Department of Veterinary Public Health, Faculty of Veterinary Science, Chulalongkorn University

Antibiotic resistance in bacteria: Background and Significance

Rungtip Chuanchuen

Broilers serve as sources for widespread of resistant bacteria.

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

Co-selection

Cross-resistance

Chromosomal mutation

Horizontal transfer

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

S

S

S

S

R

R

R

RR

R

R

S

S

R

S

R

R

R

S

S

S sensitiveR resistant

I don’t think so.

I don’t think so.

Goal: minimize development &

spread of antibiotic resistance.

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

•National Bureau of Agricultural Commodity and

Food Standards (ACFS)