the authors of the paper agree that the paper be retracted from Microbiology Australia. Reason: irresolvable authorship dispute.

128 10.1071/MA13046retract MICROBIOLOGY AUSTRALIA * SEPTEMBER 2015 Under the Microscope

Coupling anaerobic bacteria and microbial fuel cells as whole-cell environmental biosensors

Ashley E Franks Lara T Bereza-Malcolm Applied and Environmental Microbiology Applied and Environmental Department of Physiology, Microbiology Anatomy and Microbiology Department of Physiology, School of Life Sciences Anatomy and Microbiology College of Science, School of Life Sciences Health and Engineering College of Science, La Trobe University Health and Engineering Melbourne, Vic. 3086, Australia La Trobe University Tel: +61 3 9479 2206, Melbourne, Vic. 3086, Australia Email: [email protected]

Microorganisms have evolved to respond to environmental microbial community driven oxidation-reduction reactions, neces- factors allowing adaption to changing conditions and mini- sary for biochemical cycling, and transformation of elements, such misation of potential harm. Microbes have the ability to as carbon (C), nitrogen (N), sulfur (S) and Iron (Fe), through the sense a wide range of biotic and abiotic factors including biosphere12. MFC designs typically consist of electrodes that act as nutrient levels, analytes, temperature, contaminants, com- electron acceptors and donors, allowing redox reactions to occur munity quorum, and metabolic activity. Due to this ability, at a distance while creating an electrical current (Figure 1). In the the use of whole-cell microbes as biosensors is attractive as it environment, MFCs take advantage of the microorganisms associ- can provide real-time in situ information on biologically ated with biogeochemical cycles that are able to utilise electrodes as relevant factors through qualitative and quantitative out- electron acceptors or donors. The electrode-associated microbes puts. Interestingly, many of the environments where these are able to extracellularly transfer electrons directly from their biosensors will be of most of use lack oxygen; and as such the central metabolism to an electrode surface through a network of use of anaerobic microorganisms to sense environmental shuttles, cytochromes, and electrical conductive nanowires11. Since factors with easy to use outputs is essential. Furthermore, the electrons come directly from central metabolism, the current sensing of contaminants can be linked with bioremediation produced is a direct measurement of metabolic activity of the of known contaminated environments, allowing a flexible, electrode associated community11. However, such processes can multiplexed device. be limited by factors, such as O2 availability, pH levels, available substrates and electron donor availability12,13. Nevertheless, there Traditionally, a whole-cell microbial biosensor is described as an has been interest in utilising naturally existing anaerobic microbial analytic device consisting of microorganisms that produce a mea- communities as biosensors in the environment where a correlation surable output in response to a particular stimulus1. In the current can be determined between available substrates and current literature, microbial biosensors are developed in laboratory based production14. E. coli strains, consisting of a cloned gene pathway incorporating regulatory and reporter genes1, e.g. gfp expression under regulation by an arsenic sensitive regulatory protein2. Microbial fuel cell (MFC) Electrically integrated microbial biosensor technology however, is now being incorporated with both naturally Organic matter content in an environmental system is normally existing and synthetic microorganisms to produce novel biosensing evaluated in terms of the biochemical oxygen demand (BOD). BOD devices, for detection of environmentally relevant compounds is a measurement of the required dissolved oxygen to completely (Table 1). Initially MFCs were developed for energy production via degrade the organic matter15. MFCs allow the use of the electrode the oxidation of organic matter, utilising anaerobic bacteria such for anaerobic respiration to gain similar information about organic as Geobacter, Shewanella, Desulfuromonas, Rhodopseudomonas, oxidation rates in real-time. A single-chamber MFC containing a and Desulfovibrio11. Species from these genera are essential in mixed anaerobic community has been used as a biosensor for the

MICROBIOLOGY AUSTRALIA * SEPTEMBER 2015 10.1071/MA15045 129 Under the Microscope

Table 1. Examples of microbial fuel cell based biosensors that utilise current as the output. Microorganism(s) Inputs and Detection Reactor Lab or field Time for Reference used as sensor outputs limits type based? detection unit

Mixed community from artificial BOD 20-100 mg L–1 Two chamber MFC Lab 60 min Chang et al.3 wastewater BOD

Mixed community from artificial BOD ~100-350 mg L–1 Single chamber Lab 40 min Di Lorenzo et al.4 wastewater BOD MFC

Shewanella oneidensis MR-1 Fumarate 10 mM operational Single chamber Lab 100 mM detectable Si et al.5 concentration MFC after 1 min 0.83 mMLOD

Mixed microbial communities Acetate metabolism, 10 mM LOD Single chamber Lab Not reported Wardman et al.6 (3 soil sites used with known Fe (III) correlated to either Fe MFC reducers, sulfate reducers (III) or sulfate or methane producers) reduction or methane production

Mixed microbial community Sodium acetate, 5 mM acetate Embedded Field (Rifle site, Not reported Williams et al.7 including Geobacter species correlating to U (VI) resulting in electrodes Colorado, USA) reduction 50 mA/m2 in soil

Shewanella oneidensis First input: IPTG Not reported Two chambered Lab Not reported Hu et al.8 MR-1 DmtrA Second input: MFC 3-oxo-C6-HSL Activates: MtrA

Shewanella oneidensis MR-1 DMSO 8.3 mM Cathodic MFC Lab >45 h Arugula et al.9 DcyaAC, operational DcyaA and DcyaC concentration

Pseudomonas aeruginosa First input: 3-oxo- 15 mM operational MFC Lab Not reported Li et al.10 Dlacl/rhlI C12-HSL concentration Second input: C4-HSL Activates: phz operon

BOD, biological oxygen demand; LOD, limit of detection; MFC, microbial fuel cell; operational concentration, concentration for optimal current output.

(a) Ex situ biosensor with (b) Naturally existing (c) Synthetic microbes mixed communities or anaerobic microbial cultured in situ to detect synthetic microbes community specific analytes

Resistor

Bore hole to inject organics Resistor H2O O2 Resistor electron donors

Proton exchange U (IV) membrane

U (VI) Acetate

Enclosed system

CO2

Figure 1. Current possible applications of microbial fuel cell technology. (a) Soil samples can be tested ex situ in single or double chamber (pictured) MFCs. Reduction of organics can be by either the resident or transplanted microbial community, producing a current output. Synthetically derived microbes may also be incorporated in this design. (b) Electrodes can be implanted into sub-soil zones where the naturally existing anaerobic community can reduce any present compounds, resulting in a detectable current. These designs can use organic (e.g. acetate) or inorganic (electrode) electron acceptors to drive bioremediation of toxic contaminants such as the reduction of U (VI) to U (VI). (c) MFCs may be implanted into the sub soil andsupplied withmicrobesexpressing syntheticsensingpathways.Genepathwaysmay be simpleor basedonBooleanlogic gate design(pictured)to detect specific compounds which may diffuse into the anodic chamber via a proton exchange membrane.

130 MICROBIOLOGY AUSTRALIA * SEPTEMBER 2015 Under the Microscope

quantification of glucose. The sensor response was linear for monitoring of changing subsurface water flow through the use of concentration of glucose between 0.025 g LÀ1 up to 25 g LÀ1. microbial activity measurements, in a grid pattern well set up. Thus Unfortunately, such sensors are difficult to implement outside of this multiplexed sensor allows for monitoring of uranium reduction, laboratory conditions due to issues with the reliability of the while acting as an on-site bioreactor, removing the need for con- biosensors, as a new anaerobic community is required for each tinuous sampling and off-site testing7. The use of a cathode alone sample tested15. can provide the redox potential required for promoting microbial activity, while simplifying the system. In this case, the cathode acts Investigation into developing a cost-effective MFC biosensor has as an electron donor and provides metabolic energy to the subsoil resulted in a Subsurface Microbial Activity in Real Time (SMART) microbial population. These devices can be solar powered, left in system, which allows constant monitoring of anaerobic microbial remote locations indefinitely, and not require any further input. It is activity in soils6. Three sediment samples were tested with known important to note that in subsoil systems undergoing bioremedi- anaerobic communities responsible for either Fe(III) reduction, ation, a good understanding of electron donor or acceptor limita- sulfate reduction, or methane production. Electrical current pro- tions, metabolic activity and community function are essential for duction was correlated with the degradation of tracer acetate efficient remediation. [uniformly 14C labelled] with Fe (III), sulfate, or methane acting as a terminal electron acceptor6. These lab-based MFC biosensors thus allow the ex situ monitoring of organic compound degradation from Synthetically derived electric biosensors added sediment samples (Figure 1a). When placed in situ, these Currently, anaerobic biosensors rely on naturally existing microbial devices will primarily be useful for monitoring relative changes in communities to degrade organics, however there has been inves- microbial activity in response to environmental perturbations. tigation into incorporating synthetically derived biosensing path- These microbial systems are sensitive to environmental variations, ways into MFC devices. Such systems have utilised Boolean logic such as temperature and seasonal changes, and may not be ideal to gate ideas, which are based on modular computer based decision be implemented as in situ standalone devices. However, investiga- circuits, such as AND, NOT and OR gates10,19. For example, for an tion of current production in these anode-resistor-cathode systems AND gate, two inputs must be present for a target gene to be provides insight into microbial activity in sediments, and may allow transcribed. A synthetic biosensor has been produced that allows monitoring of the microbial activity of communities responsible incorporation of a range of known regulator genes, and is based on a for the transformations of important organic compounds in anoxic quorum sensing system. In a demonstration system, IPTG and environments. quorum sensing modules were created through the use of lacl and luxR8. In the presence of both IPTG and 3-oxo-C6-HSL inducers, an Bioremediation of contaminants output module would be activated. To integrate this reporter system Direct or mediated electron transfer between microbes and an to an electrode, MtrA was utilised as an output in a S. oneidensis electrode may allow degradation or transformation of pollutants DmtrA mutant strain. This mutant is unable to produce an electrical in bioremediation processes16. Compounds such as metals are current due to disruption of the Mtr extracellular electron transfer unable to be degraded, but can have their solubility reduced. This (EET) system8. Thus, to return this pathway back to functionality, is essential in preventing their spread and contamination16,17. For both IPTG and 3-oxo-C6-HSL need to be present to activate the logic example, Geobacter sulfurreducens is capable of dissimilatory gate and MtrA production. This synthetic biosensor demonstrates metal reduction, a process where energy is conserved through the potential to clone alternate input/output systems, in particular oxidizing organic or inorganic electron donors while reducing a regulator elements with a high specificity for dangerous contami- metal or metalloid18. Geobacter species capable of dissimilatory nants in place of Lacl and LuxR, with the detection as an electrical metal reduction have been applied in a practical on-site in situ MFC output8. However, problems can arise through mutations to the biosensor, using acetate, an intermediate compound produced sensors through random mutation events, or selection via the 7 during metabolic processes , to drive reduction of soluble U(VI)to contaminants present, which may affect functionality. Further re- less-soluble U(IV). Anodes were installed down-gradient of the search is also necessary to incorporate safety mechanisms into the site of bioremediation and a cathode was embedded on the soil synthetic microbes to deter fears of accidental release of genetically surface (Figure 1b). This allowed a correlation to be determined modified microorganisms. However with further research synthetic between the injections of acetate at the site of contamination, with microbes may effectively be utilised in ex situ (Figure 1a)orin situ a significant increase in current7. This spike in activity also allowed (Figure 1c) MFC biosensing systems.

MICROBIOLOGY AUSTRALIA * SEPTEMBER 2015 131 Under the Microscope

Future directions and conclusions 13. Rivett, M.O. et al. (2008) Nitrate attenuation in groundwater: a review of biogeo- chemical controlling processes. Water Res. 42, 4215–4232. doi:10.1016/j.watres. Anaerobic bacteria may provide value for biosensing technologies to 2008.07.020 6,20 monitor anoxic zones . Their ability to naturally cycle contami- 14. Bond, D.R. and Lovley, D.R. (2003) Electricity production by Geobacter sulfurre- – nants, along with survivability in contaminated soils and waterways is ducens attached to electrodes. Appl. Environ. Microbiol. 69, 1548 1555. doi:10.1128/AEM.69.3.1548-1555.2003 allowing incorporation into both biosensing devices and bioreac- 15. Kumlanghan, A. et al. (2007) Microbial fuel cell-based biosensor for fast analysis of tors. Many anaerobic biosensors developed are generally reliant on biodegradable organic matter. Biosens. Bioelectron. 22,2939–2944. doi:10.1016/ j.bios.2006.12.014 MFC type designs because the ability to utilise an external electron 16. Lovley, D.R. (2003) Cleaning up with genomics: applying molecular biology to acceptor provides an easy-to-monitor output, in the form of current. bioremediation. Nat. Rev. Microbiol. 1,35–44. doi:10.1038/nrmicro731 Furthermore, these designs have already been linked to degradation 17. Anderson, R.T. et al. (2003) Stimulating the in situ activity of Geobacter species to remove uranium from the groundwater of a uranium-contaminated aquifer. of organics, or immobilisation of metals. However, such outputs are Appl. Environ. Microbiol. 69, 5884–5891. doi:10.1128/AEM.69.10.5884-5891. subject to environmental fluctuations and as thus may not always be 2003 a reliable detection method. Further investigation by monitoring 18. Caccavo, F. et al. (1994) Geobacter sulfurreducens sp. nov., a hydrogen-and acetate-oxidizing dissimilatory metal-reducing microorganism. Appl. Environ. 21 the metabolism of microbes associated with anodes is also still Microbiol. 60, 3752–3759. necessary. Currently, there is promise in incorporating synthetic 19. Wang, B. et al. (2013) A modular cell-based biosensor using engineered genetic microbes into the anode compartment to produce a biosensing logic circuits to detect and integrate multiple environmental signals. Biosens. Bioelectron. 40, 368–376. doi:10.1016/j.bios.2012.08.011 device for a range of contaminants. Hence, whole-cell microbial 20. Coates, J.D. et al. (1997) Anaerobic degradation of polycyclic aromatic hydro- biosensors based in anaerobic microbes may provide a cost effective carbons and alkanes in petroleum-contaminated marine harbor sediments. Appl. Environ. Microbiol. 63, 3589–3593. means of detection and bioremediation, allowing long-term mon- 21. Aracic, S. et al. (2014) Investigating microbial activities of electrode-associated itoring that may be deployed in a variety of environments. microorganisms in real-time. Front. Microbiol. 5,663.

References Biographies 1. Bereza-Malcolm, L.T. et al. (2015) Environmental sensing of heavy metals through Lara Bereza-Malcolm is a PhD candidate at Dr Ashley Franks whole cell microbial biosensors: a synthetic biology approach. ACS Synth. Biol. 4, 535–546. laboratory in the College of Science, Health and Engineering, at 2. Liao, V.H.C. and Ou, K.L. (2005) Development and testing of a green fluorescent La Trobe University. Her research focus is on the development of protein-based bacterial biosensor for measuring bioavailable arsenic in contam- inated groundwater samples. Environ. Toxicol. Chem. 24, 1624–1631. novel whole-cell microbial biosensors for the detection of heavy doi:10.1897/04-500R.1 metals in the environment. 3. Chang, I.S. et al. (2004) Continuous determination of biochemical oxygen demand using microbial fuel cell type biosensor. Biosens. Bioelectron. 19, Ashley E Franks is an Associate Professor in the Department of 607–613. doi:10.1016/S0956-5663(03)00272-0 Physiology, Anatomy and Microbiology at La Trobe University and 4. Di Lorenzo, M. et al. (2009) A single-chamber microbial fuel cell as a biosensor for wastewaters. Water Res. 43,3145–3154. doi:10.1016/j.watres.2009.01.005 head of the Applied and Environmental Microbiology research 5. Si, R.W. et al. (2015) A whole-cell electrochemical biosensing system based on Laboratory. He conducted his doctorate research as part of the bacterial inward electron flow for fumarate quantification. Biosens. Bioelectron. Centre of Marine Biofouling and Bioinnovation at the University of 68,34–40. doi:10.1016/j.bios.2014.12.035 6. Wardman, C. et al. (2014) Real-time monitoring of subsurface microbial metab- New South Wales by investigating antifungal compounds produced olism with graphite electrodes. Front. Microbiol. 5, 621. doi:10.3389/fmicb.2014. by marine bacteria in biofilms. During his PhD he spent 4 months at 00621 the University of Exeter in the UK on an Adrian Lee fellowship to 7. Williams, K.H. et al. (2010) Electrodic voltages accompanying stimulated biore- mediation of a uranium-contaminated aquifer. J. Geophys. Res. 115, G00G05. develop dual bacterial/yeast biofilm systems. On graduating he 8. Hu, Y. et al. (2015) Programming the quorum sensing-based AND gate in moved to the Biomerit Research Centre in Cork, Ireland to work Shewanella oneidensis for logic gated-microbial fuel cells. Chem. Commun. 51,4184–4187. doi:10.1039/C5CC00026B on bacterial plant interactions at as a Government of Ireland Fellow 9. Arugula, M.A. et al. (2012) Molecular AND logic gate based on bacterial anaerobic in Science Technology and Engineering. This research looked at – respiration. Chem. Commun. 48, 10174 10176. doi:10.1039/c2cc35595g how to use bacteria to help plant growth. He then took a position as 10. Li, Z. et al. (2011) Bacteria-based AND logic gate: a decision-making and self- a Senior Scientist and Research Professor within the Geobacter powered biosensor. Chem. Commun. 47, 3060–3062. doi:10.1039/c0cc05037g 11. Semenec, L. and Franks, A.E. (2014) The microbiology of microbial electrolysis Project at the University of Massachusetts Amherst in the USA where cells. Microbiol. Aust. 35, 201–206. doi:10.1071/MA14065 he worked on microbes that make electricity. His lab currently has 12. Paerl, H.W. and Pinckney, J.K. (1996) A mini-review of microbial consortia: research interests in ecology, electric microbiology and microbiome their roles in aquatic production and biogeochemical cycling. Microb. Ecol. 31, 225–247. doi:10.1007/BF00171569 research.

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Interesting anaerobes in the environment

Microbiologically influenced corrosion (MIC) Corrosion of pure metals or their alloys involves ionisation of the metal (the oxidative anodic reaction) coupled with the cathodic reaction (a chemical species reduction), is relatively well under- Linda L Blackall stood and accounted for in design and maintenance of metallic structures. Metals are used in many terrestrial (e.g. buried pipes for School of Science Faculty of Science, Engineering and transmission of many different liquids) and aquatic, particularly Technology marine (ships hulls, steel sheet piles; Figure 1), structures. MIC is Swinburne University of Technology Email: [email protected] also known as biocorrosion and involves microbial processes on metal surfaces that lead to localised material degradation at rates that are orders of magnitude higher than would normally be expected from standard, abiotic corrosion7–9. No new electrochem- Prokaryotes (Bacteria and Archaea) have a wide range of ical processes are involved in MIC, simply corrosion is accelerated capacities to survive by generating energy in environments and occurs under conditions that would not normally favour it. MIC and situations lacking oxygen, which abound on Earth. can lead to premature and unpredicted failure of a wide range of Anaerobic metabolic strategies include anaerobic respira- important structures that can be costly to repair and, in some cases, tion (numerous types – e.g. nitrate reduction – Paracoccus can have fatal consequences. The overall cost of corrosion has been denitrificans; sulfur respiration – Desulfuromonadales; estimated to be between approximately 2 and 5% of the gross methanogenesis – Methanosarsina spp.; iron reduction – national product in a range of countries, with a global value of Geobacter spp.; dehalorespiration – Dehalococcoides ethe- nogenes) and fermentation (sugars converted to simpler organic compounds like acids, gases and alcohols – e.g. Lactobacillus spp.). Relatively novel environmental anaer- obic strategies include anaerobic ammonium oxidation (Anammox – e.g. Brocadia spp.) and anaerobic methane oxidation (AMO)1, which is a syntrophic association between anaerobic methanotrophic archaea (ANME) and sulfate-, iron-, manganese- or nitrate-reducing bacteria2. The classic anaerobic synthrophic example is interspecies hydrogen/ formate transfer between a hydrogen/formate producing fatty acid oxidising bacterium (the syntroph) and a hydrogen/ formate consumer (methanogen or sulfate-reducer)3. Microbes vary in their oxygen tolerance and are described as obligate anaerobes if they are killed by atmospheric levels of oxygen due to the lack of catalase and superoxide dismutase that provide oxygen radical protection.

A recent Microbiology Australia issue (The Microbial Ecology of the Environment; November 2014) covered some environmental anaerobic microbiology topics including sulfidic sediments4, elec- tromicrobiology (typically comprising an anaerobic anode and an oxic cathode)5 and chloroethene degradation (dehalorespiration)6.

This short review provides a brief overview of three environmental anaerobic metabolic topics and demonstrates metabolic and envi- Figure 1. An example of late stage microbiologically influenced corrosion of a sheet metal structure in a shallow marine environment. ronmental diversity employed by the microbes. (Photo credit: Dr Scott Wade, Swinburne University of Technology.)

MICROBIOLOGY AUSTRALIA * SEPTEMBER 2015 10.1071/MA15046 133 Under the Microscope

10 + – US$50–80 billion per annum and approximately A$18 billion (NH4 +NO2 –> N2 +2H2O) from nitrogen and redox balances in suggested for Australia in 200611. a denitrifying bioreactor treating effluent from a methanogenic bioreactor17 reported a novel biochemical pathway. Although the Microbes mass at the metalic surface to produce a ‘biofilm’ and the discovery was in a bioreactor with Anammox-enriched biomass, collective action of the different microbial species in the biofilm theoretical calculations had previously been used to hypothesise along with abiotic features result in the phenomenon of biocorro- that Anammox could occur18 and it has been found to be environ- sion. Corrosion kinetics are modulated by the surface physico- mentally widespread. Despite that for the majority of the 20th chemical properties including oxygen, ions, pH, redox, conductivity century it was thought that the nitrogen cycle was fully understood, and more. Various microbial species have been singled out as major Anammox has been found to be responsible for up to: culprits in enhancing corrosion, notably sulfate reducing bacteria 12 * 70% of marine nitrogen cycling, and (SRB) . However, in a natural corrosion site, there will be a complex 19 * 50% of marine N2 production . community of microbes present with some involved in setting up and facilitating the appropriate conditions for the anaerobic corrosion organisms. Thus, although many microbes might not Anaerobic methane oxidation (AMO) directly cause the corrosion, their properties might be vital for The possibility of AMO (CH +SO 2– +H+ –> CO +HS– +2H O) the maintenance of the corrosion species. Major biocorrosion 4 4 2 2 only developed through the 1970s from coupled methane and mechanisms include acid attack, creation of anodic sites and ca- sulfate profiles in marine sediments20. AMO was not available in thodic depolarisation12. enriched biomasses and experimental evidence for AMO and its In MIC of steel surfaces, SRB energy production relies on elemental syntrophic SRB came by implementing a combination of method- iron oxidation to release soluble ferrous iron (Fe –> Fe2+ +2e–) ological procedures including lipid biomarking, clone libraries and and typically sulfate is used by the SRB as their electron acceptor fluorescence in situ hybridisation (FISH)21. Subsequently, second- 2– + – – (SO4 +9H +8e –> HS +4H2O). Acid producing bacteria and ary ion mass spectrometry and FISH were used in re-confirming methanogens are also corrosive anaerobes, the former by way of AMO in marine methane-seep sediments, for revealing two distinct their corrosive acid metabolites. A recent microbiological study of AMO archaeal groups called ANME-1 (a distant relative of Metha- ‘accelerated low water corrosion’ using high throughput DNA nosarcinales and Methanomicrobiales) and ANME-2 (in the Metha- sequencing determined that Methanococcus maripaludis, which nosarcinales), and for demonstrating that the AMO in the syntrophic comprised 31% of retrieved sequences, could extract electrons association likely employs a ‘reversed methanogenesis’ pathway22. 13 directly from steel and use CO2 as a carbon source . A third ANME-3 group (in the Methanosarcinales), typically found near mud volcanoes has been discovered23. The ANME clades form How to mitigate MIC involves numerous strategies including use of syntrophic associations with specific partners and most that have biocides, promoting non-corrosive biofilms on surfaces to mask it been reported are with SRB, which are in the Deltaproteobacteria. from the MIC organisms and manipulation of electron acceptors to These syntrophic associations are highly relevant to global carbon preclude MIC organisms14. Recently reported steel MIC control cycling since although oceans contribute only 2% of the global methods involve: methane budget, it has been found that this low net total is due to 2+ * microbiological production of an Fe -phosphate layer that has a the fact that AMO utilise 70–300 teragrams of methane per year24. reduced corrosion potential15, and 16 ‘ * the use of graphene coatings . The AMO-SRB synthrophic associations typically form microbial reefs’ in marine sediments at the sulfate-methane transition zone – However, more research into the microbiological mechanisms of critical for the metabolism of both syntrophic partners25. MIC (anode/cathode metabolism, acid production) is required for additional novel, targeted, reliable control strategies to be devel- Recently, from bioreactors, a single organism (ANME-2d, ‘Candidatus oped. These studies will likely involve controlled experimental Methanoperedens nitroreducens’) has been reported to be capable of conditions that simulate the natural settings10 and determination independent AMO through reverse methanogenesis using nitrate as of specific corrosive strains and their mechanisms. the terminal electron accepter26:

– – o –1 * CH4 + 4NO3 -> 4NO2 +2H2O(DG = –503 kJ.mol CH4)

Anaerobic ammonium oxidation (Anammox) Another string to the AMO bow is the star-like shaped ‘Candidatus Ammonia oxidation has been very well studied and the biochemistry Methylomirabilis oxyfera’ (in the bacterial candidate phylum NC10), understood in aerobic bacteria (e.g. Nitrosomonas spp.) and more which is appears solely responsible for nitrite-dependent AMO recently in the abundant, ammonium-scavenging, aerobic, marine (N-DAMO) in fresh and marine waters27. The fine balance of the archaeon Nitrosopumilus spp. However, the finding of Anammox global carbon budget and its relationship with the nitrogen cycle is

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substantially held by the AMO and their syntrophic associations. 10. Marty, F. et al. (2014) Identification of key factors in accelerated low water fl Since many aspects of the biochemistry are not fully elucidated, corrosion through experimental simulation of tidal conditions: in uence of stimulated indigenous microbiota. Biofouling 30,281–297. doi:10.1080/0892 25 including definitive proof of the reverse methanogensis ,itis 7014.2013.864758 imperative that urgent research addresses this lack of knowledge2. 11. Wade, S.A. et al. (2011) Microbiologically influenced corrosion in maritime vessels. Corrosion and Materials. 36,68–79. Summary 12. Gu, T. (2012) New understandings of biocorrosion mechanisms and their classifications. Journal of Microbial and Biochemical Technology. 4, iii–vi. Anaerobic processes (those lacking the requirement for or intoler- doi:10.4172/1948-5948.1000e107 ance of oxygen) have many industrial applications (e.g. wastewater 13. Usher, K.M. et al. (2014) Marine rust tubercles harbour iron corroding archaea and sulphate reducing bacteria. Corros. Sci. 83,189–197. doi:10.1016/j.corsci. treatment) and implications (e.g. MIC) and also are critical to the 2014.02.014 global cycling of many elements (two relevant to nitrogen and 14. Little, B. et al. (2007) A review of ‘green’ strategies to prevent or mitigate microbiologically influenced corrosion. Biofouling 23,87–97. doi:10.1080/ carbon are briefly described). Despite that microbes were implicat- 08927010601151782 ed in corrosion at least by 1910, due to the multidisciplinary nature 15. Cote, C. et al. (2015) Geobacter sulfurreducens: an iron reducing bacterium that of the field and the lack of connection between experts in the can protect carbon steel against corrosion? Corros. Sci. 94,104–113. doi:10.1016/ j.corsci.2015.01.044 disparate fields like electromicrobiology, corrosion and process 16. Dumée, L.F. et al. (2015) Growth of nano-textured graphene coatings across engineering, mechanisms involved in MIC are not well under- highly porous stainless steel supports towards corrosion resistant coatings. – stood12. Detailed studies into the role of relevant microbes in MIC Carbon 87, 395 408. doi:10.1016/j.carbon.2015.02.042 17. Mulder, A. et al. (1995) Anaerobic ammonium oxidation discovered in a denitrying and of smart biomimetic surface control strategies are required to fluidized-bed reactor. FEMS Microbiol. Ecol. 16,177–183. doi:10.1111/j.1574- abate this problem. 6941.1995.tb00281.x 18. Broda, E. (1977) Two kinds of lithotrophs missing in nature. Z. Allg. Mikrobiol. 17, The relatively novel processes of Anammox and AMO are common 491–493. doi:10.1002/jobm.3630170611 in the environment but their existence was largely predicted from 19. Strous, M. and Jetten, M.S.M. (2004) Anaerobic oxidation of methane and ammonium. Annu. Rev. Microbiol. 58,99–117. doi:10.1146/annurev.micro.58. thermodynamic calculations. Their studies have been prolific since 030603.123605 they are relevant to marine nutrient cycling where >75% of methane 20. Zehnder, A.J.B. and Brock, T.D. (1980) Anaerobic methane oxidation – occurrence and ecology. Appl. Environ. Microbiol. 39,194–204. and 30–50% of ammonium are oxidised by them24. Industrial 21. Boetius, A. et al. (2000) A marine microbial consortium apparently mediating application of Anammox in the wastewater treatment sphere facil- anaerobic oxidation of methane. Nature 407,623–626. doi:10.1038/35036572 itates cost and CO2 emissions reductions but the environmental 22. Orphan, V.J. et al. (2001) Methane-consuming archaea revealed by directly coupled isotopic and phylogenetic analysis. 293,484–487. doi:10.1126/ relevance of Anammox in marine and freshwater ecosystems is Science science.1061338 profound. Future research elucidating connections between sulfate, 23. Niemann, H. et al. (2006) Novel microbial communities of the Haakon Mosby mud nitrite/nitrate, and metal ion (manganese and iron) electron accep- volcano and their role as a methane sink. Nature 443,854–858. doi:10.1038/ nature05227 tors in AMO will be valuable in improved understanding of global 24. Reeburgh, W.S. (2007) Oceanic methane biogeochemistry. Chem. Rev. 107, nitrogen and carbon cycles. 486–513. doi:10.1021/cr050362v 25. Mueller, T.J. et al. (2015) Methane oxidation by anaerobic archaea for conversion References to liquid fuels. J. Ind. Microbiol. Biotechnol. 42,391–401. doi:10.1007/s10295-014- 1548-7 1. Knittel, K. and Boetius, A. (2009) Anaerobic oxidation of methane: progress with an unknown process. Annu. Rev. Microbiol. 63, 311–334. doi:10.1146/annurev. 26. Haroon, M.F. et al. (2013) Anaerobic oxidation of methane coupled to nitrate – micro.61.080706.093130 reduction in a novel archaeal lineage. Nature 500,567570. doi:10.1038/ nature12375 2. Cui, M. et al. (2015) Anaerobic oxidation of methane: an ‘active’ microbial process. 27. Shen, L.-d. . (2015) Nitrite-dependent anaerobic methane-oxidising bacteria: MicrobiologyOpen 4,1–11. doi:10.1002/mbo3.232 et al unique microorganisms with special properties. Curr. Microbiol. 70,562–570. 3. Sieber, J.R. et al. (2012) Genomic insights into syntrophy: the paradigm for doi:10.1007/s00284-014-0762-x anaerobic metabolic cooperation. Annu. Rev. Microbiol. 66, 429–452. doi:10.1146/annurev-micro-090110-102844 4. Stroud, J.L. and Manefield, M. (2014) The microbiology of acid sulfate soils and Biography fi – sul dic sediments. Microbiol. Aust. 35, 195 198. doi:10.1071/MA14063 Linda L Blackall is a microbial ecologist who has studied many 5. Semenec, L. and Franks, A.E. (2014) The microbiology of microbial electrolysis different complex microbial communities ranging from host asso- cells. Microbiol. Aust. 35,201–206. doi:10.1071/MA14065 6. Patil, S.S. et al. (2014) Microbiology of chloroethene degradation in groundwater. ciated through to free living in numerous environments. Her Microbiol. Aust. 35,211–214. doi:10.1071/MA14067 research has covered environmental microbiomes spanning waste- 7. Beech, I.B. and Gaylarde, C.C. (1999) Recent advances in the study of water treatment (aerobic and anaerobic), solid waste digestion biocorrosion – an overview. Rev. Microbiol. 30, 177–190. doi:10.1590/S0001- fi 37141999000300001 (land ll and composting), bioelectric systems and microbiologically 8. Little, B.J. et al. (2008) The influence of marine biofilms on corrosion: a concise influenced corrosion and the methods used allow elucidation of – review. Electrochim. Acta 54,2 7. doi:10.1016/j.electacta.2008.02.071 massive microbial complexity and function in these diverse biomes. 9. Usher, K.M. et al. (2014) Critical review: microbially influenced corrosion of She is a Professor of BioSciences at Swinburne University of Tech- buried carbon steel pipes. Int. Biodeterior. Biodegradation 93,84–106. doi:10.1016/j.ibiod.2014.05.007 nology in the Faculty of Science, Engineering and Technology.

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Report from ASM 2015: One Microbiology

Dee Carter Chair of Scientific Program committee

July 2015 has seen another highly successful meeting of the monitoring, next generation sequencing, imaging and proteomics. Australian Society for Microbiology, this time in our capital city at These were run with a high level of professionalism by dedicated QT Canberra, with over 460 microbiologists converging on the city volunteers and were very well attended and received. for three days of science, networking and socialising around our The meeting itself got off to a bang, quite literally, on Sunday evening common love of microbes. at the Shine Dome, with a Questacon presentation of a sneeze, fi Our theme this year was One Microbiology, and our aim was to where giant microbes were red into the air with the aid of a liquid present a highly integrated program that worked across the tradi- nitrogen bomb! We were then treated to a highly entertaining public tional divisions to bring us together to discuss some of the big issues lecture on Microbiology from Guts to Great Oceans, presented by and opportunities in microbiology, ranging from animal microbiol- our international guests Janet Jansson and Stephen Giovannoni, fi ogy and ecosystem health, across host-pathogen interactions, anti- along with organiser and LOC Chair Mike Mane eld. Mike presented ‘ ’ biotics, and clinical microbiology, and to some of the modern uses of an excerpt from his book Esher Surfs the Sewer aimed at promot- microbes in synthetic and industrial microbiology. ing microbiology to preschoolers and young children. We then got down to some more serious science with Bazeley Orator, Professor The meeting began on Saturday 11 July with the EduCon meeting, Yoshihiro Kawaoka, taking us through the harrowing issues of which continued from its inaugural meeting at ASM 2014 with a working with Ebola and providing us with an update on progress highly stimulating program covering current topics in microbiology in working with and understanding this very challenging infectious ‘ ’ education, ranging from big picture issues such as graduate disease. employability and adaptive learning, to the nitty-gritty of teaching Three days of microbiology followed, with wonderful presentations methods, such as writing better multiple choice questions, and from our international speakers and terrific talks from our locals. engaging students with social media tools. A major outcome of Particular highlights were the Rubbo Oration by Janet Jannson on this year’s EduCon was the drafting of a national microbiology the gut microbiome and Crohn’s Disease, the astonishing advances curriculum, which aimed to put in place some universal learning made by young scientist and Fenner Award winner Gene Tyson, outcomes for tertiary teaching in microbiology across Australia. and the fascinating world of giant viruses presented by Chantel Thanks go to Josie Lategan and the EduCon team for inspiring us Abergel. And while we were all very impressed by our visitors, they with their dedication to teaching our future microbiologists. in turn commented on how impressed they were with the quality Workshops continued on Sunday, attracting delegates interested of talks by our symposium speakers, and we can all be very proud of in updating their skills in antimicrobial resistance assessment and the standard of microbiology research that is going on in Australia.

Ruth Hall, Ricardo Guzman, Slade Jensen, Tim Newsome, Nick Coleman, Dee Carter, Tom Jeffries, Mike Manefield, Kara Taglieri (ASN organiser) and Mitchell Brown. Not present: Christel Armstrong, Maurizio Labbate, Deborah Lum, Naresh Verma and Charlotte Webster.

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The talks were complemented with plenty of opportunities for networking over posters and in the trade area, and our intimate and comfortable venue in QT Canberra, combined with the chilly Can- berra weather outside, promoted friendly and fruitful interactions.

A particular highlight of our social program this year was the inclusion of events targeted at our newest members: the students and early career researchers, with a dedicated student breakfast, student and ECR lunches, and a fabulous social night at Bent Spoke Brewery. The latter attracted over 90 student and ECR participants, who enjoyed the local hand-crafted fermentation products until the early hours of the morning. There was an educational component too, with the local brewers giving guided tours of their fermentation facilities, and describing the ins and outs of applied microbiology in Andrew Butcher, Mohammad Katouli, Mike Manefield, Harsha Sheorey, Diane Lightfoot, Dee Carter and Megan Lloyd. this context. An excellent sense of camaraderie filled the room at this brewery event, and the degree to which groups from different Teachers Travel Award: Megan Lloyd institutions mingled was notable; many new friendships and colla- Megan Lloyd started working as a borations were forged onthis evening,which will surely lead to more trainee clinical scientist at Fair- useful synergies as these students and ECRs continue to link up to field Infectious Diseases Hospital discuss their projects and their careers in microbiology. whilst completing an undergrad-

Many people and organisations helped to make this meeting a uate degree from RMIT in 1989. fi success. I would like to thank the trade for their financial support, She worked at Fair eld Hospital ASN forits superb professional organisation of all logistical aspects of (both Clinical Bacteriology and the meeting, the Academy of Science for use of the Shine Dome for Rickettsial Research Laboratory) the public lecture, the helpful staff at QT Canberra, and the ASM until 1992 and then worked in the executive for help and support leading up to the meeting. Very pathology laboratory at Kather- special thanks go to the LOC based in Sydney and Canberra, and to ine District Hospital (NT). She moved to Perth in 1995 and began the Scientific Program Committee, who worked for nearly 2 years working in cytomegalovirus research at the University of Western before the meeting to come up with the scientific and social Australia and completing her PhD in 2007 in the laboratory of program that we all enjoyed. We can now all look forward to a trip Professor Geoffrey Shellam (Mechanisms of Virally Vectored Immu- out west for ASM 2016 in beautiful Perth. nocontraception). She wrote and delivered a new 2nd year unit for Edith Cowan University in 2014, Medical Microbiology. New Fellows of the Australian Society for Microbiology Burnet Hayes Postgraduate Award: Matthew Johnson Andrew Taylor-Robinson, CQ University The Hayes-Burnet award en- Nevada Pingault, Department of Health, WA abled me to experience work in

Christopher McIver, St George Hospital a UK laboratory at the University of Birmingham. During my time Charlene Kahler, University of Western Australia in the laboratory, I developed new skills that I will be able to Adam Polkinghorne, University of the Sunshine Coast utilise back in Melbourne. Work- ing in a different laboratory has ASM Distinguished Service Awards broadened my horizons and I Harsha Sheory, Louise Roddam, Mohammad Katouli, Diane Light- have learnt new ways to tackle foot and David Dickeson were acknowledged with ASM Distin- laboratory problems. In addition, the guest laboratory was very guished Service Awards. friendly and accommodating. I hope that the working relationships

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that were set up during this trip will lead to a strong collaboration in same thing. I certainly needed the extra exercise to compensate for the near future. all the oysters, jambalaya and gumbo I was eating.

After 3 weeks of laboratory work at the University of Birmingham, The Ebola outbreak in West Africa was the hot topic of the meeting, I attended the Society for General Microbiology spring meeting in with several sessions focusing on Ebola virus detection, outbreak Birmingham city centre. The abstract booklet was littered with world management and vaccine development. Dr. Ian Crozier, who con- leading molecular microbiologists from across the globe. Notable tracted Ebola while treating patients in Sierra Leone, gave us a speakers include Prof Regine Hengge from Germany and Prof personal and touching insight as to what it was like for him to Eduardo Groisman from Yale US, both gave incredible talks that I suddenly find himself as the patient, fighting for his life. I don’t think found fascinating. It was also great to see two speakers from there was a dry eye in the house as he paid tribute to those who were Melbourne, Dr Kathryn Holt and Dr Eva Heinz who gave Impressive not lucky enough to survive. talks on K. pneumoniae epidemiology and comparative genomics One of the great features of the ASM meeting was the strong focus on respectively. learning. There were plenty of engaging workshops, learning labs At the meeting I presented a poster that was well received. The and networking and career opportunities. It was such a stimulating feedback and discussion from fellow attendees regarding my work environment for an early career researcher to be in, and I tried to was invaluable. Moreover, several posters and presenters from soak up as much as I could. UK universities were on similar subject to my own. I found the discussion of experiments and methods with my peers to be very Prior to the meeting I was lucky enough to spend a couple of ’ informative. weeks with Craig Roy s group at the Department of Microbial Pathogenesis at Yale University in Connecticut. Craig’s group was This was an exciting time to visit Prof Ian Henderson’s laboratory. very welcoming, and it was energising to be around new people who Currently, they are setting up a new sequencing facility to incorpo- work in a similar field. During my time in Craig’s lab, I used a yeast rate multiple high throughput sequencing technologies and se- lethality screen to try and identify eukaryotic processes that are quence bacterial genomes at low cost. This facility, named altered by effector proteins secreted by C. burnetii. While MicrobesNG, launched at the SGM. It was a pleasure to meet the two weeks does not seem like long to be in a new lab, it was long organisers and speak to experts about the future of high throughput enough for me to learn the technique and to cement an important sequencing and its applications. collaboration.

Millis Colwell Postgraduate Award: Now that I am back at the Doherty Institute for Infection and Jennifer Moffat Immunity in Melbourne, I am continuing my post-doctoral work with a renewed passion. It really is an incredible experience to This year I was fortunate enough present your work overseas, and to engage and collaborate with to receive the Millis-Colwell international colleagues. I sincerely thank Dr Hayley Newton, Pro- award, which allowed me to pres- fessor Craig Roy and the Australian Society of Microbiology for ent my work on the intracellular funding and supporting this incredible opportunity. pathogen Coxiella burnetii at the 115th American Society for David White Excellence in Teaching Award: Microbiology General Meeting. This year the meeting was held Helen Agus in New Orleans, a city close to Helen currently works in the my heart. Despite the summer Faculty of Science, University of heat, New Orleans proved to be the perfect backdrop to an action Sydney as a Pro-Dean. Her Micro- packed conference. Having never attended an American Society for biology teaching career began Microbiology meeting, I wasn’t really sure what to expect. I heard it as a new Microbiology graduate was going to be big, but it certainly exceeded my expectations! With demonstrating in a University of five concurrent sessions, I was overwhelmed with choice. I would Sydney (USYD) dentistry course, have looked utterly ridiculous running up and down the corridors of progressing to encompass major the conference centre trying to catch presentations in different roles in development, imple- sessions if wasn’t for the fact that everyone else was doing exactly the mentation of curricula and

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teaching in 30 units of study across the Faculties of Science, own research group within the Advanced Water Management Medicine, Pharmacy and Agriculture in five different tertiary institu- Centre (AWMC) at The University of Queensland. Later that year tions. She has held a USYD Microbiology academic teaching fo- he was awarded a Queen Elizabeth II Fellowship as part of a cussed position in the School of Molecular Bioscience for the past successful ARC Discovery grant. A/Professor Tyson’s research has 22 years, whilst also being active in the research area, supervising been published in more than 30 articles in the mainstream science and mentoring many Honours and Masters research students. She media and has presented invited talks at more than 14 international has a general Microbiology capability and specialist expertise in meetings. medical Microbiology. bioMe´rieux ASM Identifying Resistance Award: ASM Lyn Gilbert Award David Wiley Professor Theo Sloots is the Director of Research at the David Whiley is the senior Queensland Paediatric Infectious research scientist & Specialist Diseases (QPID) laboratory at the Microbiologist at the Queensland Queensland Children’s Medical Paediatric Infectious Diseases Research Institute. Over the past (QPID) Laboratory, Sir Albert 25 years, his professional activi- Sakzewski Virus Research Centre ties have resulted in the establish- and Queensland Children’s Med- ment of a pathology service in ical Research Institute, The Uni- Queensland, which is amongst versity of Queensland, at the the best in Australia, and recog- Royal Children’s Hospital. nised internationally. His skills have been applied to develop an He has been at the forefront of developing molecular methods for extensive translational research program, which have made a major direct detection of antimicrobial resistance for Neisseria gonor- contribution to our understanding of infectious disease, and have rhoeae, an organism now ranked as an ’urgent’ antimicrobial significantly contributed to enhance clinical management. Over the resistance threat, the highest level designated by the CDC and past 10 years Professor Sloots has attracted research funding in shared with only two other organisms, Clostridium difficile and excess of $14 million from external granting bodies such as the carbapenem-resistant Enterobacteriaceae. He has 17 years’ expe- NHMRC, ARC, NIH and the Children’s Health Foundation. He has rience in clinical microbiology research and is a leading authority in published extensively with 205 scientific articles in peer-reviewed molecular detection and characterisation of infectious diseases, journals and books. particularly sexually transmitted infections and novel or emerging agents.

ASM Frank Fenner Award: Gene Tyson Gene W. Tyson is an Associate Professor at the Australian Centre for Ecogenomics (ACE) and Advanced Water Management Centre (AWMC), University of Queensland. He completed his PhD at The University of Califor- nia, Berkeley in 2006, earning himself a reputation among colleagues as a ‘pioneer of metagenomics’. In May 2009 he returned to Australia to start his Mike Manefield, Dee Carter, Lyn Gilbert and Theo Sloots.

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BD ASM Student Travel Awardees Victoria Sushama Telwatte, Burnet Institute. South Australia Hayden Thain, Flinders University. New South Wales Amelia Hynen, University of Technology Sydney. Tasmania Christopher Atkinson, University of Tasmania. Queensland Agathe Colmant, University of Queensland. Western Australia Lucy Furfaro, Amelia Hynen, Sushama Telwatte, Christopher Atkinson, Lucy Furfaro, University of Western Australia. Hayden Thain and Agathe Colmant.

Science meets Parliament 2015

Hayley J Newton A and Kate L SeibB ADepartment of Microbiology and Immunology at The Peter Doherty Institute for Infection and Immunity, University of Melbourne BInstitute for Glycomics, Griffith University

Science meets Parliament is an annual two-day event conducted Department of Industry and Science) talked about ‘The messy nature of the policymaking process’. by Science and Technology Australia (STA). This workshop brings * Professor Graham Durant AM (Director, Questacon) asked us approximately 200 Australian scientists, from all fields, to the ‘Who is Inspiring Australia?’ and talked about Questacon’s fi Australian Parliament for a series of workshop and networking initiatives to bring scienti c achievement into the public eye. * Professor Brian Schmidt AC (Nobel Laureate and ARC Laureate events that aim to unravel the mysteries of the parliamentary Fellow, ANU) and Professor Hugh White AO (Professor of Strategic and policy-making process while also providing an opportunity to Studies, ANU) reflected on their own experiences in a session on ‘How to talk/think like a policymaker’. meet with the nation’s top parliamentarians and impress on them * Dr Rod Lamberts and Dr Will Grant (Centre for the Public the importance of supporting scientific research. Awareness of Science, ANU) concluded the day with an interactive session on how to pitch your research in two-minute The Australian Society for Microbiology was represented at Science presentations aimed at ‘Getting your science out of the lab’. meets Parliament 2015 by Dr Kate Seib and Dr Hayley Newton. Day one concluded with a Gala dinner in the Great Hall at Parliament This was the 15th Science meets Parliament and it was jam-packed House. Many MPs attended the event where we heard from Ms with panel discussions, presentations and networking opportunities Catherine Livingstone AO, President Business Council of Australia, that allowed the scientific audience to come to terms with how about issues including the importance of knowledge infrastructure different our world is from the world of politics. and STEM education. The Hon Ian Macfarlane, Minister for Industry Day one focused on developing skills for communicating science. and Science, discussed the importance of science in our lives There were multiple panel discussions from those involved in the on a day-to-day basis, and to our national prosperity. He also media, policy making and lobbying parliament, discussing philo- highlighted Australia’s poor performance in terms of collaboration sophical and operational obstacles that scientists must be aware of between business and research organisations, and the need to when communicating with politicians. work together across disciplines and sectors to translate research

* MsAlison Carabine (Political Editor, ABC Radio National Breakfast) into goods and services, new technologies and life improvements. and Mr James Massola (Political Correspondent, Fairfax Media) Similarly, the Hon Bill Shorten, Leader of the Opposition, discussed gave an overview of ‘A day in the life of a journalist’ highlighting the tight time frames involved in the press world and what they the importance of developing a National STEM Strategy and his need to turn our science into news. view that it is time to make science a national political priority. * Ms Jannette Cotterell (Managing Director, Executive Counsel Australia) and Mr Simon Banks (Managing Director, Hawker Britton) discussed ‘The art of the political meeting’, while Among the diverse events of Day two was lunch at the National Mr Martin Hoffman (Deputy Secretary, Science Group, Press Club with an address from Australia’s Chief Scientist Professor

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Ian Chubb. Professor Chubb reflected on his time in this position development for several infectious diseases. Mr Wood also outlined and talked of his vision for the future of Australia whereby some of the environmental issues in his electorate that he was science education was prioritised in schools not only to nurture interested in, including weed and pest management. curiosity but also to develop a much higher societal scientific Attending question time in the House of Representatives during the literacy. Professor Chubb stressed the notion that science is final afternoon was an interesting experience. From a scientist’s not only valuable but has quantifiable value as demonstrated perspective it is hard to understand how this process is particularly by his release of a report quantifying the huge contribution of productive. physics, chemistry, earth science and mathematical sciences to fi our economy over the past 20 years. He also reminded his scienti c Overall, the two-day program provided the scientists involved fi audience to be consistently relentless in our scienti c pursuits with new insights into the workings of another ‘3P’s’ – Parliament, ‘ ’ ’ and stressed the importance of the 3Ps : Passion, Patience and Politicians and the Press. Hopefully, the politicians involved also Persistence. gained an increased understanding of science and its benefits to

Day two also included meetings between scientists and parliamen- the community as a whole. tarians at Parliament House. Hayley met with the Shadow Minister for Agriculture and Rural Affairs and Federal Member for Hunter, Joel Fitzgibbon. They discussed the impact of microbiological pathogens in the agriculture setting and the importance of understanding these animal and sometimes-human pathogens that threaten our important farming industry as well as our native wildlife. Mr Fitzgibbon, as a former Minister for Defense, also expressed a keen interest in opinions of microbiologists in our biosecurity policies. Mr Fitzgibbon holds a genuine appreciation and interest in science and acknowledges the vital role science plays in our society. Kate met with the Federal Member for La Trobe, Jason Wood, and discussed the growing problem of antibiotic Professor Brian Cooke, Dr Kate Seib and Dr Hayley Newton attending the Science meets Parliament 2015 Gala dinner. Photo courtesy of resistant bacteria and the need for an increased focus on vaccine Lorna Sim.

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Visit from Turkish Society of Microbiology

As part of ASM’s interactions with other microbiology societies, Also during his visit he, along with Jon Iredell, Peter Traynor and Prof Ahmet Basustao¸ glu was welcomed to the ASM Conference Colonel Yıldırım Günes,¸ the Turkish Defence Attaché, laid a wreath in Canberra. Prof Basustao¸ glu, representing the Turkish Society at the Australian War Memorial to honour the fallen soldiers. Also of Microbiology, wrote two articles for last November’s issue of present were Ipek Kurtböke, and Ian and Jo Macreadie. Microbiology Australia. The theme of that issue was ‘Microbial The wreath laying was part of the daily Last Post Ceremony. The full diseases and products that shaped world history’. ceremony can be viewed at: https://www.awm.gov.au/collection/ PAFU2015/304.01/ and https://static.awm.gov.au/video/PAFU2015/ 304.01.mp4.

Yıldırım Gu¨ nes,¸ Peter Traynor, Jon Iredell and Ahmet Basustao¸ glu.

Jon Iredell and Ahmet Basustao¸ glu.

In Canberra he delivered a moving talk about the infections and infectious diseases affecting Turks in WWI and Turkey’s contribu- tions to the development of vaccination practices. His talk was of great interest and attended by Turkish Embassy officials and guests. His powerpoint presentation has now been edited by Dr Ipek Peter Traynor, Ahmet Basustao¸ glu Yıldırım Gu¨ nes,¸ Ipek Kurtbo¨ ke, Jo Kurtböke and is available for viewing at www.theasm.org.au. Macreadie and Ian Macreadie.

142 10.1071/MA15051 MICROBIOLOGY AUSTRALIA * SEPTEMBER 2015 ASM Affairs

Vale Stephen Davis

Compiled by the SA NT Committee

It is with great sadness that we adjunct lecturer in the School of Pharmacy and Medical Sciences acknowledge the passing of Mr at the University of South Australia. He presented and supervised Stephen Davis who passed away many specialist Mycology lectures and practical workshops for on Thursday 16 July after a long Laboratory Medicine students as well as providing much of the illness. materials for the workshops. In doing so he has given many students the benefit of his years of experience in diagnostic microbiology and Stephen was a Senior Medical encouraged them to pursue careers in this area. Teaching Mycology Scientist and Mycology specialist was a particular love for Steve. He was a great teacher, delivering in the Microbiology and Infectious his lectures and workshops in a down to earth fashion and with Diseases Laboratories at the great passion. Women’sandChildren’sHospital/ SA Pathology in Adelaide. Steve was a long time ASM member and a stalwart supporter of the Steven worked in various roles in diagnostic Microbiology for almost society. He was highly involved in the SA NT branch spending 40 years. He began his working life in the Microbiology laboratory at many years on the local branch committee as an enthusiastic the then Adelaide Children’s Hospital in 1975. He soon developed a committee member. He took on the role of Scientific Meetings keen interest in Mycology and became part of the Mycology Refer- Convenor for a number of years and was branch Chair from 2007 to ence Laboratory as the Microbiology department evolved into the 2009. He supported and included all areas of Microbiology and Women’s and Children’s Hospital. members found him extremely approachable and supportive. He loved sharing his knowledge and was a regular speaker at SA He devoted much of his working life specialising in Mycology, NT Branch scientific meetings giving interesting and entertaining particularly identification and differentiation of problematic fungi. Mycology special interest and clinical case presentations. He re- Steve’s contributions to Microbiology in South Australia have been ceived an ASM Distinguished Service Award in 2014. significant. Steve will be greatly missed by his many work and ASM friends and In addition to his professional position and contributions to the colleagues. science of Mycology (medical and non-medical), he was also an

MICROBIOLOGY AUSTRALIA * SEPTEMBER 2015 10.1071/MA15052 143 Hot Topic

Out of Africa: response to Ebola in the developed world; lessons for the future

Lyn Gilbert Peter Collignon Centre for Infectious Diseases and Microbiology ACT Pathology Westmead Hospital and Canberra Hospital University of Sydney Garran, ACT, Australia Email: [email protected] Email: [email protected]

The unprecedented, ongoing Ebola virus disease (‘Ebola’) outbreak security’ and established the Mission for Ebola Emergency Re- in West Africa has caused nearly 28 000 cases and more than 11 000 sponse (UNMEER), with powers similar to that of a peacekeeping deaths between December 2013 and July 20151, including 876 cases mission – the first of its kind for a health emergency8. and 509 deaths among healthcare workers (HCW). It is over 60 times Meanwhile, the American aid-workers had recovered after larger than any of 26 previous outbreaks, since 1976 when Ebola was several weeks’ treatment in the high security containment facility first recognised, the largest of which involved ~450 cases (Figure 1). at the Emory University Hospital, which had been built by the It is the first to have occurred in West Africa and spread across Centers for Disease Control (CDC) to care for returning aid-workers national borders, to major cities and beyond Africa. with highly transmissible diseases. The CDC had reassured Amer- It came to attention in March 2014, when the Guinean Ministry of icans that there was minimal public risk (Figure 2)9,10, since Ebola fi ‘ ’ Health noti ed WHO of a mysterious disease that had been spreads only by contact with blood or body fluids of patients in the occurring in Guéckédou province, since December. Although it ‘wet’ stage of the disease – not by the respiratory route – and fi had not previously occurred in the region, Ebola was con rmed hospital spread could be prevented by strict droplet and contact 2 a week later . By the end of March, cases were reported in neigh- precautions. bouring Liberia and, later, Sierra Leone3. This reassurance was first challenged when a nurse became ill, after Despite warnings from Médicins sans Frontières (MSF), in June caring for a Spanish priest, who had died from Ebola in Madrid after 2014, that the outbreak was out of control4 and increasingly urgent being evacuated from West Africa11. Then, a Liberian-American man, calls for international help, there was little response until August, who had recently returned from Liberia, was admitted to hospital in when two American aid-workers were evacuated to USA from Dallas, Texas, seriously ill with abdominal pain and diarrhoea; Ebola Liberia, with Ebola. A week later (8 August), WHO belatedly ac- was diagnosed two days later. When two nurses who had cared for knowledged that the outbreak was ‘a public health emergency of him became ill with Ebola there was a storm of recrimination and international concern’5. In early September, when the grossly fear in America12. The CDC Director, Tom Frieden, suggested, inadequate local health workforce had been further depleted by reasonably, that the nurses’ infections had resulted from breaches HCW deaths and patients were being turned away from over- of infection control (IC) protocol13,14; this infuriated the nurses’ crowded hospitals, an increasingly frustrated MSF President de- union, who claimed the hospital had no protocols15. Frieden nounced the ‘dangerously inadequate’ international response as a apologised for apparently blaming the nurses, but pointed out ‘coalition of inaction’6 and challenged the West to provide people that all hospitals should have appropriate transmission-based IC rather than financial promises. Within two weeks, the US president protocols16. announced deployment of 3000 military personnel to build treat- ment centres and train HCWs7. On 18 September the UN Security Nevertheless, CDC changed their IC guidelines and recommended Council declared the outbreak ‘a threat to international peace and personal protective equipment (PPE) to include respiratory route

144 10.1071/MA15053 MICROBIOLOGY AUSTRALIA * SEPTEMBER 2015 Hot Topic

Guinea, Liberia, Sierra Leone, 2013-15, 27,000 cases

60 x larger Nigeria, 2014 (20 cases) than any previous outbreak

Figure 1. Ebola cases by location in Africa. protection by using P2/N95 mask or powered air-purifying respirator African HCWs indicated increased virulence and/or transmissibility (PAPR), rather than just a water-repellent surgical mask. They also of Ebola virus and cited unconfirmed reports of possible respiratory recommended complete coverage of all mucous membranes, skin transmission. Others strongly advocated ‘routine’ contact and and hair and scrupulous care with removal17. It was not clear exactly droplet precautions because (a) PPE was widely available and HCWs how the nurses became infected, but hasty removal of PPE carries a were familiar with its use; (b) there were other explanations for high risk of skin and/or mucous membrane contamination. CDC high HCW infection rates in Africa and no evidence of increased recommended intensive and repeated training of staff, short shifts transmissibility or virulence; and (c) the diagnosis was far more likely (to prevent excessive sweating and fatigue) and supervision by a to be a common traveller’s infection, such as malaria, and excessive ‘buddy’, to provide reassurance, check integrity of PPE and ensure caution could delay appropriate diagnosis and management20.In its removal in correct sequence18. general, the latter, moderate view prevailed.

In the circumstances, these recommendations were reasonable There was further controversy, when a MSF volunteer, developed for care of patients with ‘wet’ Ebola and were reflected in the IC Ebola 6 days after returning to New York from Guinea. According guidelines of most western countries, including Australia. However, to protocol he had taken his temperature twice daily and immedi- there was controversy about what PPE should be used for initial ately reported to hospital when it rose. Although quarantine was management of people who had recently been in an affected not required while he remained well – asymptomatic people do country, possibly had contact with Ebola, had ‘dry’ symptoms, such not spread Ebola virus – some media and politicians accused him as fever, headache, malaise but not vomiting, diarrhoea or bleeding of recklessly endangering public safety21. The New York and New and were awaiting test results. In Australia19 and elsewhere, some Jersey governors announced that, in future, anyone returning authorities, evoking the precautionary principle, advocated full from an Ebola-affected country, after possible contact, would be PPE, as for care of a ‘wet’ case; they feared that the high toll among quarantined for the 21-day incubation period22. After a volunteer

MICROBIOLOGY AUSTRALIA * SEPTEMBER 2015 145 Hot Topic

Figure 2. CDC fact sheet on Ebola in the USA. nurse was summarily quarantined in New Jersey, MSF objected Liberia was declared Ebola-free but, at the time of writing (July 2015), strongly because, like CDC and the White House, they pointed out cases are still occurring – albeit in much reduced numbers – in Sierra that it was unnecessary and would discourage HCWs from Leone and Guinea and six new cases have occurred in Liberia1. The volunteering23. threat is not over, but the turnaround confirms past experience, that Ebola outbreaks can be controlled relatively quickly, once the Meanwhile, the tide had begun to turn in Africa, due to the co- necessary ‘staff, stuff, space and systems’25 are available. A rapid ordinated efforts of UNMEER, local and foreign government agen- response to MSF’s warnings and pleas for help, in June 2014, could cies and NGOs which had established treatment units, community have prevented much of what followed. Criticism of WHO for its care centres and laboratories and trained many more foreign and slow response was justified and has been acknowledged by the local HCWs, contact tracers and safe burial teams24. On 9 May 2015, Director-General26; but WHO was not – and still is not – adequately

146 MICROBIOLOGY AUSTRALIA * SEPTEMBER 2015 Hot Topic

27 resourced to respond, rapidly, to infectious disease emergencies . 10. Centers for Disease Control Guidelines for Evaluation of US Patients Suspected of A concerted international effort will be needed to establish a global Having Ebola Virus Disease. (2014) 1 August 2014. https://www.dallascounty.org/ department/hhs/documents/CDCEbolaHealthAdvisory08-01-2014_000.pdf 28 emergency response workforce and build resilient health systems 11. World Health Organization. Spanish nurse diagnosed with Ebola virus disease. in developing countries, including hospitals with adequate supplies http://www.euro.who.int/en/health-topics/emergencies/pages/news/news/2014/ 10/spanish-nurse-diagnosed-with-ebola-virus-disease of PPE and appropriately trained healthcare workers. 12. Burrough, B. (2015) Trial by Ebola. Vanity Fair February. Available at: http://www. vanityfair.com/news/2015/02/ebola-us-dallasepidemic (accessed 24 February 2015). The threat to HCWs in developed countries from imported Ebola 13. Centers for Disease Control. (2014) CDC update on Dallas Ebola Response, 10-12- and the high mortality among African HCWs focussed attention on 2014. http://www.cdc.gov/media/releases/2014/t1012-ebola-reponse-update.html hospital IC like nothing before. Many hospitals spent a fortune 14. http://www.cbsnews.com/news/cdc-chief-on-second-ebola-case-there-was-a- breach-in-protocol/ preparing facilities and staff for a possible Ebola case, often in the 15. http://www.cbsnews.com/news/nurses-union-dallas-hospital-lacked-ebola- context of previously dwindling IC budgets and poor staff compli- protocols/ 29 ance with IC practices . Has this experience, taught us how to stop 16. http://www.pbs.org/newshour/rundown/cdc-hold-news-conference-ebola-re- the next infectious disease emergency at its source and avoid the sponse-u-s/ 17. http://www.cdc.gov/media/releases/2014/fs1020-ebola-personal-protective- desperate scramble, elsewhere, to train and equip hospital staff equipment.html for what should be ‘business as usual’? Without strong political 18. Centers for Disease Control and Prevention. (2014) Guidance on personal and professional leadership there is a risk that, once the crisis protective equipment to be used by healthcare workers during management of patients with Ebola virus disease in US hospitals, including procedures for putting passes, lessons will be forgotten, funds redeployed and bad habits on (donning) and removing (doffing). Atlanta, GA: CDC. reinstated30. 19. Department of Health Australia. (2015) Infection prevention and control princi- ples and recommendations for Ebola virus disease. http://www.health.gov.au/ internet/main/publishing.nsf/Content/D9CE7F7977BFB6A8CA257D8D00834F53/$ References File/ebola-infections-prevention-final-Mar2015.pdf 1. World Health Organization. (2015) Ebola situation report – 15 July 2015. http:// 20. Gilbert, GL and Kerridge, I (2015) Communication and communicable apps.who.int/ebola/current-situation/ebola-situation-report-15-july-2015 disease control: lessons from Ebola virus disease. Amer. J. Bioethics 15,62–65. 2. World Health Organization. (2015) Ground zero in Guinea: the outbreak doi:10.1080/15265161.2015.1009564 smoulders – undetected – for more than 3 months. A retrospective on the first 21. Spencer, C. (2015) Having and fighting Ebola — public health lessons from a cases of the outbreak. http://www.who.int/csr/disease/ebola/ebola-6-months/ clinician turned patient. N. Engl. J. Med. 372,1089–1091. doi:10.1056/NEJMp guinea/en/ 1501355 3. WHO Ebola Response Team. (2014) Ebola virus disease in West Africa – the first 22. http://www.usatoday.com/story/news/2015/02/25/craig-spencer-ebola-doctor/ 9 months of the epidemic and forward projections. N. Engl. J. Med. 371, 24004889/ – 1481 1495. doi:10.1056/NEJMoa1411100 23. Medicins sans Frontiere. MSF nurse held in isolation in New Jersey. http://www. 4. Médicins sans Frontières. (2014) ‘The epidemic is out of control.’ 23 June 2014. doctorswithoutborders.org/article/msf-nurse-held-isolation-new-jersey http://www.msf.ca/en/article/ebola-west-africa-epidemic-out-control 24. United Nations. (2015) Global Ebola response. Making a difference – progress 5. World Health Organization. (2014) Statement on the 1st meeting of the IHR report 2015. https://ebolaresponse.un.org/progress-2015 Emergency Committee on the 2014 Ebola outbreak in West Africa. 8 August 2014. 25. Farmer, P. (2014) London Review of Books. Diary 23 October 2014. http://www.lrb. http://www.who.int/mediacentre/news/statements/2014/ebola-20140808/en/ co.uk/v36/n20/paul-farmer/diary 6. Médicins sans Frontières. (2014) Global bio-disaster response urgently needed in 26. WHO Director-General’s speech at the sixty-eight World Health Assembly. http:// Ebola fight. 2 September 2014. http://www.msf.org/article/global-bio-disaster- www.who.int/dg/speeches/2015/68th-wha/en/ response-urgently-needed-ebola-fight 27. Wilkinson, A., and Leach, M. (2015) Briefing: Ebola—Myths, realities and structural 7. Cooper, H. et al. (2014) U.S. to commit up to 3,000 troops to fight Ebola in Africa. violence. African Affairs 114/454,136–148. doi:10.1093/afraf/adu080 New York Times 15 September 2014. http://www.nytimes.com/2014/09/16/world/ africa/obama-to-announce-expanded-effort-against-ebola.html 28. Gates, B. (2015) The next epidemic – lessons from Ebola. N. Engl. J. Med. 372, 1381–1384. doi:10.1056/NEJMp1502918 8. UN News Centre. (2014) UN announces mission to combat Ebola, declares outbreak ‘threat to peace and security’. 18 September 2014. http://www.un. 29. Drazen, J. M., et al. (2015) Ebola in West Africa at one year—From ignorance to fear org/apps/news/story.asp?NewsID=48746#.Va3PO7c0H-Y to road blocks. N. Engl. J. Med. 372, 563–564. doi:10.1056/NEJMe1415398 9. Centers for Disease Control. Facts about Ebola in the US. http://www.cdc.gov/vhf/ 30. Gostin, L.O. et al. (2015) The President’s national security agenda: curtailing ebola/pdf/infographic.pdf Ebola, safeguarding the future. JAMA 313,27–28. doi:10.1001/jama.2014.16572

Future issues of Microbiology Australia

November 2015: Congenital cytomegalovirus and congenital infections Guest Editor: Bill Rawlinson

March 2016: Parasitology Guest Editor: Harsha Sheory and Richard Bradbury

May 2016: Education: graduate employability and curriculum design Guest Editor: Danilla Grando

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