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

Siriraj Hospital, Mahidol University

chanwit.tri@mahidol.ac.th

ASM Ambassador to Thailand

www.asm.org

การเฝ้าระวังโรค : การปฏิบัติการ 2 Surveillance : Workshop II

mailto:chanwit.tri@mahidol.ac.th

Patient’s history

A 37 years old Thai man

Acute Leukemia

Long-term hospitalization

Consumed food obtained from

street vendor before developing

bacteremia and diarrhea

2

Methods

2 isolates of V. cholerae non-O1, non-

O139 have been recovered from

blood and stool culture

Antibiotic susceptibility test indicated

an ESBL production

Genetic confirmation was performed

by PCR amplification

3

ESBL Screening in V. cholerae

non-O1, non-O139

4

Horizontal Gene Transfer From

Enteric Bacteria?

Stool examination for other multidrug resistant bacteria was done

On 100 mg/L of ceftazidime MacConkey agar plate, Aeromonas sobria and 2 strains of Klebsiella pneumoniae were identified

PCR amplifications identified a blaVEB-1 and int genes in A. sobria

blaSHV and blaTEM were identified in K. pneumonia, but no blaVEB-1

5

ESBL screening with A. sobria and K. pneumoniae

6

Plasmid and conjugation assay

7

Liquid phase conjugation

Laboratory strain of E. coli was used

as a recipient (TOP10, Invitrogen)

Donor : Recipient = 1 : 10

Selection of transconjugants on 100

mg/L of ceftazidime MacConkey agar

plate

8

TOP10 E. coli Transconjugants

9

Plasmid profiles of AmpC hyperproducers

M 108 T108 No.58 108 215 232 197

Single AmpC plasmid has been isolated

Disk Diffusion of TOP10(p108)

15 mm

23 mm

25 mm

Cefotaxime

Cefotaxime/clavulanate

Cefoxitin

Gene Orientation in pT108

•99% DNA sequence matched with plasmid from Taiwan

(Deposited in GenBank in Oct, 2006)

The dot-blot hybridizations were performed on

247 strains of ESBL producing strains

(A) dhps probe (B) ColE1 ori probe

A B

dhps probe ColE1 probe

86 kb Resistance Island Acinetobacter baumannii

Total of 45 resistance genes

PLoS Genetics: www.plosgenetics.org January 2006, Vol. 2, Issue 1 14

Conventional PCR amplification of 5’ and 3’ junctions

PCR amplification Positive (No. of isolates/%)

5ʹand 3ʹ Junctions 23/63.8

5’ Junction 3/8.3

3’ Junction 7/19.44

Junction absent 3/8.3

OriC

AYE

5’ ATPase 3’ ATPase

AbaR1

AbaR2/ACICU

PCR amplification Positive (No. of isolates/%)

intI1 13/36.11

blaVEB-1 7/19.44

arr-2 10/27.77

cmlA 5/13.8

blaOXA-10 9/25

blaIMP All negative

blaOXA-23 36/100

blaVIM All negative

blaTEM-1 14/38.8

Amplification of variable region

and characterization of Integron

carrying isolates 32/36

5’CS 3’CS

intI1

Variable region

5ʹCS cmlA arr-2 aadB blaVEB-1 3ʹCS

5ʹCS-VEB-1R VEB-1F-

arr2R arr2F-cmlAR

cmlAF-3ʹCS

Ab03_168

Headline in Hong Kong March 11, 2010

Real Life Situation (1)

Long-term admission patient

– Unconscious

– Incontinence

– Catheterized

– Tracheostomy

– Bed sore

– History of MRSA and Pseudomonas

aeruginosa

19

Real Life Situation (2)

Diaper change by one medical personnel

Soap and water

Baby powder (talcum) for odor masking

Patient was coated with feces containing

105 CFU/g of MRSA/Pseudomonas

IV injection introduces 1 bacteria into

blood stream

Bacteria divide every 30 minutes

20

Real Life Situation (3)

Next day (24 hours later)

– There are 248 bacteria (THAT IS

281,474,976,710,656 cells!) in patient’s blood

The care team still didn’t wash their hands

The resistant organisms spread further

21

Conclusions

There were evidences of horizontal gene

transfer of resistance genes from

nosocomial pathogens to virulent

pathogen of community origin

Prohibition of food consumption not

prepared by the hospital should be

enforced

22

Conclusions Physicians should pay attention on whereabouts the reservoirs of multi-drug resistant bacteria

Awareness of unusual resistant phenotype also should be raised.

Reservoirs of multi-drug resistant bacteria exist

Decolonization of resistant bacteria may be needed in certain patients.

23