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Department of Veterinary Public Health, Faculty of Veterinary Science, Chulalongkorn University
Antibiotic resistance in bacteria: Background and Significance
Rungtip Chuanchuen
What are drug resistant bacteria?
How do drug resistant bacteria occur?
What do antibiotics do?
What should we do?
Resistance determinants:
DNA segments encoding
for antibiotic resistance
Ex. transposons, resistance genes,integrons
Drug resistant bacteria:
Certain bacterial strains that are capable of
withstand specific antibiotics to which theyonce were susceptible.
Time of exposure
Natural
Susceptible
bacteriaDrug exposure
RR
S
SS
S
Resistant
bacteria
S RR R R RR R R
Dominant
Drug
exposure
SS SS
Rare
Dead!!
Pseudomonas Enterobacteriaceae
Vibrio cholerae
Campylobacter
Resistance
transferR RS R
Resistant Susceptible Resistant Resistant
Survival of the fittest:
“Individuals having the highest level of fitness for
particular environment tend to survive longer in it.
http://www.mbedard.com/scripts/prodView.asp?idproduct=330
Some bacteria were
born to be resistant.(Intrinsic resistance)
Some bacteria develope
resistant later through
mutations or obtaining
resistance determinants
(Acquired resistance)
Erythromycin
Citracin,
Novobiocin,
Rifampin, Trimethoprim
Streptogramin B
Salmonella:
Campylobacter:
Susceptible bacteria
S
RXX
S SS
RXX New resistant
bacteria
Mutations
Antibiotic exposure R
RXX
RXX
R R
Resistance gene
transfer
Antibiotic exposure
MDR bacteria:
Certain bacterial strains that are capable of
resistant to many antibiotics of different classessimultaneously.
http://gardenrain.wordpress.com/2009/03/26/vancomycin-resistant-enterococcus-vre/
VRE DT104 MDR ST131
Different resistance determinants are present on
the same genetic element.
A single antibiotic coselect for several resistance genes
resulting in MDR phenotype.
TetRGenR
AmpR
KamR
R-plasmid
intl1 qacE∆1 sul1
Integrase
gene
Gene cassettes in
variable region
GenR TetR StrepR
GenR Genamicin resistance gene; TetR Tetracycline resistance gene; StrepR Streptomycin resistance gene
Horizontal transfer in the presence of
antimicrobials
Confer resistance to multiple antibiotics
Coselection of many resistance genes by a single antibiotic
intl1 qacE∆1 sul1aadA StrepRGenRTetR
Most commonly assocaited with MDR
GenR Genamicin resistance gene; TetR Tetracycline resistance gene; StrepR Streptomycin resistance gene
Salmonella enterica
Commensal E. coli
Campylobacter coli
Aeromonas hydrophila
Vibrio species
Lukkana et al (2012) J. Vet. Med. Sci.
Khemtong & Chuanchuen (2008) Microb Drug Resist.; Chuanchuen et al (2010) Foodnorne Pathog. Dis.
Wannaprasat et al (2011) Int. J. Antimicrob. Agents
Ekkapobyotin (2008) Int. J. Food Microbiol.; Lay et al (2012) Foodnorne Pathog. Dis.(submitted)
Kitiyodom et al (2010) FEMS Microbiol. Eco.
Species Source Class 1 integrons Transfer
Salmonella Poultry & pigs 33/54 14/33
Pork & humans 18/84 5/18
Healthy cow 2/2 0/2
Commensal E. coli Pigs 46/251 8/46
C. coli Pigs 4/4 nd
Vibrio species P. monodon 0/5 0/5
A. hydrophila Nile tilapia 14/23 3/14
* No. of isolates carrying class 1 integrons with resistance gene cassettes/No. of intl1-positive isolatesClass 1 integrons play a role in dissemination of
resistance among bacteria of food animals.
dfrA12 encoding resistance to trimethoprim
aadA2 encoding resistance to streptomycinHorizontal transfer of resistance has taken place.
intl1 qacE∆1 sul1aadAdfrA12 aadA2
dfrA12-aadA2
Salmonella
Salmonella
SalmonellaE. coli
Salmonella
A.hydrophila
Salmonella
A. baumannii
P. aeruginosa
Resistance determinants circulate among
bacterial pathogens in Thailand.
Thailand
( Salmonella from
poultry, pig pork,
chicken human)E. coli from pigs
Korea
(Salmonella
from
poultry;
E. coli from humans )
Vietnam
(Salmonella from poultry)
Norway
(E. coli from cattle )
Taiwan
(Salmonella from pigs)
Germany(E. coli from pigs)
dfrA12-aadA2
A resistance-virulence
plasmid
Salmonella
Virulence gene(spvC)
Class 1 integrons
A single drug selects for both resistance and
virulence genes.
The same genetic determinant responsible for
resistance to many antibiotics.
A single antibiotic could promote resistance to many
drugs simultaneously resulting in MDR phenotype.
Bacteria expose to
an antibiotic
Bacteria develop
resistance to
the antibiotic.
Bacteria develop
resistance to multiple
antibiotics.
Mex
Low intracellular-
drug concentration
Resistant to many
antimicrobials
“Cross-resistance”
MDR bacterial strainsMultidrug efflux pumps
AMG
TET
ERY
FLU
ampicillin, chloramphenicol, erythromycin, bile salts, triclosan,
chloroxylenol, chlorhexidine
Porin
H+
OM
H+
IM AcrB
AcrA
TolC
drugsdrugs
Wannaprasat and Chuanchuen (submitted)
AcrAB-TolC
expresses
all the times in Salmonella.
Lin J et al. Antimicrob. Agents Chemother. 2002;46:2124-2131
erythromycin, ciprofloxacin, detergents and dyes
Porin
H+
OM
H+
IM CmeB
CmeA
CmeC
drugsdrugs
Species Source No. Resistance
(%)
Salmonella Poultry & pigs 211 28
Pork & humans 183 48
Healthy cow 160 2
Commensal E. coli Pigs 344 79
Chloramphenicol resistance gene may be co-selected
by other antimicrobials.
An efflux pump that also extrudes chloramphenicol
may be turned on by other antimicorbials.
Study 1:
Test for horizontal transfer of resistance
Study 2:
Test for cross-resistance among antibiotics used in broilers
Study 1 . Test for horizontal transfer of resistance
Resistant S. enterica from broilers & farm
environment (n=65)
ChiangMai (n=33), 2549-2550, Khon Khan (n=32), 2551-2553
Antibiotic susceptibility test
ampicilin, chloramphenicol, gentamicin, streptomycin, sulphonamides, tetracycline & trimethoprim
Test for resistance transferRecipient: E. coli MG1655rifr
Conjugation experiment
+
R
R
S
S
Resistant
SalmonellaDonor
E. coli
Recipient
R resistantS Suceptible
1
2
+ Resistance transfer
Drug No. of resistant
isolates (n)
No. of
transfer (%)
Ampicillin
Streptomycin
Chloramphenicol
Gentamicin
Tetracycline
Sulphamethoxazole
Trimethroprim
52
60
42
37
40
62
41
3(5.8)
10 (16.7)
8(19.0)
9 (24.3)
13(32.5)
8 (12.9)
12 (29.3)
The Salmonella isolates tested could transfer
resistance to the E. coli recipient.
Study 2: Test for cross-resistance among antibiotics
S. enterica from broiler & farm environment:
sensitive to antibiotics tested (Khon Khan, 2012)
Antibiotic susceptibility test
ampicilin, chloramphenicol, gentamicin, streptomycin, sulphonamides, tetracycline & trimethoprim
In vitro exposure experiment
In vitro exposure experiment
………
1/4 MIC 1/2 MIC
MIC
4 MIC
0
20 days
……
…
1 5
………
MIC
Day
-Antibiotics
2
Exposure to certain antibiotics could promote
cross-resistance to other antibiotics in Salmonella.
Strain ExposureAntibiotics
exposedCIP TET CHP NAL TRI CEF
H1/1 Pre 4 128 32 32 4 >64
H1/1 CIPR Post CIP 64 256 32 >256 64 >64
H1/2 Pre 4 4 32 16 1 16
H1/2 NALR Post NAL 4 64 32 256 4 8
S6 Pre 8 64 128 128 0.5 8
S6 CEFR Post CEF 8 128 128 >256 64 64
S7 Pre 1 64 32 8 >64 32
S7 CIPR Post CIP 64 128 32 >256 >64 32
S7 NALR Post NAL 1 64 64 >256 >64 32
S10 Pre 4 16 128 128 1 8
S10 TETR Post TET 32 256 64 256 256 16
S16 Pre 4 128 32 16 1 16
S16 CHPR Post CHP 2 128 256 >256 256 32
S16 NALR Post NAL 2 256 64 256 256 16
Strain Exposure Antibiotics exposed
CIP TET CHP NAL TRI CEF
HE1 Pre 4 64 128 16 2 2
HE1 NALR Post NAL 4 256 128 256 256 32
HE2 Pre 4 64 128 128 64 2
HE2 CEFR Post CEF 4 128 128 >256 64 64
HE4 Pre 8 64 32 32 1 8
HE4 CHPR Post CHP 4 128 256 >256 256 8
HE4 TRIR Post TRI 8 64 32 32 256 8
HE4 CEFR Post CEF 64 256 128 >256 32 64
HE5 Pre 8 64 64 32 64 2
HE5 CHPR Post CHP 4 128 256 >256 256 16
E14 Pre 16 64 64 128 2 2
E14 TRIR Post TRI 16 64 128 256 256 8
Exposure to certain antibiotics could promote
cross-resistance to other antibiotics in E. coli
Infection prevention
Research intervention
Rational use & Regulation
Surveillance & monitoring
Clean animal husbandry
Prudent use:
Usage of antimicrobials, which maximizes therapeutic effect
and minimizes the development of antimicrobial resistance
(WHO, 2000)
Responsible use:
The use of animal medicines carries with it responsibilities
(Veterinarians’ and Farmers’) (RUMA, 2005).
Judicious use:
Antibiotics should be used to maximize the benefits of the
therapeutic antibiotic use while minimize the development of resistance
Goal:
Maximize the benefits of the therapeutic antibiotic use,
while minimize the development of resistance
Suggestion for the guideline development:
Species-specific clinical practice guidelines on the responsible use of veterinary antimicrobial drugs
1. Increased expenses
2. Time consumption
3. Laborious task
1. Protect consumer health by
ensuring the safety of food of
animal origin
2. Maintain the efficacy of antibiotics
in both human and veterinary
medicine
3. Prevent & reduce development &
transfer of resistant bacteria &
resistant determinants
4. Support international trade
(Prepare for possible trade barriers)
Judicious use of Antimicrobials in Poultry. AVMA (2007)
Antibiotic Resistance & Prudent use of Antibiotics in Veterinary Medicine. Federation of Veterinarians of Europe (FVE,1999)
Draft position paper on responsible use of antimicrobials: The Global
Basic Principles. World Veterinary Association (WVA, 2011)
Antimicrobial resistance: responsible and prudent use of antimicrobial
agents in veterinary medicine. Anthony et al (2001) (OIE)
RUMA (Responsible Use of Medicines in Agriculture Alliance) (2005)
Responsible use of antimicrobials in poultry production.
WHO Global Principles for the Containment of Antimicrobial
Resistance in Animals Intended for Food. WHO (2000)
Guidelines for Prescribing, Authorizing and Dispensing Veterinary
Medicines. Australian Veterinary Association (AVA, 2005)
AVPA code of practice for the use of antibiotics in the poultry industry.
Endorsed by the Australian Chicken Meat Federation and the Australian Egg Industry Association. AVPA (2001).
The use of antibiotics in food-producing animals: antibiotic-resistant bacteria in animals and humans. JETACAR (1999).
European Platform for the Responsible Use of Medicines in Animals (EPRUMA) (2008)
2. นิยาม4. สัตวแพทยแ์ละผูไ้ดรั้บมอบหมาย7. เกณฑก์ารใชย้าตา้นจุลชีพอยา่งสมเหตุสมผล (ภาคผนวก ก.1)8. การเลือกใชย้าตา้นจุลชีพในการรักษาโรคในไก่เน้ือ (ภาคผนวก ก.2)9. การใชย้าตา้นจุลชีพ (ภาคผนวก ก.3)
(เอกสารส าหรับจัดท า)ร่างแนวทางการใช้ยาต้านจุลชีพอย่างสมเหตุสมผลในฟาร์มไก่เนือ้
(เอกสารส าหรับจัดท า)ร่างแนวทางการใช้ยาต้านจุลชีพอย่างสมเหตุสมผลในฟาร์มไก่เนือ้
10. การเกบ็ตวัอยา่งเพื่อตรวจความไวต่อยาตา้นจุลชีพ (ภาคผนวก ก.5.1)- เพื่อเป็นแนวทางในการรักษา- เพื่อเฝ้าระวงัและตรวจติดตามการด้ือยา
11. การแยกและพิสูจน์เช้ือจากตวัอยา่งในหอ้งปฏิบติัการ12. การตรวจความไวต่อยาตา้นจุลชีพของแบคทีเรียในหอ้งปฏิบติัการ
- เพื่อเป็นแนวทางในการรักษา- เพื่อเฝ้าระวงัและตรวจติดตามการด้ือยา
13. การรายงานผล
ภาคผนวก กก.1 เกณฑก์ารใชย้าตา้นจุลชีพอยา่งสมเหตุสมผลในการเล้ียงไก่เน้ือ
ก.2 ยาตา้นจุลชีพท่ีแนะน าใหใ้ชรั้กษาและ/หรือควบคุมโรคติดเช้ือ แบคทีเรียในสตัวปี์กก.4 แนวทางการใชย้าตา้นจุลชีพในการรักษาโรคติดเช้ือแบคทีเรีย ท่ีส าคญัในสัตวปี์กก.5 การเกบ็ตวัอยา่งก.7 ยาตา้นจุลชีพท่ีแนะน าใหต้รวจความไวเพื่อการเฝ้าระวงัและการ ตรวจติดตามการด้ือยา
(เอกสารส าหรับจัดท า)ร่างแนวทางการใช้ยาต้านจุลชีพอย่างสมเหตุสมผลในฟาร์มไก่เนือ้