ANNALS OF MICROSCOPY Vol 9, April 2009

Histopathologic and Scanning Electron Microscopic Observations of Changes

in Experimental Colonic Tumors Induced by Streptococcus bovis and Chemical
Carcinogenesis

Al-Jashamy K.1*, Murad A.2, Samiah Yasmin AK.1, Rajab, W.J.3 Zeehaida M.2, and Hasnan
Jaafar2
1
Department of Medical Sciences, Faculty of Health and life Sciences Management and Sciences University/ Shah
Alam
2
Universiti Sains Malaysia, Kota Bharu, Kelantan
3
College of Pharmacy, Al-Zaytoonah Private University of Jordan
*Corresponding email: Jashamy@yahoo.com*

ABSTRACT

Histopathological and scanning electron microscopy (SEM) studies have been used
to compare the appearances of the colonic mucosal surface and overlying mucus layer of rats
during S. bovis and chemical carcinogenesis with Azoxymethane (AOM) with normal colons and
colorectal cancer. The normal colonic mucosa had characteristic orderly arrangement of crypts
and was completely covered by an intact mucus layer. The normal colon mucus layer had a
dense homogeneous appearance and provided a complete cover for the mucosal epithelium. The
histopathology and SEM of treated group in both of S. bovis and AOM showed the epithelial
and cryptal irregularities were seen on the colonic surface, and in addition, the mucus layer was
fragmented, leaving areas of the underlying epithelium exposed. These changes were more marked
in colorectal cancers and were present to a lesser degree in the apparently normal mucosa adjacent
to tumors sites. At high magnifications, tiny fenestrations could be seen in the mucus layer. During
carcinogenesis these fenestrations enlarged, increased in number, coalesced, causing focal defects
in the mucus layer, which eventually broke into strands and clumps of mucus. The findings indicate
that colonic mucus layer develops progressive abnormalities during carcinogenesis, which result
in breakdown of its integrity and exposure of the mucosal epithelium to colonic contents. The
SEM can detect minor subtle irregularities on the surface of the colon and so may be useful in
detecting preneoplastic and early neoplastic changes.

KEYWORDS: Histopathology, SEM, Colonic Tumor, S. bovis, Chemical Carcinogenesis

INTRODUCTION

The genus Streptococcus comprises a large number of species that are morphologically
Gram-positive-cocci (Facklam, 2002). Streptococcus bovis is a normal inhabitant of the intestinal
tract and may be isolated in 5–16% of fecal samples in normal adults (Alazmi et al. 2006). There
are some reports confirming the presence of S. bovis bacteremia and endcarditis with various
forms of gastrointestinal diseases, such as inflammatory bowel diseases (IBD), primary colonic
cancer (Teitelbaum and Triantafyllopoulou, 2006), and colorectal cancer (CRC) (Osada et al.
2001; Apsingi et al. 2007).
Colorectal cancer is characterized by uncontrolled growth of neoplastic cells developing in
the lower segment of the digestive tract, with the potential to invade and spread to other sites (Gold
et al. 2004). CRC is the second carcinoma in incidence among females after breast cancer and
third in incidence among males behind prostate cancer and lung cancer. The current hypothesis is
that adenomatous polyps and some pathogenic bacteria are the precursors of the vast majority of
colorectal cancers (Mager, 2006, Yang and Pei, 2006). The first report about this topic in Malaysia

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as a case report study on colonic cancer associated with S. bovis was in 2005 (AL-Jashamy et al.
2005). The main purpose of the present study was to observe the surface features of aberrant and
normal rats’ colonic surface and crypts using Light and scanning electron microscopy in induced
CRC in rats with S. bovis and AOM.

MATERIALS AND METHODS

Bacterial Strain

The S. bovis biotype II/1 strain used in this experimental study was isolated from colorectal
cancer case in hospital Universiti Sains Malaysia.

Experimental Challenge

A total of 24 male Sprague-Dawley rats at four weeks old were used in this study. The rats
were obtained from the Animal Unit, Universiti Sains Malaysia, and randomly divided into two
main groups: Azoxymethane (AOM) group and non AOM group, each group consisted of 12
rats. All animal in AOM group received intraperitoneum (i.p.) injections of 15mg AOM/kg body
weight (Ellmerich et al. 2000) once a week for two weeks. Then each group was randomly divided
into two subgroups with six rats each. The first subgroup of non AOM received 1ml of Trypticase
soya bean (TSB) by oral gavage only as control group. The second subgroup of non AOM received
only 1ml of S. bovis of bacterial suspension (1010). The third subgroup of AOM received 1ml of
TSB orally and AOM (i.p.) and last subgroup received both of AOM (i.p.) and 1ml of S. bovis
of bacterial suspension (1010). All rats were killed at 14 weeks of experimental challenge and
the colon (sigmoid portion) specimens were collocated for histopathology and SEM studies. The
experimental animal protocol was conducted in compliance with humane animal care standards
outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals,
the experimental study was with ethic number PPSG/07(A) 044.

Collecting Specimens and Histopathology Processing

The colonic specimens were collected from all rats and fixed in 10% neutral buffer formalin
overnight. The specimens were sent to histopathology laboratory for processing. The specimens
were embedded in paraffin, sectioned, and stained with hematoxylin and eosin stain. The slides
were observed under the light microscope.

Collecting Specimens and Scanning Electron Microscopy Procedure

Colonic specimens were collected from the same lesion site, were washed in 1% phosphate
buffered saline (PBS), and then the tissue specimens were fixed in 2.5% glutaraldehyde, dehydrated
through a graded ethanol series, immersed in hexamethyldisilazane (HMDS) for 5 minutes 3 times,
and air dried. Finally removed as much of the HDMS as possible from the specimen was allowed
to be air-dried. The samples were then ready to be mounted and sputter coated with gold for
scanning electron microscopy.

RESULTS

Histopathology

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The histopathological results of control group showed normal structural the colon specimens
(Fig. 1a), while the rats’ colons treated with AOM and S. bovis showed that adenomas with
glandular formation, containing nests of colonic epithelium within the lamina propria of the
neoplasm and hyperchromasia. The cytologic changes were accompanied by increased nuclear

1a 1b
Histopathogical Micrographs of rats’ colons.
Fig. 1a. Showing normal structures of colon H&E X 4.
Fig. 1b. Rats treated with S. bovis and AOM showing
aberrant crypt foci (ACF) arrows H&E X 10.
Fig. 1c. Rats treated with S. bovis and AOM showing
uncontrolled growth of neoplastic which chracterized
by adenomas demonstrate glandular formation and
contain nests of colonic epithelium within the lamina
propria of the neoplasm hyperchromasia, the cytologic
changes are accompanied by increased nuclear
pleomorphsim, loss of nuclear polarity, atypical mitotic
H&E X40

1c

pleomorphsim, loss of nuclear polarity, atypical mitotic and increased aberrant crypt foci (ACF)
(Figs. 1b & c).

Scanning Electron Microscopy

The SEM of rats’ colons in control group showed normal structural characterizations (Figs
2a & b). Meanwhile, rats’ colons in the group treated with S. bovis showed irregularities of the
epithelial and cryptal surface of colons with fragmentation of the mucus layer was fragmented (Figs
3 a & b). Rats’ colons in the group treated with S. bovis and AOM showed progressive distortion
and erosion of the crypts, development of pericryptal fissures, mucosal surface irregularities, and
formation of focal protuberances of colons (Figs. 4a & b).

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DISCUSSION

The results of this study showed highly significant pathological changes which were induced
by S. bovis and AOM under light and scanning electron microscopy. Also the results of present
study revealed that S. bovis can promote carcinogenesis of colon cancer without carcinogenic
factors such as AOM especially in chronic colonic infection by S. bovis. An association between S.
bovis and CRC neoplasia has been recognized since the first reports of S. bovis endocarditis related
to CRC in 1974. The etiology of colon cancer can be related to environmental and hereditary factor.
Bacteria have been linked to cancer by two mechanisms: chronic inflammation and production
of carcinogenic metabolites by intestinal microflora has been reported to have an action on the
carcinogenesis of colon cancer (Schlegel et al. 2003; Gold et al. 2004; Tafe and Ruoff, 2007).
The colonic neoplasia specifically allows for the overgrowth or translocation of S. bovis and that
S. bovis is in fact causative of neoplasia itself (Yang and Pei, 2006, Tafe and Ruoff, 2007). Some
authors refer that alteration in local conditions and disruption of capillary channels at the site of
neoplasm allowed S. bovis to proliferate and gain entry into the blood stream. Local actions of
cytokines or chemical mediators able to promote vasodilatation and the enhancement of capillary
permeability may support bacterial adherence to various cells. It has also been speculated that S.
bovis produce a carcinogen that induces intestinal cancer (Ellmerich et al. 2000; Tafe and Ruoff,
2007).
As mentioned, there is correlation between S. bovis and colon cancer, since S. bovis is
occasionally present as human colonic flora, and it has been reported that fecal carriage of the
bacteria is increased among colon cancer patients. Various studies have shown that about 80% of
patients with S. bovis also had colorectal adenomatous polyps, aberrant crypt foci and extracolonic
malignancy. It has been demonstrated that S. bovis or its wall extracted antigens (WEA) were
able to promote carcinogenesis and progression of preneoplastic lesions in rats (Gold et al. 2004),
it has been found that to promote the increasing of formations of hyperproliferative aberrant
colonic crypts, enhanced expression of proliferation markers and increased production of IL-8 in
the colonic mucosa (Gold et al. 2004, Mager, 2006).
Many studies showed a trend of increasing incidence of S. bovis in adult patient with
IBD (Teitelbaum and Triantafyllopoulou, 2006). CRC is a worldwide problem with an annual
incidence of approximately one million (Winawer, 2007). CRC is on a rapidly rising trend in
Asia, countries such as China, Japan, South Korea, and Singapore have witnessed a two- to four-
fold incidence in the past decades (Sung, 2007). The bacterial association was also significant
with convincing evidence has linked Helicobacter pylori with both gastric cancer and mucosa
associated lymphoid tissue lymphoma. However other Salmonella typi and gallbladder cancer, and
Chlamydia pneumoniea with lung species associated with cancer include Streptococcus bovis and
colon cancer, cancer. Important mechanism by which bacterial agents may induce carcinogenesis
was chronic infection, immune evasion and immune suppression (Mager, 2006).

CONCLUSION

The findings indicated that there was definite pattern of progressive mucosal abnormalities
commencing long before the development of focal colon tumors, SEM is useful in the detection
of these abnormalities even in minor subtle irregularities on the surface of the colon and so may
be useful in detecting pre-neoplastic and early neoplastic changes. This result suggested that all
patients with S. bovis bacteremia need aggressive evaluation of the gastrointestinal tract especially
the colon.

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2a 2b

3a 3b

4a 4b
SEM Photographs of rats’ colons.
Figs. 2 a & b. Control group showed normal structural characterizations.
Figs. 3 a & b. Rats’ colons treated with S. bovis showing irregularities of the epithelial and cryptal surface of
colons with mucus layer fragmented.
Figs. 4a & b. Rats’ colons treated with S. bovis and AOM showing progressive distortion and erosion of the
crypts, development of pericryptal fissures, mucosal surface irregularities, and formation of focal protuberances of
colons. Lead citrate and uranyl acetate.

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ACKNOWLEDGEMENTS

This study was supported by Universiti Sains Malaysia short term grant No 304/
PPSP/6131439.

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