nghiên cứu kiểu hình kháng kháng sinh của một số vi khuẩn thường trú trong đường tiêu hóa người tình nguyện khỏe mạnh đến khám sức khỏe tại trung tâm y tế dự phòng huyện hóc môn trong năm 2015

khang cephalosporin the h� 3 chiJm 85,48 % trong t6ng s6 420 nguai khoe ml.lnh tham
gia nghien cfru nay.

i'u nhung chimg vi khutn khang thu6c tren, sau qua trinh sang I9c va xac dinh

chung toi thu duqc ket qua nhu sau:
+ Chung vi khufin sinh enzym ESBL: 261 chung E. coli (chi"8m 62,14 %), 53
chung Klebsiella spp. (12,62 %), 3 chung Enterobacter spp (chiSm �q1 %).
+ Chung vi khufin sinh enzym AmpC: 69 chung E. coli (chiSm 16,43 %), 41 chung
Klebsiella spp. (9,76 %),
+ Chung vi khufin sinh_enzym carbapenemase:-31 chung E.coli (chiSm 7,38%), 22
chung Klebsiella spp. (chiSm 5,24%), 18 chung Acinetobacter spp. (chiSm 4,29%) va 6
chung P.aeruginosa (chiSm 1,43 %).
- Tir cac kSt qua knao sat vS d�c diSm dan s6 cfing nhu d�c diSm kham chfra b�nh
,;·

Cua nh6m nguai tham gia nghien ClIU, chung toi nh�n thfiy vi�c SU' d1_mg khang sinh trong
vong 3 thartg tru6c khi tham gi{ nghien CUU, cfing nhu SU'. d1_mg khang sinh khong dung
liSu hay bo liSu, h.r ,;, mua thu6c ho�c mua thu6c do SlJ tu vfin cua nhan vien ban thu6c tai
nha thu6c tu nhan dSu lam gia tang ty 1� nguai mang vi khuAn duang ru(H dS khang
khang sinh. Ben cl_lnh d6, ty 1� nguai nh�p vi�n trong vong 3 thang tru6c khi tham gia
nghien cuu cfing lam gia tang ty 1� nay.
--- -Cac chung·vi khufin khang thu6c cho thfiy cac chung vi khufin nay dSu la nhung
chung vi khufin da khang thu6c, c6 kha nang khang tir 2 lol_li khang sinh tr& len
5. San phftm:

- Chung vi khufin E.coli, Klebsiella spp., Acinetobacter spp., P.aeruginosa dS
khang khang sinh duqc phan l�p tir nhfrng nguai tinh nguy�n khoe ml_lnh.

- Dao tl_lO duqc 02 CU' nhan cong ngh� sinh h9c kh6a 2011.
6. Hi�u qua, phrrong thrrc chuy�n giao k�t qua nghien CU'U va kha nang ap dl}ng:

.

.

1



Mau 12. Thong tin kit quii nghien cfru bdng tilng Anh
MINISTRY OF EDUCATION AND TRAINING
HO CHI MINH CITY OPEN UNIVERSITY

INFORMATION ON RESEARCH RESULTS
1.

General information
Project Title: Research phenotypic antibiotic resistance of some bacteria
residence on gastrointestinal of healthy volunteers to a medical examination
_attheJ:�_reventive-Medicine_Center__ ofHoc_MonJ�r:ovince-in--2015-- Project Code: T2015.1.180
- Coordinator: Duong Nhat Linh
Project Owner: Hp Chi Minh City Open University -

- Project time: July 2(fl2 -April 2016
2. Purpose of the Study

,-

Research phenotypic antibiotic resistance of some bacteria residence on gastrointestinal
of healthy volunteers to a medical examination at the Preventive Medicine Center of Hoc
Mon Province in 2015
3. Creation and innovation
Identify antibiotic resistance phenotypes of E. coli, Klebsiella spp., Acinetobacter
spp., P.aeruginosa isolated from healthy volunteers. From there, help give warnings and
measures, specific communication to help curb the spread of the lowest of antibiotic
resistant bacteria into the community and aims to protect the community.
4. Research results
From 420 stool samples collected from healthy people to visit at the Preventive
Medicine Center of Hoc Mon Province, we identified 359 stool samples generation 3rd
cephalosporin-resistant bacteria, synonymous with healthy people strong bearing
generation 3rd cephalo.sporin-resistant bacteria accounted 85,48% of 420 healthy people
participat_ed in this study.

.

.




1.5.2. Vift Nam ...............................................................................................44
CHlfllNG 2. VJT LI}JU VA PHUONG PHAP ..................................................46
2.1. VJT Lli;;U......................................'. ...................................................... ; ......47

a•,:

2.1.1. D•1a rem va'th'•
• ngh•A

, .......................................................47
a1 gzan
1en ctrn
2.1.2. D2.1.3. Thilt bj, d1:1ng Cl:l, moi truimg .................................
�.......................... .47
··:�
.

"Ii.-,.

2.2. PHUONG PHAP NGHIEN C tfU' ..............................:·............................... .48
,

A

2.2.1. B6 tri thi nghifm...................................................................................48
2.2.2. Quy trinh lciy mtiu va phiin lt;ip ........................................................... .49
2.2.3. -Quy trinh iljnh-danh ........................................................................... '..51
2.2.4. Ky thut;it khang sinh

i18

bling phuang phap khuich tan khang sinh

trong thqch tit ilia giciy tdm khang sinh ........................................................ 56
2.2.5. Ky thflt;it xac ilfnh vi khulm iluang rU2.2.6. Ky thu'i;t sang l9c vi khulm iluang ru9t tiit enzym AmpC ...................59
2.2. 7. Ky thut;it xa� iljnh vi khudn tilt enzym carbapenemase.......................60
2.2.8. Xu ly st5 li¢u ... ... ... ... ... ... ... ...... ... ... ... ... ....... ... ... ... ... ... ... ... ... ... ...62

2.2.8. vdn iii y iluc ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... .... ... ... ... .....62
CHUONG 3. KET QUA VA BI}JN LUJN.�......................................................... 63
3.1. DJC TiNH MAU NGHIEN c(!u VA DJC DIEM KHAM CHUA Bi;;NH.64
3.1.1. Dijcctinhmtiu nghien cuu .....................................................................64
3.1.2. Di;ic ilidm kham chfta bfnh...................................................................64
3.2. KET QUA PHAN LJP VA DJNH DANf[. ...................................................65
3.2.1. Kit qua phiin-ft;ip ...................,...............................................................65
3.2.2. Kit qua iltnh danh ................................................................................67
3.3. X4C D/NH TY Li;; VI KHUAN E. COLL KLEBSIELLA SPP. SINH
ENZYM ESBL, AmpC, CARBAPENEMASE ......................................................68
3.3.1. Xac iljnh ty If E. coli, Klebsiella spp. sinh ESBL ...............................68
3.3.2. Xac ilinh ty If E. coli, Klebsiella spp. sinh enzym AmpC ....................73
3.3.3. Xac iljnh ty If E. coli, Klebsiella.spp. sinh carbapenemase................ 77

iv



DANH MUC cAc TU VIET TAT
CDC: Center for Disease Control and Prevention
CFU: Colony Forming Unit (s6 dan vj khmin li.ic)
CLSI : Clinical and Laboratory Standards Institute
DNA: Deoxyribonucleic aicd
ESBL: Extended spectrum beta- lactamase
GARP: Global Antibiotic Resistance Partnership
MIC : Minimum Inhibitory.Concentration
RNA: Ribonucleic acid
VSV : Vi sinp v�t
..:-

PBP : Pencillin - blinding protein
PABA: Parraminooenzoic acid

-.
vi



DANH MUC HINH ANH
Hinh 1.1 Ty !¢ ESBL tu cac tac nhlin gliy nhiim khulm 6 bimg theo khu VlfC tu niim
2002 ain niim 2011. ..........................:........................................................................
·
23
Hinh 1.2 Tyli ESBL tu cac tac nhlin gliy nhiim trung iluang tiit niiu theo khu VlfC
. tu niim 2009 ain niim 2011. .......................................................... ::........................... 24
\�
"Ii,.

Hinh 1.3 Hinh anh nhu9m gram vi khudn Escherichia coli (Ngu6n: Internet) ........30
Hinh 1.5 Hinh thai khudn lqc Klebsiella ,spp. tren moi truang Maconkey.
(Ngu6n: Internet) ........................................................................................................34
Hinh 1.6 Hinh anh nhu9m gram v{khudn P.aeruginosa (Ngu6Y1:_: Internet) .............39
Hinh 1.5 Hinh thai khudn lqc P. aeruginosa tren moi truang MacConkey (Ngu6n:
Internet) ..................·-.....................................................
:7: .............................................. 40
.
-:·
Hinh 2.1 Thu tlf ma khang sinh ... ... �·· ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..... 61

,,.

..

Hinh 2.2 Phuang phap Hodge test biin il6i ... ... ... ....... ... ... ... ... ... ... ... ... ... ... ... ....62
'•

Hinh 3.1 Phan l(lp mJu phan tren moi truang tren ChromID ESBL (A) va m6i
truang ChromID C(J ban (kh6ng khang sinh) (BJ ....············ .................····················· 66
Hinh 3.2 Hinh thai' khudn lqc cua cac vi khudn phlin l(lp

a mdu phlin tren m6i

truang ChromID ESBL............................................................................................... 67
Hinh 3.3 Kit qua khang sinh il6 cua E.coli 57e sinh enzym ESBL, AmpC va
carbapenemase ................................................... !'•••••..•••.•.••••.•••••••....•.••••..••.••..... , •••••.. 69
ffinh 3.4 Kit qua khang sinh il6 cua Klebsiella spp. 12k sinh enzym ESBL, AmpC va
carbapenemase ........................................................................................................... 74
ffinh 3.5 Kit qua khang sinh il6 cua E.coli 29e sinh enzym AmpC

va

carbapenemase ........................................................................................................... 77
ffinh 3.6 Kit qua khang sinh il6 cua P.aeruginosa 86p va Acinetobacter spp. 26a
sinh.enzym carbapenemase ............................... .•........................................................80

viii



DAT VAN DE

Hi�n nay, vi�c sir d\mg khang sinh & Vi�t Nam khong duqc ki�m soat ch�t
che, phfin l&n nguai b�nh t1J

y

sir d\mg va khong theo SlJ hu&ng dfin cua bac si.

Chinh vi SlJ 1�m d1,1ng m(H each qua mire cua con nguai da khi€n vi khuftn phai
· nhi�m nhi�u v&i khang sinh dfin d�n cac chung vi khuftn khang thu6c c6 nhi�u ca
,

...

hc)i d� phat tri�n va lay Ian (GARP - Vietnam, 2010). S1J giictang tftt y€u tinh tr�ng
d� khang khang sinh dfin d�n nguy ca khong · con khang sinh d� di�u tri nhi�m
khuftn trong tuang lai. Do d6, d� khang khang sinh la vftn d� cfin duqc quan tam va
nghien cuu sau rc)ng tren th� gi&i. Trong cac vi khuftn khang thu6c hi�n nay, dang
luu

y

nhftC la cac vi khuftn Gram am; C\l th� la tf\1'£_ khuftn duong rtic)t

(Enterobacteriaceae ). Enterobacteriaceae la mc)t h9 l&n g6m nhi�u tflJC khuftn
Gram (-), chung �uqc tim thfty trong duang tieu h{$a cua nguai va dc)ng v�t (trong
s6 d6 E. coli va Kl�bsiella chi€m ty 1� cao nhftt), c6 th� gay b�nh ho�c khong gay
b�nh.(CDC, 2013).

1"'

Tir nhiing nam 2000, nhi�u nghien cuu cho thfty cac chung vi khuftn thm)c h9
Enterobacteriaceae co kha nang sinh ra cac enzym

fl -

lactamase phf> rc)ng
)

(Extended-Spectrum Beta-Lactamases; ESBLs) ly giai hfiu h�t cac khang sinh ph6
rc)ng thm)c nh6m cephalosporin (Pitout J. D. va Laupland K. B., 2008). D�n nam
2007, & New York va mc)t s6 khu VlJcJ<liac & Hoa Ky da_bung nf> nhfrng_tr�n dich
nhi�m khuftn Gram am khang thu6c trong b�nh vi�n, sau d6 Ian khip th€ gi&i
(Robledo va cs., 2010). d Vi�t Nam, nhi�u ba9 cao cho thfty tinh tr�ng khang khang
sinh·& mire bao dc)ng t�i tftt ca cac b�nh yi�n, cac vi khuftn Gram am gay b�nh dang
chi€m uu th� v&i ty I� khoang 70% va thuong g�p la h9 Enterobacteriacae (E.coli,
Klebsiella pneumoniae, .. ), Acinetobacter baumanii, Pseudomonas aeruginosa. Cac
vi khuftn nay c6 th� sinh beta - lactamase phf> rc)ng (ESBL) d� khang tftt ca cac
khang sinh nh6m beta - lactam trir carbapenem, nhung d�n nay mc)t s6 chung da c6
kha nang ti€t ra carbapenemase d� khang carbapenem, nliu NDMl - New Deli
M�talo - beta - lactamase. Nhi�u chung vi.lgmftn gay nhi�m khuftn b�nh vi�n la da



•!• Noi dung thu·c hien bao g6m
- Khao sat d?c di€m mfru (gi&i tinh, d(> tu6i) va d?c di€tn kham chfra chfra
b�nh (sir d1.mg khang sinh trong vong 3 thang, each sir d1.mg khang sinh, ngu6n
thu6c sir d1.mg, nh�p vi�n trong vong· 3 thang).

- Xac djnh m(>t s6 vi khufin khang thu6c thuong tru duong tieu h6a nguoi tinh
. nguy�n khoe mi,mh (E.coli, Klebsiella spp., P.aeruginosa vaAcinetobacter spp.).

- Xac djnh

'�

ty 1� vi khufin khang thu6c thuong tru duitng tieu h6a nguoi tinh

nguy�n khoe mi,mh (E. coli, Klebsiella spp., P.·aeruginosa va Acinetobacter spp.).

- Xac dinh ty 1� E. coli ti�t enzym /J- lactamase ph6 n)ng (ESBL), AmpC va

carbapenemase theo d?c di€m mfru, d?C di€m chfra b�nh va th6i quen sinh ho?t.
- Xac -dinh

ty 1� Klebsiella

spp. ti�t enzym

fJ -

lactama§e ph6 r(>ng (ESBL),

AmpC va carbapenemase theo d?c di€m mfru, d?c di€m chfra b�nh va th6i quen sinh
ho?t.

- Xac · dinh t§. 1� P. aeruginosa ti�t enzym carbapenemase theo d?c di€m mfru,

d?c di€m chfra b�nh va thfi quen sinh ho?t.

- Xac djnh Acinetobactf!r spp. ti�t enzym carbapenemase theo d?c di€m mftu,

d?c di€m chfra b�nh va th6i quen sinh ho?t.
- Xac djnh ty 1� d€ khang khang sinh cua cac chung E. coli, Klebsiella spp., P.
aeruginosa va Acinetobacter spp. sinh enzym khang thu6c tren .

.

.
3







• Cdc p

.."

I Pseudomonas).

- lactam khdc

Nh6m carbapenem
Nghien cuu bi€n d6i cfru true h6a h9c cua penicillin va cephalosporin da t�o

. thanh m()t nh6m khang sinh p

- lactam m&i, c6 ph6 khang kh�dn r(mg, d�c bi�t c6

ho�t tinh rfrt m�nh tren vi khufin Gram (-), _d6 la khang �!1h nh6m carbapenem.
Nh6m carbapenem g6m cac khang sinh: _imipenem, meropenem, doripenem,
ertapenem (B(> Y t€, 2015).
Nh6m monobactam
Khang sinh monobactam la khang sinh ma cong thirc.phiin tir c6 chira /3

-

lactam dan vong. Chftt di�n hinh cua nh6m nay la aztreonam (B(> Y t�, 2015).
Ph6 khang lmufin cua aztreonam kha khac bi�t v&i cac khang sinh h9 p ...
,
' �
.,
lactam va c6 ve giin han v&i pho cua khang sinh nh6m aminoglycosid. Thuoc chi co
tac dl)ng tren vi khufin Cram ( -), khong c6 tac d\l�g tren vi khuftn Gram (+) va vi
khuftn kY. Khi. Tuy nhien, ho�t tinh rfrt m�nh tren Enterobacteriaceae va c6 tac dl)ng
df>i v&i P. aeruginosa (B(> Y t€, 2015).
Cac chftt ire ch� 8 - lactam
Cac chfrt nay ciing c6 cfru true p - lactam, nhung khong c6 ho�t tinh khang

khuftn, ma chi c6 vai tro ire ch€ enzym f3 - lactamase do vi khufrn ti�t ra. Cac chllt
hi�n nay hay dUQ'C SU' dl)ng tren lam sang la acid clavulanic, sulbactam va
tazobactam (B(> Y t€, 2015).
1.1.3. CO' ch� tac d<}ng cua khang sinh
£)� c6 th� ire ch� hay gi�t ch�t vi khmin, khang sinh phai c6 tac d(>ng tqrc ti�p
tren sµ t6ng hgp hay ho�t d(>ng cua cac cfru true lien quan d€n sµ sf>ng con cua tS
bao vi khuftn. C6 bf>n ca ch� tac d()ng chu y�u cua khang sinh tren t� bao vi khufin
(Ph�m Hung Van, 2013).
.

.
9



"*-

ac chi chuc nt1ng cua mang

ti bao

T€ bao ch.it cua cit ca t€ bao s6ng d�u duqc bao b9c b&i m<)t mang t€ bao ch.it.
Mang nay duqc xem nhu la hang rao c6 kha nang thdm th.iu ch9n l9c, thgc hi�n
chuc v�n chuySn chu d<)ng va nhu v�y kiSm soat cac thanh ph!n &.ben trong & ben
trong mang t€ bao. N€u sg toan v�n chuc nang mang t€ bao ch.it bi pha va thi nhfrng
dc;1i phan tu va nhfrng ion se thoat khoi t€ bao (Nguy€n Thanh Bao), d6ng thai nuac

·�

tir hen ngoai se di vao hen trong lam t€ bao ch€t (B<) Y t€, 2°b'15).
OS c6 thS tac d<)ng len mang t€ bao, khang sinh phai c6 c.iu true luang C\l'C
(m<)t d!u ai nu&c va m<)t d!u lq nu6c) dS chen vao duqc c.iu true hai lap
phospholipid cua mang t€ bao vi khuftn. Polypeptide (polymyxin) c6 ca chi tac
d<)ng ire ch€ chuc nang mang t€ bao vi khudn la nha c.iu truc-nhu v�y (Phc;1m Hung
Van, 2013). V6i chinh nha ca ch€ tac d<)ng nay, polymyxin c6 tac dl)ng di�t khudn
tuy�t d6i, rue la gi€t ca t€ bao dang nhan len va cat€ bao a trc;1ng thai nghi - khong
, ,_:,-.

nhan len (B<) Y te, 2015).
.. ,

,
.,
.,
Uc che Slf tong hr;,p protein cua vi khuan

"*

,.

C6 b6n nh6m khang sinh c6 ca ch€ tac d<)ng ire ch€ sg tfmg hqp protein cua t€
bao vi khudn:
Khang sinh nh6m aminoglycosides: giai do�n d!u: thu6c g�n vao m<)t lo�i
grotein la thl) th8 chuyen bi�t & tren ti8u dan vi 30S cua ribosome. Giai doc;1n hai:
thu6c phong b€ hoc;1t tinh cua phuc hqp d!u tien cua qua trinh thanh l�p chu6i
peptide (mRNA + formyl methionine + tRNA). Giai do�n ba: thong tin mRNA bi
d9c sai & vung nh�n di�n · cua ribosome, k€t qua la m<)t acid amin khong phu hgp
duqc dua vao chu6i peptide, t�o ra m<)t protein khong ch(rc nang. Giai doc;1n b6n: SlJ
g�n CUa thu6c lam VO' cac polysome thanh cac monosome khong CO kha nang tf>ng
hqp protein. Cac tac d<)ng nay xay ra it hay nhi�u c6 tinh d6ng thai va k€t qua cu6i
cung la ts bao vi khudn bi gi€t.
.

.
11