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TRANSCRIPT
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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 [email protected]
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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
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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.
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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
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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
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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)
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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
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Coagulase (coa) Gene Polymorphism
Primers
Coa2:
5′-CGAGACCAAGATTCAACAA G-3′
Coa3:
5′-AAAGAAAACCACTCACATCA-3′
Followed by AluI endonuclease restriction
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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/
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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
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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
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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.
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Of the 72 MRSA isolates,
• 50 (69.4%) nontypable,
• 19(26.4%) Lytic group III
• 3 (4.2%) Mixed group.
Phage Typing
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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
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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
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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
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Rsa I digested-spa PCR product
All of 72 strains revealed A-pattern
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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
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Alu I digested-coa PCR product
Pattern A = 1 strain
Pattern B = 69 strains
Pattern C = 1 strain
Pattern D = 1 strain
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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
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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
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• 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)
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PFGE patterns 72 isolates
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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
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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)
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ST239 and their complex
ST239
8
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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
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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.