Khảo sát đậm độ và khả năng sinh độc tố của vi khuẩn S. aureus

BỘ GIÁO DỤC VÀ ĐÀO TẠO
TRƢỜNG ĐẠI HỌC NÔNG LÂM TP.HCM
BỘ MÔN CÔNG NGHỆ SINH HỌC
***000***




PHẠM TRẦN XUÂN HIỀN



KHẢO SÁT ĐẬM ĐỘ VÀ KHẢ NĂNG SINH ĐỘC TỐ
CỦA VI KHUẨN Staphylococcus aureus
TRÊN MÔI TRƢỜNG NUÔI CẤY





Luận văn kỹ sƣ
Chuyên Ngành: Công nghệ sinh học







T

9/2006

BỘ GIÁO DỤC VÀ ĐÀO TẠO
TRƢỜNG ĐẠI HỌC NÔNG LÂM TP.HCM
BỘ MÔN CÔNG NGHỆ SINH HỌC




KHẢO SÁT ĐẬM ĐỘ VÀ KHẢ NĂNG SINH ĐỘC TỐ
CỦA VI KHUẨN Staphylococcus aureus
TRÊN MÔI TRƢỜNG NUÔI CẤY



Luận văn kỹ sƣ
Chuyên ngành:Công nghệ sinh học




Giáo viên hƣớng dẫn: Sinh viên thực hiện:
ThS. NGUYỄN ĐỖ PHÚC PHẠM TRẦN XUÂN HIỀN
Khóa: 2002-2006







9/2006

MINISTRY OF EDUCATION AND TRAINING
NONG LAM UNIVERSITY, HCMC
DEPARTMENT OF BIOTECHNOLOGY









RELATIONSHIPS BETWEEN THE BACTERIA DENSITY
AND ABILITY PRODUCING STAPHYLOCOCCAL
ENTEROTOXIN (SEs) OF Staphylococcus aureus
IN TSGM AND BHI BROTH



Engineer Thesis
Major: Biotechnology




Research adviser Researcher
 
Term: 2002 - 2006

HCMC, 09/2006


LỜI CẢM ƠN



 



           

 
- 



















TÓM TẮT KHÓA LUẬN

           
 
Staphylococcus aureus 

ThS. 
Staphylococcus aureus 



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
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


 
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
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



 
trong 
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
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


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

 

 

 , 
S. aureus
S. aureus              
S. aureus 
   S. aureus         (Tecra
Staphylococcal Growth Medium)  (Brain Heart Infusion)
 



S. aureus  
(
S. aureus 
 
                
 


S. aureus.
S. aureus 


ABSTRACT

Staphylococcus aureus is a Gram-possitive coccus having ability to produce
enterotoxin. It is reponsible for one of the most common types of food poisoning. In
testing food and finding the reason causing food poisoning nowadays, we have mainly
testsed the present of S. aureus, not enterotoxin which is the primary caussative agent
of Staphylococcal food poisoning. Therefore, in oder to get information of producing
enterotoxin, we carry on examining S. aureus growth and its ability to produce
enterotoxin in culture medium that contributes practically in testing food, especially in
Staphylococcal food poisoning outbreaks. S. aureus strains are cultured in TSGM and
BHI medium; then we examine their growth and test SE by ELISA method with
TECRA kit after 16, 24, 48 and 72 hours.
The results we find:
Among 36 strains surveyed, there are ten ones having ability to produce
SE (27,8%). In these ten strains, the ones from clinical samples have the highest rate
(50%).

S. aureus growth and SE amount are not correlative. SE amount
increases with time. After 16 hours culture, on TSGM, as the cell concentation reaches
7,86 log10 cfu/ml, OD ELISA value is 1,464; on BHI, as the cell concentation reaches
8,13 log10 cfu/ml OD, E
TSGM and BHI medium are the same influence for S. aureus growth
and ability to produce enterotoxin.
These S. aureus strains producce SEA, SEB, SEC. Among them, the rate
of SEA is 80%, of SEB is 10% and of SEC is 10%.









MỤC LỤC


ĐỀ MỤC TRANG

 ................................................................................................................ iii
 ..................................................................................................................... iv
Abstract .................................................................................................................... v
 .................................................................................................................... vi
 ....................................................................................... ix
 ................................................................................................. x
 .................................................................................................. xi
1. MỞ ĐẦU

1.1.  ............................................................................................... 1
1.2.  ................................................................................................. 1
1.3.  ................................................................................................... 2
2. TỔNG QUAN TÀI LIỆU
2.1. GiStaphylococcus .................................................................. 3
2.1.1.  ......................................................................................... 3
2.1.2.  ......................................................................................... 3
2.1.3.  ......................................................................................... 4
2.2. Staphylococcus aureus ...................................................... 4
2.2.1.  .......................................................... 4
2.2.2. .............................................. 6
2.2.3.  ......................................................... 7
2.2.4. S. aureus .......................................................... 8
2.3. Staphylococcus aureus ..................................... 8
2.3.1. Nh .................................................... 8
2.3.2. S. aureus ................................................... 9
2.4. Staphylococcus aureus...................................... 11
2.4.1.  ........................................................... 11
2.4.1.1.  ................................................... 11

2.4.1.2.  ................................................ 12
2.4.2.  .................................................................. 13
2.5. Staphylococcus aureus ...................................... 14
2.5.1.  .............................................................................. 14
2.5.2.  ............................................................................. 14
2.5.3.    .............................. 16
2.6. Staphylococcus aureus ............................... 18
2.6.1.  ........................................................................................ 18
2.6.2.  ...................................................................................... 18
2.6.3.  ...................................................................................... 19

2.6.4. 
 .......................................................................... 19
2.6.5.  .................................................... 20
2.6.5.1.  ......................................... 20
2.6.5.2.  ........................................................... 21
2.6.6.  ................................................ 22
2.6.6.1.  ................................... 22
2.6.6.2. - RIA ....................... 22
2.6.6.3.  ....................................................... 23
2.6.6.4.  .......................................................... 23
2.6.6.5. P ...................................................... 24
3. VẬT LIỆU VÀ PHƢƠNG PHÁP TIẾN HÀNH
3.1.   ............................................................................. 26
3.1.1.  ............................................................ 26
3.1.2.  ....................................................................... 26
3.2.  .................................................................................................... 26
3.2.1. S. aureus .......................................................................... 26
3.2.2.  ...................................................................... 26
3.2.3.  ....................................................................................... 26
3.3.  ........................................................................................... 27
3.3.1.  .......................................................................... 27
3.3.2.  ..................................................................... 27

3.3.2.1. g S. aureus .................................. 27
3.3.2.2.  ...................... 29
3.3.2.3. 
 ......................................................... 30
4. KẾT QUẢ VÀ THẢO LUẬN
4.1. S. aureus ......................... 36
4.2. S. aureus ....... 39

4.2.1.  ......................................................................................... 39
4.2.1.1.  ...................................................... 39
4.2.1.2.  ........................................................... 40
4.2.2.  ................................................................... 42
4.3.  .......................................... 44
4.3.1.  ......................... 44
4.3.2.  ................ 47
4.4. SE .................................................................... 47
5. KẾT LUẬN VÀ ĐỀ NGHỊ
5.1.  ................................................................................................... 50
5.2.  .................................................................................................... 50
6. TÀI LIỆU THAM KHẢO ............................................................................... 51
PHỤ LỤC .......................................................................................................... 56













DANH SÁCH CÁC CHỮ VIẾT TẮT

BHI : Brain Heart Infusion
BP : Baird Parker

cfu : colony form unit
EIA : Enzyme Immunoassay
ELISA : Enzyme Linked Immunosorbent Assay
FAME : Fatty Acid Modifying Enzyme
KL : 
MSA : Manitol salt agar
MPN : Most Probable Number Method
MRSA : Methicilin resistant Staphylococcus aureus
PCR : Polymerase Chain Reaction
pNPP : -nitrophenyl photphatase
RIA : Radio Immunoassay
RFLP : Randomly Fragment Length Polymorphism
RLPD : Randomly Amplified Polymorphic DNA
RPLA : Reversed Passive Latex Aggulutination
S. aureus : Staphylococcus aureus
SE : Staphylococcal enterotoxin
S. epidermidis : Staphylococcus epidermidis
TSA : Tryptic soy agar
TSB : Tryptic Soy Broth
TSGM : Tecra Staphylococcal Growth Medium
TSST-1 : Toxic shock syndrom toxin
VRSA : Vancomycin Resistant Staphylococcus aureus
(+) : 
(-) : 





DANH SÁCH CÁC BẢNG

BẢNG TRANG
S. aureus, S. epidermidis Micrococci ................ 6
S. aureus .............................. 36
S. aureus ..................................................... 37
S. aureus
 ........................................................................ 39
4 S. aureus
 ............................................................................. 41
5.  ................................. 42
S. aureus ch .......................... 44
7. S. aureus  .... 44
8. S. aureus  ........ 45
9S. aureus ................................................. 48

DANH SÁCH CÁC HÌNH

HÌNH TRANG
Staphylococcus aureus ............................................................ 4
 Staphylococcus aureus  .............. 5
3Staphylococcus aureus ....................................... 14
4 S. aureus ...................................... 17
nh 2.5 ................................................................... 21
 .................................................................... 29
2 ................................................... 35
3 .................................................. 35
S. aureus  ................................... 38
2S. aureus  ............................................. 38
S. aureus .............................................................. 38
4. S. aureus  .............. 40
5. S. aureus  .................. 42
 ................................. 43
7.   ..... 46
8.   .......... 46
 ......................... 48
.  .......................................................................... 49

PHẦN 1. MỞ ÐẦU

1.1. Đặt vấn đề
               Tuy

 Theo
  
s. 
Staphylococcus aureus S. aureus 

S. aureus  enterotoxin 

o
C tron
S. aureus 3-
   
 -  





S. aureus

S. aureus


 Khảo sát đậm độ và khả
năng sinh độc tố của vi khuẩn S. aureus trên môi trƣờng nuôi cấy
1.2. Mục đích
Kh s   v kh nng sinh  t c vi khu S. aureus trn
mi tr   c kho th gian kh nhau.



1.3. Nội dung nghiên cứu
o S. aureus 
o  S. aureus  
               
 Australia).
o               
S. aureus.
o 

PHẦN 2. TỔNG QUAN TÀI LIỆU

2.1. Giới thiệu về Staphylococcus:
Staphylococcus staphylo coccus

Staphylococcus 
o 
o 
o 
o 
o Staphylococcus
2.1.1. Hình thái
Staphylococcus - 




-
 Staphylococcus

Staphylococcus pyrogen aureus (
2000).
2.1.2. Tính chất
Staphylococcus 
 



               

Staphylococcus 
-37
o



               
Iandolo, 2000).
2.1.3. Phân loại
         Staphylococcus    
               
Staphylococcus 
- 
- 
- 
Staphylococcus Staphylococcus aureus 

2.2. Giới thiệu về Staphylococcus aureus
2.2.1. Hình thái, đặc điểm sinh hóa
Staphylococcus aureus   Staphylococcus     
staphylococcus. S. aureus 
-

.


Hình 2.1. Hình thái Staphylococcus aureus


Hình 2.2. Tụ cầu Staphylococcus aureus gram dƣơng dƣới kính hiển vi
 />age/imagky.html>)
S.aureu

catalase
H
2
O
2
H
2
O + O
2

S. aureus 
S. aureus
bi S. aureus            
            
               

             


ctv, 1995).
            
S. aureus 


S. aureus 


 S. aureus 
-
          


S. aureus 
--
5 mm (do k
2
TeO
3


              

 (Mary K. Sandel

S. aureus 

o
  -37
o
C

Bảng 2.1. Những đặc tính của S. aureus, S. epidermidis và Micrococci


2.2.2. Điều kiện tăng trƣởng và sự phân bố
Staphylococcus aureus 
S. aureus 
-48
o
-45
o

4,2--J
 S. aureus S. epidermidis Micrococci
Catalase + + +
Coagulase + - -
Thermonuclease + - -

Lysostaphin
+ + -
 + + -
 + - -


- 
Shigella Salmonella 



S. aureus 
S. aureus 
           
J Harvey
, 2000)S. aureus 
o
C
-

- S. aureus 
S. aureus 

 
S. aureus 


S. aureus 
S. aureus               
Lancette, 2001).
2.2.3. Tính kháng thuốc kháng sinh
S. aureus 

Staphylococcus aureus

Enterococcus faecalis 
S. aureus 
a, S. aureus 







      Staphylococcus aureus)
(Kenneth Todar , 2005).
2.2.4. Sự sinh trƣởng của S. aureus
    S. aureus 


Phase chậm

Phase tăng trƣởng cấp số
              


Jawetz, 1989).
Phase dừng
 
Phase suy vong 

2.3. Ngộ độc thực phẩm do Staphylococcus aureus
2.3.1. Những triệu chứng thƣờng gặp
Staphylococcus aureus  
         Salmonella  Clostridium perfringens

J Bremer 

          
-




-8 gi-2




G. Normanno , 2004).



                
-48
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


L. McKillip, 2002).
2.3.2. Tình hình ngộ độc do Staphylococcus aureus

 
          u USD. Trong





-
Staphylococcus aureus  


 







Staphylococcus aureus 

          


 
              

  

                 







         

 


                 



S. aureus 


S. aureus 





S. aureus 

-
--


S. aureus 

-

          

2.4. Phƣơng pháp phát hiện Staphylococcus aureus
2.4.1. Phƣơng pháp truyền thống
2.4.1.1. Phƣơng pháp định tính

o



o

S. aureus  -


o



S. aureus khi


Staphylococcus aureus Staphylococci coagulase
 
   S. aureus 



2.4.1.2. Phƣơng pháp định lƣợng
Phƣơng pháp đếm khuẩn lạc
o  
-
1
, 10
-2
, 10
-3


o 
       
37
o
C trong 24-S. aureus -
-4

(
o 
 TSA (Tryptic soya

o


o 
S. aureus 








Phƣơng pháp MPN (Most Probable Number Method)
S. aureus S.
aureus 








o


       
o
     
S. aureus 
     S. aureus  


S. aureus 
2.4.2. Phƣơng pháp hiện đại
        

-S.
aureus 
sin



S. aureus
nuc 
, gen tst eta etb 
-


-IS256 PCR, spa t-
         
       S. aureus    -RFLP
aro
ctv, 2004).
2.5. Các yếu tố độc lực của Staphylococcus aureus