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.. - Tir cac kSt qua knao
Trang 4khang 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 �q 1 % )
+ 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 1liSu 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:
Trang 6
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 HocMon 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
Trang 9
1.5.2 Vift Nam 44
CHlfllNG 2 VJT LI}JU VA PHUONG PHAP 46
2.1 VJT Lli;;U ' ; 47
2 1 1 D• 1a rem va a1 gzan ng 1en ctrn a•,: 'th'• • h•A , 47 2.1.2 D<Ji tu()'ng nghien cuu ; .47
2.1.3 Thilt bj, d1:1ng Cl:l, moi truimg � ··:� 47
, A tfU' "Ii.-, 2.2 PHUONG PHAP NGHIEN C :· .48
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 rU<Jrtiit ESBL 58
2.2.6 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
Trang 11DANH 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
:-P ABA: :-Parraminooenzoic acid
-
vi
Trang 13DANH MUC HINH ANH
Hinh 1.1 Ty !¢ ESBL tu cac tac nhlin gliy nhiim khulm 6 bimg theo khu VlfC tu niim
Hinh 1.2 Tyli ESBL tu cac tac nhlin gliy nhiim trung iluang tiit niiu theo khu VlfC
\�
"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 1.5 Hinh thai khudn lqc P aeruginosa tren moi truang MacConkey (Ngu6n: Internet) ·- :7: 40
Hinh 3.3 Kit qua khang sinh il6 cua E.coli 57e sinh enzym ESBL, AmpC va
Trang 15DAT 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 vanghien cuu sau rc)ng tren th� gi&i Trong cac vi khuftn khang thu6c hi�n nay, dangluu 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
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
Trang 17•!• 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 chfrab�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 vacarbapenemase 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 thf i 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
Trang 23Cac 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
"
Trang 25"*- 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
Trang 31tren gfrn vi tri tam ho�t d<)ng cua enzym (Bradford, 2001 ), hay la gen m&i xufrt hi�n cfing tren plasmid, ho�c la tren gen nhay a nhi�m s�c thS cua vi khufin (Ph�m Hung Van, 2013) E>i�u nay canh bao m<)t nguy ca gia tang vi khufin d� khang khang sinh
va h€t khang sinh di�u tri trong m<)t tuang lai rfrt gfrn
, ho�t tinh cephalosporinase c6 kha nang pha huy cac cephalosporins th€ h� 3 va
\�
cephamycin, nhung khac v&i ESBL, AmpC cam ung kh'tJng pha buy dugc cac cephalosporin th€ h� 4 va khong bi ire ch€ b&i cac chfrt ire ch€ fJ - lactamase nhu acid clavulanic, sulbactam, tazobactam (Manchanda va cs, 2003; Shahid va cs, 2004; Thomson, 2010) AmpC cam ung dugc quy djnh b&i nhfing gen niim tren
Iihi�m s�c thS va cfihg c6 m<)t s6 vi khu§n mang gen amp{; cam irng niim tren
plasmid (Manchanda va cs, 2003; Livermore va Brown, 2001 ) M<)t s6 dong vi
khufin E coli va • K pneumoniae ti€t AmpC nha -gen tren plasmid Tuy nhien, vi
•
khufin ti€t AmpC qtia gen tren plasmid thi it c6 y nghia lam sang vi it ph6 biJn han
so vqi vi khufin ti€t ESB),,; han nfra vi khufin ti€t AmpC qua gen tren plasmid c6 ,thS di�u tri d� dang do nh�y cam v6i ceftriaxone, ·cefotaxime, ceftazidime (CLSI, 2013)
'* Enzym carbapenemase: la enzym thu<)c hQ fJ - lactamase bfrt ho�t dugc tftt ca cac lo�i � - lactam nhu pencillin, cephalosporin ph6 r<)ng va kS cakhang sinh m�nh nhftt hi�n nay la carbapenem, vfi khi khang sinh duy nhftt giup cac bac sidi�u tri chi dinh nhi�m khufin do vi khufin ti€t ESBL (Queenan va cs, 2007; Phi;im Hung Van, 2013) Carbapenemase pha huy dugc carbapenem v6i ngu6n g6c
· gen niim tren plasmid hay integron cua nhi�m s�c thS do v�y ma c6 kha nang lay Iancao Cac carbapenemase hi�n nay da x�frt hi�n a nhi�u nai tren th€ gi6i va dangdugc xem la "ac m<)ng" d� khang khang sinh d6i v6i con nguai (Phi;im Hung Van,
2013) C6 hai loi;ii carbapenemase dugc canh bao gfrn day, d6 la blaKPC va NDM
-1 KPC la m<)t loi;ii fJ - lactamase 16p A ngu6n g6c plasmid phat hi�n tren K.
pneumoniae, Iuu hanh a Hoa Ky, Nam My va Chau Au va hi�n cfing lay Ian ra m<)t
sf> loai khac (Yigit va cs, 2001) NDM - 1 dugc canh bao la nguy hi€m nhfrt vi Ian-
truy�n cao, thu<)c 16p B, metallo -fJ - lactamase, ngu6n g6c plasmid va intergon
17
Trang 41�
aeruginosa va Acinetobacter, m<)t khi da tiet AmpC cam (mg thi c6 th� khang dugc ��
nhfrng khang sinh thay the nhu carbapenem va fluoroquinolone (Livermore va
Brown, 2001 ) Ngoai ra, m�c du plasmid truyen dugc m9t sf> vi khufin nhu E coli,
va cs, 2002) nhung gen nay thuong niim tren nhi�m sic th� hon nen xet & muc d<)lay Ian vftn chua dang bao d<)ng nhu f3 - lactamase ph6 r<')ng (Ph?m Hung Van,2013)
1.3.3 Carbapenemase
De khang n6i tr<)i hi?n nay tren vi khufin Enterobacteriaceae la ESBL va ke d6
la /3 -4actamase-AmpC Hi?n nay_J?Lc6-them m<)t nguy ca-moi xmlt hi?n do la
Enterobacteriaceae khang dugc carbapenem, vfi khi khang sinh duy nhfrt giup cac
bac si' dieu trj ebi dinh tren cac nhi�m khufin do vi khufin tiet ESBL C6 hai lo?i
carbapenemase dugc canh bao gkn day, do la blaKPC va NDMl KPC la m<)t lo?i f3
- lactamase l&p A ngu?m gf>c plasmid, luu hanh & Hoa Ky, Nam My va Chah Aufren K pneumoniae va hi?n cfing lay Ian ra m9t sf> loai khac NDM - 1 dugc canh
bao la nguy hi�m nhfrt vi Ian truyen cao, thu9c l&p B, metallo - /3 - lactamase,
ngu6n gf>c plasmid va intergon, luu hanh cao & An D<), Pakistan, phat hien thfry & E.
- lactamase) ket hgp v6i d9t bien mfrt kenh porin Ngu6n g6c gen ctia cac CO' ch€nay thuong khong niim tren plasmid hay transposon do v�y ma tinh de khang kh6lay Ian trong cung loai hay khac loai Chinh vi v�y, phat h?n chinh xac vi khudn ti8tcarbapenemase la rfrt quan tn;mg d� canh bao nguy CO' lay Ian tinh d€ khang nguyhi�m nay trong b?nh vi?n (Ph?m Hung Van,•2011 )
27