การสอบสวนโรคติดเชื้อในโรงพยาบาล MRSA Model in Rajavithi Hospital

by

Chanwit Tribuddharat, M.D., Ph.D. Department of Microbiology

Faculty of Medicine Siriraj Hospital

Mahidol University

E-mail sictb@mahidol.ac.th

Study Plan

Infectious Disease experts from universities

Microbiological experts from Thai NIH

Volunteer hospital (Rajavithi Hospital)

Infection Control personnel (Very very strong

group of determined staff)

Supporting budget from the Government

(700,000 bahts)

Accepted for publication in European Journal

of Clinical Microbiology & Infectious Diseases

on 30 April 2010

Study population and sample collection

619 in-patients

19 different wards

During November 9 – 10, 2006

All participants have signed informed

consent and agreed to be screened for the

presence of MRSA from their stools nasal

swabs

The study has been approved from the

ethical committee of the Rajavithi Hospital.

Microbiological Methods

Nasal swabs, feces or rectal swabs were

cultured on selective media (blood agar +

6 μg oxacillin + 4% NaCl)

Feces or rectal swabs also were cultured

on PEA (Phenylethyl Alcohol Agar)

Confirmation of methicillin resistance was

based on a 30 µg cefoxitin disk on Muller-

Hinton agar and a 6 µg oxacillin disk

Phage typing

72 MRSA was performed by heat-shock

technique (heating a culture at 55C for 3

min immediately before phage typing)

International phage typing set issued by

the International Center, Colindale, UK

was used

Determination of Hypervarible

Region downstream of mecA Gene

primer HVR1:

5′-ACTATTCCCTCAGGCGTCC-3′

(position 338-356)

Primer HVR2:

5′-GGAGTTAATCTACGTCTCATC-3′

(position 912-892)

(Using GenBank accession number X52594)

Typing by Surface Protein A (spa) Gene

spa forward primer:

5′-TGTAAAACGACGGCCAGTGCTAAA

AAGCTAAACGATGC-3′

spa reverse primer:

5′-CAGGAAACAGCTATGACCCCACCA

AATACAGTTGTACC-3′

Following by RsaI endonuclease restriction

of the PCR product

Coagulase (coa) Gene Polymorphism

Primers

Coa2:

5′-CGAGACCAAGATTCAACAA G-3′

Coa3:

5′-AAAGAAAACCACTCACATCA-3′

Followed by AluI endonuclease restriction

Multi-locus sequence typing (MLST)

Approximate 400-500 bp PCR fragments

of the seven housekeeping genes

arc, aroE, glpF, gmk, pta, tpi, and yqiL

Allelic profiles were obtained from the

MLST website http://saureus.mlst.net

http://saureus.mlst.net/

Total number of patients 619

Total identified carriers 57

MRSA in nose and stool 7

MRSA in nose only 45

MRSA in stool only 5

Results

Confirmation

72 MRSA were isolated and confirmed by

Chrom-agar

coagulase production

Latex Agglutination Test for identification of S. aureus

(Pastorex Staph-plus : Bio-Rad)

Latex Agglutination kit for the rapid detection of PBP2’ (MRSA-Screen : DENKA SEIKEN CO.,LTD.)

Oxacillin disk agar diffusion technique

Oxacillin sreening agar test

Phage Typing Phage typing of 72 MRSA was performed by Heat-shock technique (heating a culture at 55C for 3 min immediately before phage typing), using the international phage typing set issued by the International Center, Colindale,UK.

The phage typing set consisted of – Lytic group I : 29, 52, 52A, 79, 80 ;

– Lytic group II : 3A, 3C, 55, 71;

– Lytic group III: 6, 42E, 47, 53, 54, 75, 77, 83A, 84, 85 ;

– Lytic group V : 94, 96

– Miscellaneous group : 81, 95.

Susceptibility to phages was determined by standard routine test dilution (RTD) at 1,000 x RTD.

Of the 72 MRSA isolates,

• 50 (69.4%) nontypable,

• 19(26.4%) Lytic group III

• 3 (4.2%) Mixed group.

Phage Typing

PCR typing methods

SCCmec PCR

– Type I = 613 bp

– Type II = 398 bp

– Type III = 280 bp

Spa gene PCR

– 504 bp

Coa gene PCR

– 800 bp

HVR PCR

– 291, 371,411, 451, 491, 531, 691, 771 bp

SCCmec PCR Primers – Type I-F = 5’- gct tta aag agt gtc gtt aca gg -3’

– Type I-R = 5’- gtt ctc tca tag tat gac gtc c -3’

– Type II-F = 5’- cgt tga aga tga tga agc g -3’

– Type II-R = 5’- cga aat caa tgg tta atg gac c -3’

– Type III-F = 5’- cca tat tgt gta cga tgc g -3’

– Type III-R = 5’- cct tag ttg tcg taa cag atc g -3’

PCR condition – 95 C for 4 min

– 95 C for 1 min

– 65 C for 30 sec 10 cycles

– 72 C for 1 min

– 95 C for 1 min

– 55 C for 30 sec 25 cycles

– 72 C for 1 min – 72 C for 5 min

All of 72 strains revealed Type III SCCmec

Spa PCR

Primers

– 5’-tgtaaaacgacggccagtgctaaaaagctaaacgatgc-3’

– 5’-caggaaacagctatgaccccaccaaatacagttgtacc-3’

PCR condition – 95 C for 4 min

– 95 C for 40 sec

– 60 C for 30 sec 35 cycles

– 72 C for 1 min

– 72 C for 7 min

All of 72 strains showed a 501 bp

Rsa I digested-spa PCR product

All of 72 strains revealed A-pattern

Coa PCR

Primers

– 5’-cgagaccaagattcaacaag-3’

– 5’-aaagaaaaccactcacatca-3’

PCR condition – 95 C for 4 min

– 95 C for 40 sec

– 60 C for 30 sec 35 cycles

– 72 C for 1 min

– 72 C for 7 min All of 72 strains showed a 800 bp

Alu I digested-coa PCR product

Pattern A = 1 strain

Pattern B = 69 strains

Pattern C = 1 strain

Pattern D = 1 strain

HVR PCR

Primers

– 5’-actattccctcaggcgtcc-3’

– 5’-ggagttaatctacgtctcatc-3’

PCR condition

– 95 C for 4 min

– 95 C for 1 min

– 55 C for 30 sec 35 cycles

– 72 C for 1 min

– 72 C for 7 min

HVR-PCR

3 DRU = 1 strain 5 DRU = 5 strains

6 DRU = 1 strain 7 DRU = 48 strains

8 DRU = 1 strain 9 DRU = 1 strain 13 DRU = 4 strains 15 DRU = 10 strains

• CDC protocol.

• Extracted chromosomal DNA

• digested with the SmaI restriction enzyme.

• PFGE was performed with CHEF DRII and

CHEF DRIII system (Bio-Rad laboratories),

• running condition was 6V/cm, with switching

times of 5-40 seconds for 21 hours.

• PFGE was analyzed by Syngene Gene Directory Application – Version 1.02.0.

Pulsed field gel electrophoresis (PFGE)

PFGE patterns 72 isolates

P24, 45(72)

P33

P36

P1, 2(51), 47(48, 49, 50),

4, 5, 6, 60, 7, 8, 9(10), 13,

14, 15(64), 20, 21, 61,

62,63,16, 17, 18, 19, 29,

34, 35, 37, 38, 39, 40,

54(55), 56(57), 58(59), 65, 66, 67(68), 70(71)

P31(32)

P22

P11, 12, 30, 69, 44

P3, 53, 23, 25, 26,

27, 28, 41, 42, 46,

P43

P52

Grouping by

Combined PCR-based methods

Floor plan of a 12-floor building Ward West wards East wards

10th Floor, Ortho. (P34, ST1227), (P35, NoST ), (P37, ST1227), (P38, NoST), (P39, ST239 )

(P46, NoST), (P45(P72), NoST)

9th Floor, ENT (P3, NoST), (P52(P53), ST239)

8th Floor, Gen. Surg. (P23, ST239), (P25, ST239), (P26, ST239), (P27, ST239), (P28, NoST), (P24, ST239)

(P29, ST239), (P66, NoST)

7th Floor, Worker Healthcare/GYN

(P70(P71), NoST), (P43(P44), NoST) (P31(P32), ST239), (P33, ST239)

6th Floor, Med. (P4, NoST), (P54(P55), ST239), (P56(P57), ST1228), (P58(59), ST239)

4th Floor, Radio. (P40, ST239), (P41, NoST), (P42, NoST) 3rd Floor, ICU. Surg. (P36, NoST)

EMS Building

West wards East wards

(P67(P68), NoST), (P30, ST239), (P69, NoST)

(P16, NoST), (P17, ST343)

Internal Medicine Building West wards East wards

3rd Floor (P7, ST239), (P8, NoST), (P9(P10), NoST), (P13, NoST), (P11, NoST), (P12, ST239)

(P5, NoST), (P6, ST239), (P60, NoST)

2nd Floor (P14, NoST), (P15(P64), NoST), (P61, NoST), (P62, NoST), (P63, ST1227), (P20, ST239), (P21, NoST), (P22, ST241)

(P18, NoST), (P19, NoST)

1st Floor, ICU Med. (P1, NoST), (P47(P48, P49, P50), NoST), (P2(P51), ST239)

ST239 and their complex

ST239

8

Heteroduplex-PCR for ST239 MRSA

ST 30-like specific primers

SA0317Forward: 5′-TCGCACTCTCGTTGAACA-3′

SA0317Reverse: 5′-AAATCCGCTTCGACAAACATT-3′

ST 8-like specific primers

SA2003Forward: 5′-CACTTTAAATACTGACGAAAAT-3′

SA2003Reverse: 5′-TTGAAAATTGATCATTCAGCAA-3′

Edward J Feil et al. J Clin Microbiol. 2008 April; 46(4): 1520–1522

Heteroduplex PCR for ST239 Lineage

The identification of the ST8 and ST30 recombinant chromosome.

PCR of 484 bp = ST30-like chromosomal segment and 220 bp = ST8-

like segment.

T. Jariyasethpong, C. Tribuddharat, S. Dejsirilert, et al: Eur J Clin

Microbiol Infect Dis (2010) 29:977–9

ST-30 Like = S. aureus Cowan I

ST-8 Like = MRSA SCCmec IV

ST239 = P24

ST241 = P22

ST343 = P17

ST1227 = P37

ST1228 = P56

ST8 F: 5′-CACTTTAAATACTGACGAAAAT-3′

R: 5′-TTGAAAATTGATCATTC AGCAA-3′

ST30 F: 5′-TCGCACTCTCGT TGAACA-3′

R: 5′-AAATCCGCTTCGACAAACATT-3′

ST-30 ST-8 ST-239 ST-241 ST-343 ST-1227 ST-1228

Like Like

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Conclusions

MRSA is a major nosocomial pathogen

At the prevalence rate of 9.2%.

Risk factors for MRSA carriage were the

same as other studies (long stay,

Antibiotics, male, chronic diseases, etc.)

Long term and large scale spread of

MRSA subtypes has occurred in a single

hospital.

The predominant MRSA type in this

hospital is of ST239 clonal cluster.