The role of Bacteroides fragilis in the development of colorectal cancer. Jacqui Keenan Department of Surgery

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1 The role of Bacteroides fragilis in the development of colorectal cancer Jacqui Keenan Department of Surgery

2 Microbial dysbiosis is observed in CRC patients Sobhani et al. PLoS ONE, 2011

3 Are there strains of bacteria that drive carcinogenesis in an otherwise healthy colon?

4 Human intestinal microbiota across the life cycle Kostic A D et al. Genes Dev. 2013

5 H. pylori: a bacterial driver of gastric cancer Normal stomach Chronic gastritis Atrophic gastritis Intestinal metaplasia Adenoma /dysplasia Intestinaltype gastric cancer Risk factors - bacterial virulence factors - bacterial & host cell interaction - host reactivity Years Adapted from P. Correa. 1992

6 A bacterial driver-passenger model for colorectal cancer Years Tjalsma et al. Nature Reviews, 2012 A driver bacterium needs to be toxigenic. It also need to be persistent.

7 Bacterial toxins and cancer Campylobacter jejuni Escherichia coli Bacteroides fragilis Salmonella typhi Shigella flexneri Shigella dysenteriae Lax. Nature Reviews. 2005

8 Time-line of human history (yr bp) Effective population size Major source of microbial transmission Nature of immunity Example Persistence Most ancient (>50,000) Isolated huntergatherers (<100) Maternal/ intrafamilial Ineffective Bacteroides species, H. pylori Active Intermediate (10,000 50,000) Communicating huntergatherer groups (<100 to 10,000) Long-term carriers Containment but not elimination M. tuberculosis, S. typhi, varicella-zoster virus Latency Recent (<10,000) Large societies (>500,000) Acutely ill persons Life long Measles No Very recent (<200) >10 million Acute infection Serotype-specific Pandemic influenza No Blaser & Kirschner, Nature, 2007

9 Bacteriodes spp. B. acidifaciens B. caccae B. coprocola B. coprosuis B. dorei B. eggerthii B. finegoldii B. fragilis B. goldsteinii B. helcogenes B. intestinalis B. johnsonii B. massiliensis B. nordii B. ovatus B. plebeius B. pyogenes B. salyersai B. thetaiotaomicron B. uniformis B. vulgatus The presence of Bacteroides spp. in the colonic flora appears to be integral to human health Wick & Sears, Curr Opin Infect Dis, 2011 Bacteroides spp. contribute to the host s nutritional status by converting undigested dietary carbohydrates into readily absorbed nutrients. Bacteroides spp. may also protect against the development of colitis and diarrheal disease.

10 Enterotoxigenic B. fragilis (ETBF) 1 st discovered in 1984 in lambs presenting with acute diarrhoeal illness. Subsequently isolated ETBF from other young animals, and from symptomatic children. Like H. pylori, some strains of B. fragilis express a toxin Globally distributed and asymptomatic carriage is common.

11 Enterotoigenic B. fragilis toxin cleaves E-cadherin Sears et al. J Clin Invest. 2014

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13 Pilot study Are toxin-producing strains of Bacteroides fragilis associated with an increased risk of CRC? Community volunteers (controls) Patients with colorectal cancer, selected on diagnosis following colonoscopy and preoperation, and who had not received chemotherapy and/or radiotherapy Method PCR-based method to detect B. fragilis toxin gene in DNA extracted from a stool sample Results Controls CRC P value bft +ve 7/71 (10%) 19/71(27%) Keenan, Aitchison et al. Anaerobe, 2016

14 Are toxin-producing strains of Bacteroides fragilis associated with an increased risk of CRC? Follow-up study Biopsies collected from up to 4 sites through the colon back in 2003 (at that time looking for evidence of Helicobacter spp in the colon). The DNA extracted from these samples was kept (and survived the Christchurch earthquakes). The presence of the B. fragilis toxin gene in each tissue sample was determined using quantitative PCR, and associations with clinicopathological characteristics over time were analysed.

15 bft gene (74/150) Low grade dysplasia, p=0.007 Tubular adenoma, p=0.027 Colonisation with enterotoxigenic B. fragilis is associated with early-stage colorectal neoplasia Hyperplastic polyp/ssa, p=0.007 Any neoplastic lesion, p= Purcell et al. PLoS ONE, 2017

16 Where to from here? 1. Is carriage of ETBF a biomarker for early stage intervention? 2. Can we reduce risk in infected individuals? 3. Sheep as a model of infection?

17 1. Is ETBF a biomarker for early stage intervention? GP-derived cohort Healthy controls CRC patients Number Median age ETBF detected in stool (51-70) 14 (8%) (40-74) 5 (9%) (63-76) 17 (30%)

18 Rare Colonization Common 2. Can we reduce risk in infected individuals? Modified from Blaser, 2005

19 T o x i n t i t r a t i o n Can diet increase/decrease the risk of carcinogenesis in ETBF-infected individuals? H e m i n ( u g / m l ) BFT toxin levels increase proportional to bacterial growth in heme iron

20 Chemoprevention of colorectal neoplasia appears to be a role for aspirin in some groups Wakeman et al. ANZ J Med. 2015

21 Aspirin protects HT29 cells from BFT-mediated cleavage of E-cadherin Un-Infected 40x 40x 40x Untreated cells Cells infected with ETBF ETBF infection + 5mM aspirin green, E-cadherin Blue, nuclei

22 C D H 1 e x p r e s s i o n Aspirin increases E-cadherin expression in HT29 cells Untreated HT29 cells * * * x Magnification HT29 cells + 5mM aspirin A s p i r i n ( m M ) 40x Magnification green, E-cadherin Blue, nuclei Keenan, Aitchison et al. unpublished

23 3. Sheep as a model of infection? Normal small intestine Adenocarcinoma of small bowel (arrowed) and mesentery (asterisk), with peritoneal metastases (arrowheads) 7% of older sheep develop intestinal adenocarcinomas Munday et al. Comp. Medicine 2006

24 We find molecular evidence of the B. fragilis toxin gene in 35% of sheep with intestinal adenocarcinomas ETBF Sheep (SIA) (tumour tissue) 7/20 (35%) Human (CRC) (stool sample) 19/71 (27%)

25 Lambs, calves foals and piglets are susceptible to ETBFassociated diarrhoeal illness that is largely self-limited. Are farm animals a source of infection in young children?

26 With thanks to Univ. of Otago Christchurch Alan Aitchison Rachel Purcell James Swan John Pearson Frank Frizelle Southern Community Labs. Jacquie Leamon Canterbury Health Labs. Rosie Greenlees Massey University John Munday

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28 Global distributions and abundances of human gut microorganisms at the phylum and genus levels Wexler & Goodman, Nature, 2017

29 Impact of diet in shaping gut microbiota BF (Burkina Faso, West Africa) EU (Florence, Italy) De Filippo et al. PNAS. 2010

30 Healthy adults ETBF present in stool samples New Zealand 7/71 (10%) Turkey 5/40 (12.5%) Sweden 24/194 (12.4%) Italy 6/39 (15%) Age-matched CRC patients ETBF present in stool samples Incidence of CRC New Zealand 19/71 (27%) 33.5/100,000* Turkey 21/56 (38%) 8.1/100,000* *, World Health Organisation Cancer Country Profiles 2014