ISSN.
Bali Medical Journal (BMJ) 2012.
Volume 1.
Number 1: 6-11 Inhibition of Bifidobacterium Cell Wall 51.
74 kDa Adhesin Isolated from Infants Feces Towards Adhesion of Enteric Phatogen E.
Coli on Enterocyte Balb/C Mice Sukrama.
I D.
Microbiology Department.
Faculty of Medicine.
Udayana University Objectives: To determine 51.
74 kDa adhesin of Bifidobacterium sp cell wall isolated from infants feces as an anti adhesion of E.
coli on enterocyte mice.
Methods: Randomized Posttest-Only Control Group Design was employed to investigate adherence ability of this adhesin towards E.
coli adhesion on mice entherocyte.
Results: In this research, it was obtained, that the 51.
74 kDa adhesin cell wall of Bifidobacterium sp has an ability to inhibit adhesion of E.
coli on mice enterocyte.
The ability was increased as an increase of adhsein concentration.
Conclusions: that can be drawn from this research is the finding of 51.
74 kDa adhesin cell wall of Bifidobacterium sp isolated from infants feces that can inhibit adhseion of E.
coli on mice enterocyte.
Future work that can be carried out are further researches concerning whether these protein can be applied to inhibit adherence of other pathogen Keywords: diarrhea, adhesion.
Bifidobacterium sp.
INTRODUCTION
Diarrhea is a disease characterized by a frequent increase of defecation (> 3 time/da.
followed by changes of stools concistence, with/without gross blood and/or mucus.
Diarrhea, up to the recent year remains a cause of high morbidity and mortality worldwide, especially in developing countries including Indonesia.
Therefore, research concerning of management, prevention, and medication of the disease have been continually improved.
WHO
indicates that 4 billion cases occured worldwide during the years of 2000.
Of these, 2.
2 million were killed, whithin the most are children under 5 years.
In Indonesia, the morbidity rate of accute diarrhea are in the range of 200-400 cases per 1000 people per Of the most, 70-80% are children under 5 years.
This group is experiencing diarrhea more than once per year.
A part of this patient .
-2%) will be ended with dehydration and if there is no sufficient aid, 5060% of them could be died.
1 It was reported that the main cause of diarrhea dominated by enteropathogenic bacteria, including E.
coli diarrheagenic involving ETEC and EPEC.
Salmonella spp.
Shigella spp.
, and Vibrio spp.
3 Diarrhea caused by S.
typhi were initiated by adherence of Salmonella This adhesion induces neutrophile transepithelial migration and villi entherocyte damage followed by membrane destruction on the site of This damaging membrane subsequentially followed by endocytosis and internalization.
It was well established that there are much more bacteria including diarrhea bacteria are resitance towards 4,5 This condition has endorsed researches for establishing research to obtain an alterCorrespondence: Sukrama.
I D.
Address: Microbiology Department Faculty of Medicine Udayana University.
Bali-Indonesia.
Open access: w.
native cure to replace antibacterial that has already used clinically.
Obtaining an agent that can affect or protect bacteria adherence is one of researhers strategy to overcome this situation.
These kind of agents are known as anti-adhesion.
Other strategics are obtaining vaccine for diarrhea.
Anti-adhesion agents are not bactericide or antibiotic, therefore, their propagation and occurrence for therapy will not leading to resistence.
METHODS
Research Design Experimental study with Randomized PosttestOnly Control Group Design was employed to obtain inhibition ability of 51.
74 kDa adhesin cell wall of Bifidobacterium towards adhesion of E.
coli on mice Research Procedure Isolation of Bifidobacterium from infant feces Isolation of Bifidobacterium from infants feces was carried out following Beeren, .
in Hadadji.
About 1 g of infants feces was placed on a flask added with 9 mL NaCl .
9%) containing 0.
cystein-HCl.
This suspension was then homogenized for 2 minutes.
Around 0.
1 mL of this suspension was inoculated on MRS broth media.
All plates were then anaerobically incubated using oxoid gas jar at temperature of 37 0C with 5 % CO2 for 18-24 h.
The Bifidobacterium obtained was then separated using solid MRS broth media.
The clonal was then selected and indentified for further treatment.
Isolation of Balb/c mice enterocyte Enterocyte isolation was carried out based on Weisler method taken from Nagayama.
8 An healthy BALB/c mice age of 3 months was collected and ISSN.
Bali Medical Journal (BMJ) 2012.
Volume 1.
Number 1: 6-11 treated for this experiment.
Mice was killed using chloroform and resected.
The intestine was taken out, cleaned and cut into 5 cm.
Lumen intestine was then resected and cleaned using PBS solution containing dithiotreitol 1 mM.
The lumen was immersed on a solution containing of 1.
5 mM KCl.
6 mM NaCl.
7 mM Na-citrate.
8 mM KH2PO4 and 5.
6 mM Na2PO4, pH 7.
3 and placed on water bath at temperature of 37 0C, and shaked for 30 minutes.
All solution was drained and replaced with PBS with pH 4 containing 1.
5 mM EDTA and 0.
5 mM Intestine tissues were shaked on water bath within temperature of 37 0C for 20 minutes.
Solution was drained and the intestine was cleaned using PBS pH.
Of 7.
4 by centrifugation at 1000 rpm, at temperature of 40C for 5 minutes.
The cleansing was carried out three times and suspended on PBS pH of 7.
4 and shaked.
Solution containing enterocyte characterized by cloud filtrate was taken using sterilized pippetes and store on steril tube, counted using leucocyte counting chamber and made up the concentration of 108 enterocyte/mL.
Adhesion test of Bifidobacterium on enterocyte Nagayama method was employed for testing of Bifidobacterium adhesion on enterocyte.
8 A number of 100 AL of the Bifidobacterium suspension with concentration of 108 cells/ml mixed with 100 AL enterocyte cells of 108 cells/mL.
The mixture was then incubated on shaker water bath and shaked at temperature of 370C for 30 minutes.
Cells were collected by centrifugation at 3000 rpm for 2 The precipitate was then collected for sweft preparate and gram staining.
The preparate was observed under microscope with 1000 time zoom, to obtain the type and adhesion index.
Figure 1 Bifidobacterium sp isolated from infants feces indicated by short rod with Y and V shape .
0 X).
Enterocyte Isolation Enterocyte isolation was carried out on the basis of Weisler method in Nagayama.
8 The enterocyte obtained was pictured on Figure 2.
Figure 2 Enterocyte of BALB/c mice .
Adhesion of Bifidobacterium sp on Enterocyte Nagayama method was employed to observed adhesion of Bifidobacterium on enterocyte.
Inhibition test of 51.
74 kDa Bifidobacterium cell wall-adhesin towards Adhesion of E.
coli on mice Inhibition test of Bifidobacterium sp cell wall adhesin towards E.
coli on enterocyte was performed in the same way as inhibition test of Bifidobacterium sp by replacing the bacteria with the adhesin.
RESULTS
Isolation of Bifidobacterium from Infants feces Isolation of Bifidobacterium from infant feces was carried out following Beeren .
in Hadadji.
Fecal suspension on NaCl solution with cystein-HCl was inoculated on broth media.
The grown Bifidobacteria were separated using solid broth media.
The clonal growth were then selected for identification (Figure .
Open access: w.
Figure 3 Adhesion of bifidobacterium on enterocyte, .
oom of 1000 X).
Adhesion indices of Bifidobacterium sp on enterocyte were listed on Table 1.
ISSN.
Bali Medical Journal (BMJ) 2012.
Volume 1.
Number 1: 6-11 Table 1 Adhesion indices of Bifidobacterium sp on enterocyte Number of Bifidobacterium per 100 enterocyte Number of Bifidobacterium sp per enterocyte Average In this study, adhesion of E.
coli on enterocyte was also performed.
The result was drawn on Figure 4.
Figure 4 Adhesion of E.
coli on enterocyte, enterocyte .
oom of 1000 X).
Inhibition test of Bifidobacterium towards Adhesion of E.
coli on mice enterocyte Inhibition test of Bifidobacterium sp towards E.
coli on enterocyte was performed and the test result was presented on Figure 5.
Inhibition test of Bifidobacterium sp cell wall adhesin towards E.
coli on enterocyte was performed in the same way as inhibition test of Bifidobacterium sp (Figure .
Figure 6 Inhibition of E.
coli adhesion by Bifidobacterium sp cell wall adhesin.
adhesin of 51.
74 kDa .
oom of 1000 X).
Adherence data of E.
coli that can be inhibited by Bifidobacterium sp cell wall adhesin of 51.
74 kDa were listed on Table 2.
Data on Table 2 are normally distributed and their variance are also homogen with p>0.
Therefore, the data can be analyzed parametrically by applying one way Anova and followed by LSD posthoc test.
It was obtained, that 74 kDa adhesin exactly inhibit adherance of E.
coli on enterocyte as indicated on Table 3.
Table 2 Adhesion of E.
coli on enterocyte Average Adherance of E.
Coli on 100 enterocyte due to inhibition of 51.
74 kDa adhesin
0 Al
5AAl
10AAl
15AAl
20AAl
25AAl
DISCUSSION