The Gut Microbiome in Diverticulitis and Diverticulosis
The Gut Microbiome in Diverticulitis and Diverticulosis
Colonic diverticula are outpouchings of the large bowel, and they occur in up to 60% of
 people over 60 years of age. About 10-25% of patients with diverticula will have symptoms.
 These can range from acute diverticulitis, which can be a lethal infection to symptomatic
 diverticular disease, which involves inflammation of the bowel and altered bowel habits,
 decreasing patients' quality of life. We do not know which patients will develop acute
 diverticulitis or which patients will develop diverticula in their colon.

 We believe that diverticulitis may be associated with, or even caused by, alterations in the
 bacteria that live in the colon, known as the gut microbiome. Until recently it was too
 expensive and too complex to examine the microbiome in detail. We propose to examine for the
 first time in detail the microbiome of patients with acute diverticulitis and asymptomatic
 diverticulosis.

 Stool samples will be analysed for gut microbiome composition by 16S ribosomal RNA gene
 pyrosequencing. There is a part of the bacterial cell, the ribosome, which is the same in all
 bacteria (16S). Through PCR, polymerase chain reaction, and sequencing, we can separate out
 the different types of bacteria in a sample. We can then look at the different kinds of
 bacteria in each patient population, as well as how diverse the populations are within the
 groups, and compared to other groups.

 We hope to be able to discriminate between the microbiome of patients with acute
 diverticulitis and asymptomatic diverticulosis. This study many change how diverticulitis and
 diverticulosis are conceptualized and treated. Alterations in the microbiome in these disease
 states may be able to be treated, preventing further disease.
Diverticulosis affects 60% of people over 60 and roughly 10-25% of individuals will have
 symptoms, ranging from acute diverticulitis requiring emergent intervention to symptomatic
 diverticular disease with persistent lower abdominal pain and altered bowel habits. Up to 5%
 of patients with acute diverticulitis will die while hospitalized. In spite of the prevalence
 and severity of its manifestations, fundamental questions remain about the nature of
 diverticulitis and diverticulosis.

 The human colon contains 100 trillion bacteria, including hundreds of species and 10 times as
 many genes as the human genome. While alterations in the microbiome have been implicated in
 disease states such as Irritable Bowel Syndrome (IBS) or Inflammatory Bowel Disease (IBD),
 the microbiome has not been adequately described in relation to colonic diverticula. As
 King's College Hospital has the only known dedicated Diverticular Disease clinic, we have a
 uniquely large patient population in which to learn about this condition.

 When colonic diverticula become inflamed or perforate, patients have diverticulitis. Inciting
 factors for episodes of acute diverticulitis are not well characterized, but include age,
 lifestyle factors and certain medications. Clinicians have no way to predict which patients
 will get diverticulitis, nor how severe its presentation will be. In some cases of
 pericolonic diverticulitis (known as Hinchey I), inflammation may resolve even without
 antibiotics, while in other cases, perforated diverticulitis will include feculent
 peritonitis (Hinchey IV) and require emergent colon resection. Diverticulitis may be caused
 by alterations in the microbiome, but this has not been sufficiently addressed.

 The majority of patients with asymptomatic diverticulosis, that is, the presence of colonic
 diverticula in the absence of symptoms, will never experience an episode of diverticulitis.
 There has been, however, some limited evidence that the microbiome in diverticulosis is
 altered, and similar to that in acute diverticulitis. If that is so, then predicting which
 patients will develop acute diverticulitis will remain impossible. It will also mean that
 research which did not assess or control for the presence of diverticula in patient
 populations will need to be revisited.

 Currently, clinicians have no basis to predict (1) which patients will develop severe acute
 diverticulitis requiring surgery or (2) which patients will develop acute diverticulitis
 which will resolve with antibiotics. We believe that further insights into the gut microbiome
 will allow for better diagnostics and treatments in the future.

 There have been a few, limited studies of the microbiome in diverticulitis, but not full
 pyrosequencing. This is because research costs have fallen 5 orders of magnitude in the past
 decade while computational capacity has increased. Studies have looked at the individual
 strains of bacteria within stool or mucosal samples, which does not suffice to understand the
 complex microbiome.

 As the gut microbiome has not been thoroughly investigated in diverticular disease, this is
 an exploratory study. However, we do wish to test the hypotheses that patients with
 asymptomatic diverticulosis have a microbiome more similar to patients with acute
 diverticulitis, than to that of normal controls.

 14. Methodology Setting: King's College Hospital, a tertiary referral hospital in south
 London. King's is a referral centre for the work up of two-week wait for colorectal cancer
 (2ww) assessments, with 900 new patient referrals assessed per year. Also, the Accident &
 Emergency Department at King's admits up to 250 patients per year with acute diverticulitis.

 Patients: 4 sets of patients will be recruited. 25 patients per group will be recruited:

 Groups A and B. Patients with acute diverticulitis will be recruited from the A&E department
 at King's, prior to commencement of antibiotics. Rectal swabs will be obtained from patients
 with a chief complaint of acute abdominal pain upon initial assessment, prior to initial
 antibiotics. All specimens will be initially frozen, but only patients specimens with an
 admitting diagnosis of acute diverticulitis and colonic imaging (CT imaging or visualization
 at surgery) will be analysed. Patients with acute diverticulitis will be assessed in 2
 groups, those patients with perforated diverticulitis (i.e., Hinchey III or IV) and those
 patients with unperforated diverticulitis (Hinchey I or II).

 Group C. Patients with asymptomatic diverticulosis will be recruited from the 2-week wait
 (2ww) colorectal cancer pathway. Patients presenting with a chief complaint of fresh rectal
 bleeding, with no other complaints, will be recruited prior to diagnostic imaging, either by
 CT, CT colonography, or endoscopy. They will provide a stool sample or rectal swab prior to
 bowel cleansing, if required for their evaluation, as that may alter the microbiome. Those
 patient samples with diverticulosis (and or haemorrhoids, as the other most common cause of
 fresh PR bleeding) will be analysed.

 Group D. Patients with or without haemorrhoids, and no other colonic pathology will be
 recruited from the 2ww colorectal cancer pathway. These will be the normal controls. Patients
 presenting with a chief complaint of fresh PR bleeding, with no other complaints, will be
 recruited prior to diagnostic imaging, either by CT, CT colonography, or endoscopy. They will
 provide a stool sample or rectal swab prior to bowel cleansing, if required for their
 evaluation, as that may alter the microbiome. Those patient samples with diverticulosis (and
 or haemorrhoids, as the other most common cause of fresh PR bleeding) will be analysed.

 Stool analysis: Stool samples will be analysed for faecal microbiome composition by 16S
 ribosomal RNA gene pyrosequencing, through a cooperative agreement with the Bruce lab at
 King's College London.

 In brief, gene targeted 16S rRNA sequencing examines a structural component of the bacterial
 ribosome through targeted primers. Constant portions of the ribosomal RNA are amplified,
 which allows for identification of the variable portions. Sequences are then clustered in to
 OTUs, operational taxonomic units and may be assigned to lineages from genus all the way to
 phyla.

 Planned statistical analyses: Microbiome composition will be assessed in several different
 ways. Alpha diversity will be measured, which is a measure of the average diversity within a
 sample. Multiple assessments of alpha diversity will be made, including Chaol richness
 estimate, OTU richness, and Shannon index. Alpha diversity has been shown to be reduced in
 patients with active IBD as compared to patients with quiescent disease, but has not been
 described in diverticular disease. Beta diversity is a comparison of diversity between
 samples (that is, the separation of the phylogenetic structure of the OTUs in one sample as
 compared to all other samples) will be calculated through Unifrac distances.

 Power calculation: Metabonomic studies of the gut microbiome at this time are necessarily
 exploratory. As such power calculations have not been performed in the field. However, in
 this instance analysing 25 samples per group should be sufficient to discriminate between
 groups and assess their diversity.

 Existing infrastructure: All infrastructure needed is currently in place. The study team
 (Professor Bjarnason and Mr Papagrigoriadis) currently manage the 2ww clinics, as well as
 attend to acute admissions in A&E at King's College Hospital. A materials transfer agreement
 is in place with the Bruce lab at King's College London for transfer of other faecal samples,
 and would only need to be amended.
Inclusion Criteria:

 Groups A and B. Patients with acute diverticulitis - patients presenting to A&E at King's
 College Hospital with new onset abdominal pain, with or without a known diagnosis of
 diverticulitis

 Groups C and D. Patients with asymptomatic diverticulosis and normal controls

 - Patients being assessed through the 2ww Colorectal Cancer pathway with a chief complaint
 of fresh PR bleeding and no other 'red flag' symptoms, who are not overly anxious over
 their diagnosis as assessed by a physician outside the study team.

 Exclusion Criteria:

 Groups A and B. Patients with acute diverticulitis

 -  Patients in extremis- e.g. systolic blood pressure less than 80 on arrival or pulse
 greater than 115

 -  Patients who have received antibiotics in the three months prior to presentation.

 -  Vulnerable patient populations

 Groups C and D. Patients with asymptomatic diverticulosis and normal controls

 -  Patients with high levels of pre-test anxiety, as determined by their assessing
 physician, outside the study team

 -  Patients who received antibiotics for any reason in the 3 months prior to inclusion

 -  Vulnerable patient populations