Chanwit Tribuddharat, M.D., Ph.D.

Siriraj Hospital, Mahidol University

chanwit.tri@mahidol.ac.th

ASM Ambassador to Thailand

www.asm.org

การป้องกันและควบคุมจุลชีพดื้อยา Prevention and Control of

M.D.R.O.

mailto:chanwit.tri@mahidol.ac.th

Outline

Focussed antibiotic resistant bacteria

Horizontal gene transfer and vertical gene

transfer

Selection of Molecular (strain) typing

Resistance genes in hospital and

community

Evolutional pathway of resistance genes

Unsafe society

Horizontal Gene Transfer

Transformation

Conjugation

Transduction

Antibiotic Resistant Superbugs

The best groups of bacteria that survive

the antibiotic selection

Range from Gram positive to Gram

negative bacteria

– MRSA, Coagulase negative Staph.,

Corynebacterium sp., Pseudomonas,

Acinetobacter, other NF, etc.

Of hospital, agricultural, veterinary, and

farming (ending up as environmental)

“Gram-negative” Resistance Integron:

What we should know! “Integron” was named by H. W. Stokes and

Ruth Hall in 1989 (Australia)

Paul H. Roy et al. was also working on

these genetic elements in late 80s

(Canada)

Different resistance genes surrounded by

conserved DNA segment encoding a

putative recombinase

Integron Element

The antibiotic resistance gene

collecting machine

Most clinical (nosocomial) bacterial

pathogens have the elements

Mobile element as

– Full functional mobile elements (not so?)

– “piggybacking” in other Transposons

Detection and identification of class 1 integron

Antimicrobial Agents and Chemotherapy, Jan. 1995, p. 185–191.

Integron Element in

Pseudomonas aeruginosa

Clin. Infect. Dis. 2002;34:603-611, Girlich et al.

Identified Resistance Genes related to

Integrons Thai clinical isolates

Resistance gene Affected antibiotic Coding for

blaIMP-14 Carbapenem Metallo-ß-lactamase

blaIMP-15 Carbapenem Metallo-ß-lactamase

blaVEB-1 Cephalosporin Class A ß-lactamase

blaPSE-1 Carbenicillin Class A ß-lactamase

blaOXA-10 Oxacillin Class D ß-lactamase

Resistance Genes related to Integrons

Resistance

gene Affected antibiotic Coding for

aac(6') Amikacin Aminoglycoside

acetyltransferase

aadA2 Streptomycin Aminoglycoside

adenyltransferase

aph Kanamycin Aminoglycoside

phosphotransferase

aadB Gentamicin Aminoglycoside

adenyltransferase

arr-2 Rifampin Rifampin ADP-ribosylating

transferase

Resistance Genes Related to Integrons

Resistance

gene Affected antibiotic Coding for

dfr Sulfonamide/trimethoprim Dihydrofolate reductase

cmlA Chloramphenicol Chloramphenicol

transporter

qacED1 Quaternary ammonium

compound

Quaternary ammonium

compound resistance

sul 1 Sulfonamide Dihydropteroate synthase

qnrA Fluoroquinolone Fluoroquinolone protective

protein

Results: Percent positive Species (number) blaTEM blaSHV

blaCTX-M

blaVEB int

E. coli (100) 69 12 40 2 85

K. pneumoniae (67) 63 87 21 8 90

P. aeruginosa (31) 0 0 0 19 94 E. cloacae (13) 85 30 8 15 92 NF (11) 0 9 0 9 55 A baumannii (6) 0 0 0 50 67 P. mirabilis (5) 40 0 20 20 100 S. marcescens (5) 0 20 0 20 60

French and Thai Acinetobacter Clones

J. Clin. Microb. 2003, V. 41 p.3542

86 kb Resistance Island Acinetobacter baumannii Total of 45 resistance genes

PLoS Genetics: www.plosgenetics.org January 2006, Vol. 2, Issue 1

Mechanism of antimicrobial resistance in

Escherichia coli and Salmonella from food animals in northern Thailand

Padungtod, P.1, Tribuddharat, C.2, Chuanchuen, R.3 1 Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, Thailand.

2 Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University 3 Department of Veterinary Public Health, Faculty of Veterinary Science, Chulalongkorn

University

Widespread presence of dfrA12 and its association

with dfrA12-aadA2 cassette in Salmonella enterica

isolates from swine. Padungtod P, Tribuddharat C, Chuanchuen R.

Southeast Asian J Trop Med Public Health. 2011 Nov;42(6):1471-6.

Animals from Farms

(and their bacteria!)

Multi-drug resistant Salmonella spp. may

be spread throughout Thailand now.

– Most farms give antibiotic as feeding

supplement

– The bacteria carry integron elements

– The one identified matches with those in other

regions of the world

17

Bacterial isolates E coli Food Animal 258

Human 6

Farm env 26

Total 290

Salmonella Food Animal 117

Human 0

Farm env 167

Total 284

Chiang Mai Province

PCR of Salmonella

Antibiotic Resistance Cassettes

Salmonella spp. isolated from different animal farms

Various Class 1 Integrons

Prevalence of E. coli and Salmonella spp. in sealed packages of chicken meat from

supermarkets in Thailand

Prevalence Integrase (intI1)

E. coli 53% (106/200) 37.74% (40/106)

Salmonella 18.67% (14/75) 42.86% (6/14)

Isolate Serogrouping Serotyping RAPD-PCR Resistance

phenotype

Place of

collection

Collection date

Sal 130 Unidentified

serogroup

Kedougou I AM, GM A1 Jan 2, 2011

Sal 138 B Saintpaul II AM, GM B2 Mar 14, 2011

Sal 143 B Schwarzengrund III AM, GM B2 Mar 14, 2011

Sal 145 B Agona IV - B2 Mar 14, 2011

Sal 154 E Orion V AM, GM A3 Apr 4, 2011

Sal 160 C Manhattan VI AM, GM A3 Apr 4, 2011

Sal 162 Unidentified

serogroup

Kedougou I AM, GM, TE A1 Apr 4, 2011

Sal 164 B Saintpaul II AM, GM A1 Apr 4, 2011

Sal 165 B 4,12,27:d:- III AM, GM A1 Apr 4, 2011

Sal 178 C Albany VII AM, SXT, TE A1 Apr 19, 2011

Sal 180 Unidentified

serogroup

Albany I AM, GM, TE A1 Apr 19, 2011

Sal 186 B Kiambu VIII - C4 Apr 25, 2011

Sal 190 C Altona IX - C4 Apr 25, 2011

Sal 193 C Altona IX AM, GM A3 May 3, 2011

A, B, C = Supermarkets 1, 2, 3, 4 = Companies

intI1 5'CS-

3'CS

Resistance

cassette (s)

Resistance

phenotype

Accession

number

Dot blot

E9 (E. coli) + 1 kb aadA23 AM, CIP,

LEV, GM

FN252409 +

E9 (E. coli) + 700 bp dfrA5 AM, CIP,

LEV, GM

GU562437 +

E12

(E. coli)

+ 850 bp aadA4 CIP, LEV,

GM, SXT

FQ482074 +

E17

(E. coli)

- 500 bp lnuF AM, CF,

GM

EU118119 +

Sal130

(S. Kedougou)

- 650 bp ∆aadA2,

lnuF

AM, GM EU118119 -

Sal162

(S. Kedougou)

+ 1 kb aadA22 AM, GM,

TE

AB434537 +

Multilocus Sequence Typing (MLST)

Samples

Allelic profile ST Countries

thrA-purE-sucA-aroC-hisD-hemD-dnaN

Sal 138 17-6-12-5-9-18-21 50 Chile, Denmark, U.S.A.,

Germany, Australia

Sal 162 141-95-9-417-262-15-4 1543 Vietnam

Sal 165 3-41-15-43-49-49-47 96 Tunisia, U.S.A., Taiwan,

Denmark, Scotland

Sal 193 102-426-9-347-496-78-394 1549 Vietnam

ST11 (9)

ST34 (2)

ST469 (1)

ST96 (6)

ST469 (2)

ST11 (1)

ST139 (2)

ST11 (3)

ST34 (2) ST213 (5) ST469 (2)

ST11 (20)

ST19 (1)

ST34 (1)

ST213 (1)

ST139 (13)

ST34 (1)

Sequencing Run Performance

Shotgun run: Run yield 79MB Average read length 444bp

8Kb pair-end run: Run yield 98MB Average read length 421bp

Genome assembly:

No. of scaffolds 7 N50 scaffold size 4.7MB (the largest scaffold) No. of contigs 53

26

Whole genome sequencing

Salmonella No. 162

Genes Resistance phenotype

aac(3)-Ild

qacH

sul3

sat

aadA2

cmlA

gentamicin, tobramicin

Quaternary ammonium compound

sulfonamide

streptothricin

aminoglycosides

chloramphenicol

Genbank no.

JX101693.1

HQ386833.1

HQ386833.1

FM242709.1

JQ805121.1

HQ875012.1

Salmonella Largest Scaffold Analysis -

The Chromosome

BLAST

ANNOTATION

FUNCTION ANALYSIS

Pathogenicity island, virulence factors, and resistance genes

were identified

S.entericaserovar Choleraesuis

chromosome4.7Mbp

S. enterica subsp. enterica serovar Choleraesuis str. SC-B67 as a Reference Strain

Pathogenicity Island

Contigs

GC content

SPI

Flagellin

O-antigen gene cluster

+ strand

- strand

%GC > mean

%GC < mean

Salmonella enterica from Chicken Meat

August 2010

30

blaTEM-135 mobC mobA tnpR

Global Dissemination of IncP Plasmid Containing new blaTEM-1 variant, blaTEM-135, in Neisseria gonorrhoeae

• MLST Study: Collaboration with the

National Institute of Infectious Diseases,

Japan

• International spread among the UK,

Greece, Japan, and Thailand

• N. gonorrhoeae is transforming to ESBL

producer! • Sexual networks exist!

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Cluster A

Cluster

B

Cluster

C

* TEM-135 Asia

Nakayama et al.

Gonococci Cluster Analysis: MLST

MLST revealed some

correlations of the

isolates between

Thailand, Japan, the

United Kingdom, and Greece

J Antimicrob Chemother 2012; 67: 1769–1774

Journal of Medical Microbiology

(2011), 60, 619–624

Clin. Microbiol. Rev. 2013, 26(4):744.

ESBL carriage rates in the community

Clin. Microbiol. Rev. 2013, 26(4):744.

Myanmar

Drugs

from Thailand

CT 38

Any situation! Hospital/Community/Animals (food

and pets)/Environment/Pleasure

We are in the world of resistant bacteria

International collaboration is crucial for a possibly

controllable future

Molecular typing methods are part of evidence-

based medicine

Conclusions on Infection Control