4901

Clostridium cluster XIVa species an early fecal marker in radiation injury

Ishfaq
2 Ahmed ,

Shauna

1 McLaughlin ,

William

1 McLaughlin ,

Shrikant

2 Anant ,

Shahid

2 Umar

& Rao

V. L.

*1,2,3 Papineni .

1 Precision

X-Ray Inc, North Branford, CT; 2 Kansas University Medical Center, Kansas; 3 PACT & Health LLC, Branford, USA.
* papineni@graduate.hku.hk

Luminescence Imaging of ROS Generation

Figure-2: Reactive oxygen Species activity (ROS) in real time is imaged using L-012, an ultrasensitive analog of luminol. Robust ROS activity was observed in GI region upon 10 Gy whole body irradiation. Irradiation and luminescence imaging was done using OptiMAX-IGRT 320 (Precision Xray inc) (right panel) akin to what is observed during citrobacter infection (extreme right panel). Whole Body Irradiation: Swiss-albino Mice were subjected to 10 Gy X-ray whole body irradiation doses using X-RAD 320 irradiator (1Gy/min; F2 beam hardening filter 1.5mm Al, 0.25mm Cu, 0.75mm Sn; Precision X-ray inc USA) inc, USA Control
X-ray + Luminescence

19 day

Abstract
Radiation therapy is being used in the treatment of nearly 60% of the current cancer patients. In spite of the best efforts to provide special care in minimizing radiation exposure, significant radiation injury remains a common side-effect particularly to the gastro intestinal tract. Severe injury to GI tract may lead to multiple organ dysfunction syndrome. The status of fecal microbiota has been proposed as a rapid diagnostic platform in radiation injury. Gut microbes play a role in immunity, health, and disease. Imbalances in gut microbiota are related to numerous disorders, such as inflammatory bowel disease, colorectal cancer (CRC), and atopy. In patients with immune suppression due to high-dose chemotherapy or that undergoing radiation therapy, disruption of the microbiota through antibiotics and impairment of host immunity gives rise to perturbations favoring intestinal domination by pathogenic species, resulting in increased bacterial translocation and susceptibility to systemic infection. Here, using real-time PCR assays, we assessed the immediate changes and quantitative alterations to fecal microbiota in response to radiation injury. Swiss-albino Mice were subjected to 10 Gy X-ray whole body irradiation doses using commercially available X-RAD 320 irradiator (1Gy/min; F2 beam hardening filter 1.5mm Al, 0.25mm Cu, 0.75mm Sn; Precision X-ray inc, USA). Fecal samples collected immediately after the radiation exposure was analyzed. We show that Clostridium cluster XIVa that accounts for almost 60% of the mucin-adhered microbiota is significantly increased in the fecal samples, along with the colocalizing Firmicutes. This in comparison to the total bacteria and other bacteria that tend to enrich in the luminal region. Clostridium cluster XIVa has been proposed to be the mucosal butyrate producers producing butyrate close to the epithelium. Thereby enhancing butyrate bioavailability required in treating diseases, such as inflammatory bowel disease. These findings provide opportunities to evaluate the potential use of intestinal microbiota as biomarkers in radiation injury. Also, provides insights into the microbial changes tantamount to the radiation induced GI injury. Further, significant loss in mucus layer was observed in the alcian blue stained colon sections from swiss-albino mice subjected to 10 Gy X-ray whole body irradiation dose. We demonstrate that the similar loss in mucin during CR infection is restored using natural compounds, resulting in 3-4 fold increase in the beneficial microbiota. Thus the commonalities in mechanisms of CR infection, and Radiation Injury to GI provide opportunities in the development of better intervention strategies.

X-ray Irradiation (10 Gy)

Citrobacter rodentium (CR) infection

Papineni et al AACR Meeting 2013

Causative Agent: Citrobacter rodentium Mode of inoculation: Oral (o/n cultures; 108 CFUs) Mouse strain: NIH:Swiss (outbred)

Clostridium cluster XIVa levels are Affected During Radiation Injury as well as CR Infection
30.0

Tissue (colon)
15.0 2.5

A. Alpha Proteobacteria B. Bacteroidetes phyllum C. Bifidobacterium species

Figure-5: Hostmicrobe interactions within the glycan landscape at the epithelial interface
Ouwerkerk et al Best Pract Res Clin Gastroenterol (2013)

Fold Increase

2.0

1.5

D. Clostridium cluster XIVa

1.0

E. Firmicutes Phyllum
0.5

Inner Mucus is Compromised

N
Figure-4: Intestinal Microbiota Mucus layers and Stem cells
Papineni & Umar Transl Cancer Res 2013;2(4):359-369.

F. Gamma Proteobacteria
0.0

CR

X-ray irradiation (Gy) 0 10 A

Bacterial DNA Extraction and Quantification from the Fecal pellets collected were based on the major intestinal microbiota work in mice (Salzman et al., Microbiology, 2002). Bacterial DNA Extraction- Fresh feces from control, 10 Gy X-ray irradiated, uninfected and C. rodentium infected (Day 12 p.i) NIH Swiss mice or swiss albino mice were collected on ice and dried under vacuum, and ground to powder. Total genomic bacterial DNA was extracted using QIAmp DNA stool kit (Qiagen, Valencia, CA) following their instructions. The integrity, concentration, and quality of the total DNA were assessed by agarose gel electrophoresis, and determined by absorption at A260, and A260 to A280 ratio, respectively. DNA solutions were stored at 20C until further analysis. A branched DNA (bDNA) assay based on the 16S rDNA gene was used for quantification of individual bacteria belonging to firmicutes (total 22 species), bacteroidetes (total 14 species), and other bacteria (total 11 species). The 16S rDNA gene assay was performed using QuantiGene Plex DNA Assay Reagent System (Panomics/Affymetrix, Fremont, CA) according to the manufacturer's protocol. Because the majority of the bacteria residing in the intestine are not easily cultivable, the bacterial sequences are defined as the closest known relative in the phylogenic tree (Salzman et al. 2002). Accession numbers for the 16S-rRNA genes of the corresponding bacteria are given in the following table. Total bacterial DNA (20 ng/l, 40 l) was added to lysis buffer containing probe set and allowed to incubate for 30 mins at RT, then neutralization buffer was added to the DNA probe mixture. The neutralized DNA was added to wells containing lysis buffer with blocking reagent. Sample DNA was allowed to hybridize to each probe set overnight at 55C. Subsequently, samples were then hybridized sequentially with the preamplifier, amplifier, label probe and substrate. The resulting luminescence was quantified using a luminometer set at an integration time of 0.2 sec.

0 10 B

0 10 C

0 10 D

0 10 E

0 10 F

0 10 G

0 10 H

G. Lactobacillus group

3.5

Fecal samples
3.0

2.5

Median Fluorescence Intensity

4000

Clostridium cluster XIVa Fecal samples

*
20 X 20 X

Fold Increase

3000

2.0

1.5

2000

10Gy

Bacteria Acetivibrio cellulosolvens Bacillus mycoides Bacteroides distasonis Bacteroides distasonis Bacteroides distasonis Bacteroides distasonis Bacteroides forsythus Bacteroides forsythus Bacteroides vulgatus Bacteroides acidofaciens Bacteroides distasonis Bacteroides sp. ASF519 Bifidobacterium animalis Candidatus xiphinematobactor Clostridium absonum Clostridium celerecrescence Clostridium celerecrescens Clostridium clostridiiformes Clostridium fusiformis Clostridium methylpentosum Clostridium perfringens Clostridium polysaccharolyticum Clostridium scindens Clostridium sp Clostridium sp. ASF502 Clostridium sp. ASF502 Clostridium sp. ASF502 Desulfovibrio sp. Eubacterium desmolans Eubacterium limosum Helicobactor sp. Klebsiella granulomatis Lactobacillus acidophilus Lactobacillus murinus Lactobacillus reuteri Lactobacillus salivarius Lactobacillus sp. ASF519 Lactobaciluus acidophillus Lactococcus lactis Porphyromonas sp Porphyromonas sp. Prevotella sp. Prevotella sp. Ralstonia sp. Ruminococcus gnavus Ruminococcus schinkii SFB Streptococcus gordnii TM7 phylum sp

NCBI Accession no. AJ418058 AJ308388 AJ400243 AJ400242 AJ400241 AJ400234 AJ400249 AJ400236 AJ400246 AJ400252 AJ400254 AJ400245 AB027536 AJ400275 X77842 AJ400265 AJ400256 AJ400247 AJ400260 AJ400237 FJ384389 AJ400272 AY878326 AJ400251 AJ400255 AJ308396 AJ400261 AJ308394 AJ400270 AB298910 AJ308389 EU333881 AJ400238 AJ308393 AJ308392 FJ378897 AJ308390 AJ308391 FJ378886 AJ400264 AJ400235 AJ400267 AJ400266 AJ308395 AJ308386 AJ400250 AJ308387 EU156758 AJ400239

Clone no. clone L10-14 clone S37-11 clone L7-10 clone S30-5 clone S30-4 clone F1 clone L7-2 clone F3 clone L7-17 clone L7-8 clone F18U clone L7-16 NA clone L10-6 NA clone L10-2 clone L10-4 clone L7-1 clone L10-8 clone F13 NA clone L11-10 NA clone L7-7 clone L10-3 C13-5 clone L11-5 clone S26-5 clone L11-2 NA clone L10-17 NA clone F15 clone S25-9 clone S25-5 NA clone L10-37 NA NA clone L11-6 clone F2 clone F8 clone F5 clone S26-9 clone L10-21 clone L7-6 clone S25-6 NA clone F16

1.0

1000

0.5

0.0

X-ray irradiation (Gy) 0 10 A

0 10 B

0 10 C

0 10 D

0 10 E

0 10 F

0 10 G

0 10 H

Normal

Infection (CR)

CR+DBZ

CR+DBZ + B-Kan

CR + DBZ + P-Kan
20 X 20 X

Figure-3: Changes in the 16S rRNA levels of microbiota in the colon tissue (top panel) and the fecal samples(n=4) (upper panel). Clostridium cluster XIVa levels is decreased during infection (right panel) and natural product administration restores the butyrate producing clostridium levels.

N: Uninfected normal CR: citrobacter rodentium infected CR+DBZ: CR infected mice that received Notch inhibitor Dibenzazepine (DBZ) for 10 days. CR+D+B: CR+DBZ-treated mice on B-Kan diet (4%) CR+D+P: CR+DBZ-treated mice on P-Kan diet (6%) for 10 days.

Conclusions
Mucin adhering microbiome is an early marker in fecal samples during radiation injury. The train of events at the colon exposed either to radiation injury OR citrobacter infection seem to be identical- particularly, the ROS generation, loss of inner mucus, and modulation of butyrate producing clostridium cluster XIVa. Natural products are isolated, and they are capable of restoring such mucus loss and stabilizing the butyrate producing beneficial bacteria.

Figure-5: Inner mucus is reduced during radiation injury and also during the CR infection. Colon sections were visualized with alcian blue staining the mucus.

Courtesy: Dr. Paliy, a modified Sartor, 2008

Project Principal Investigators: Dr. Rao V. L. Papineni & Dr. Shahid Umar "Molecular Imaging - Wisdom To See For Maladies To Flee" Dr. Rao V. L. Papineni

Carestream Health, Inc.