Staphylococcal Cassette Chromosome mec Types and Antibiogram of Methicillin-Resistant Staphylococcus aureus Isolates from Maharaj Nakorn Chiang Mai Hospital ชน ดของสแตฟฟ ลโลคอคคอล คาสเซทโครโมโซมเมค เมค และแบบแผนความไวต อยาปฏ ช วนะ ของเช อสแตฟฟ โลคอคค ส ออเร ยส เร ยสท ด อต อยากล มเมทธ ท ด อต อยากล มเมทธ ซ ลล นซ ง ซ ลล นซ งแยกได จากผ ป วยใน โรงพยาบาลมหาราชนครเช ยงใหม Wanachat Chaiyasan. Code 4827035. Advisor :Assoc.Prof. Prasit Tharavichitkul. Co-Advisor : Assoc.Prof. Nuanchan Chittasobhon. : Dr. Anusorn Boonthum 7/01/08 1
Staphylococcus aureus TSA β-hemolytic colony Staphylococcus aureus. Electron micrograph from Visuals Unlimited, with permission. ~1 μm 2-4mm http://teaching.path.cam.ac.uk/microbiology/bi_bacteriology/sp_staphylococcus/m_bi_sp_10.jpg 35-37 o C Mannitol salt agar http://fr.wikipedia.org/wiki/image:staphylococcus_aureus_gram.jpg http://www.austincc.edu/microbugz/html/mannitol_salt_agar.html 2
+ - Bound coagulase Free coagulase 3
A gram positive coccus called Staphylococcus aureus that is resistant to methicillin (a type of penicillin) Resist β-lactamlactam antibiotics, marolides, lincosamides, chloramphenical, tetracyclines, aminoglycosides, sulphametoxazole and trimethoprim (Thornsberry,, 1988) MRSA Methicillin Resistant Staphylococcus Aureus The most frequent causes of nosocomial infection. 4
Mechanism of resistance to methicillin. Production of an altered drug target, called penicillin- binding protein 2a (PBP 2a or PBP 2 ) 2 PBP2a has low affinity for β-lactams Encoded by meca gene. 5
Penicillin Normal PBP Altered PBP 2a X The meca gene is found within a chromosomal DNA region called Staphylococcal Chromosome Cassette mec (SCCmec) 6
Structure of SCCmec mec. ThesizeofSCCmec ranges from approximately 21 to 67 kb, or 1%-2% of the entire S. aureus genome (Ito, T.,2001. Hiramatsu, K.,1995.) methicillin-susceptible S. aureus In addition to the mec locus, SCCmec may contain several additional resistance genes, adhesion factors and numerous open reading frames
They carry two essential genetic components mec-gene complex (mec) The mec is composed of meca, intact or truncated sets of regulatory genes, mecr1 and meci, and insertion sequences, IS431 and/or IS1272. Three classes ccr-gene complex (ccr) Class A mec: IS431-mecA-mecR1-mecI Class B mec: IS431-mecA-mecR1-IS1272 Class C mec: IS431-mecA-mecR1-IS431 (cassette chromosome recombinase) The ccr is composed of two site-specific recombinase genes, ccra and ccrb, responsible for the mobility of SCCmec, and surrounding orfs of unknown function. Four allotypes Type 1 ccr : ccrb1-ccra1. Type 2 ccr : ccrb2-ccra2. Type 3 ccr : ccrb3-ccra3. Type 5 ccr :ccrc. Ito, T, I S A A R 2 0 0 5 8
The rests of the SCCmec are designated J regions (J stands for junkyard)that contain various genes or pseudo genes whose presence does not appear essential or useful for the bacterial cell. J regions (J1,J2,J3) are located between and around the mec and ccr complexes. 5 J3 mec J2 ccr J1 3 Types of SCCmec mec. Allotypes of SCCmec elements and classified basedonthe combination of the class of mec and the type of ccr. However, other researchers classified the element by different definition. Ito, T, I S A A R 2 0 0 5 9
Types of SCCmec mec. (In the new system) Types mec-gene complex ccr-gene complex I (type 1B) II (type 2A) III (type 3A) IV (type 2B) -IVa (type 2B1) -IVb (type 2B2) -IVc (type 2B3) -IVd (type 2B4) V (type 5C) Class B Class A Class A Class B Class B Class B Class B Class B Class C Type 1 Type 2 Type 3 Type 2 Type 2 Type 2 Type 2 Type 2 Type 5 J regions* Ito, T, I S A A R 2 0 0 5 10
, Mar. 2006.
Maharaj Nakorn Chiangmai Hospital Level Year 2545 2546 2547 2548 1 E.coli P.aeruginosa P.aeruginosa P.aeruginosa 2 P.aeruginosa E.coli A.baumannii K.pneumoniae 3 MRSA K.pneumoniae K.pneumoniae A.baumannii 4 K.pneumoniae MRSA E.coli E.coli 5 A.baumannii A.baumannii MRSA MRSA Journal of Suondok nurse, Jan-June June 2006. 12
MRSA (Isolates from Maharaj Nakorn Chiangmai Hospital) HA-MRSA CA-MRSA SCCmec type? Type e I II III IV V HA-MRSA 1. SCCmec I,II,III Multiplex PCR assay 2. Multi-drug resistant CA-MRSA SCCmec IV,V Sensitive except β-lactams Antibiotic test 13
MRSA isolates (%) Type I Type IIa Type IIb Type IIIb erythromycin R(97) R(75) R(98) R(100) clindamycin R(89) R(69) R(88) R(84) rifampicin S(98) S(100) S(100) S(100) chloramphenicol S(98) S(98) R(98) S(98) ciprofloxacin S(98) R(90) R(98) R(100) gentamicin R(98) S(100) R(100) S(84) tetracycline S(98) S(100) S(98) S(100) SENTRY hospitals (http://igitur-archive.library.uu.nl/dissertations/2002-0917-090124/c3.pdf) 14
SCCmec-II strains showed greater resistance than SCCmec-IV strains to clindamycin, erythromycin, levofloxacin, gentamicin, rifampin, minocycline, and trimethoprim-sulfamethoxazole. (Kilic et al., 2006.. J. Clin. Microbiol 44;4436-4440) CA-MRSA strains, which carry SCCmec-IV, showed greater susceptibility than SCCmec-II-carried HA-MRSA strains to clindamycin, erythromycin, levofloxacin, gentamicin, rifampin, minocycline, and SXT. (Kerry et al., 2007. Pharmacotherapy 27;3-10) 15
? Antibiogram? Antibiotic test MRSA (Isolates from SCCmec type? HA-MRSA 1. SCCmec I,II,III 2. Multi-drug resistant (Isolates from Maharaj Nakorn Chiangmai Hospital) Multiplex PCR assay Type e I II III IV V CA-MRSA SCCmec IV,V HA-MRSA CA-MRSA Sensitive except β-lactams 16
Objective To determine the SCCmec types of MRSA by multiplex PCR assay To determine antibiogram of MRSA sensitivity To determine whether the relationship between antibiogram and SCCmec types 17
Approach Characterization of MRSA from clinical isolates Disk diffusion Central Lab Resistant to Oxacillin(1μg.) MRSA 2548 Jan-April 2549 Jan-April 43 isolate 108 isolate Total 151 isolate 18
S. aureus? - Gram s s stains - Mannitol test - Coagulate test MRSA? - Disk diffusion (oxacillin, cefoxitin ) - Oxacillin salt agar (Confirm) - MRSA latex agglutination test (PBP2a) 19
S. aureus? Clinical isolates Gram s s strain (+) Tryptic soy agar (TSA) 37 o C, 24 h. Mannitol solt agar (MSA) 37 o C, 48 h. (+) (+) Coagulase test (+) (-) (Plasma 1:4) (-) S. epidermidis (+) Clinical isolates 151 isolate = S. aureus 20
MRSA? Mueller-Hinton agar (MHA) (Oxacillin 1 μg, Cefoxitin 30 μg) 37 o C, 16-18 18 h. Oxacillin 1 μg g R 10, R 11-15 15, S 13S Cefoxitin 30 μg g R 19 R S 20S TSA TSB 37 o C, 24 h. (Mc Farland No.0.5) 10 μl Dilute 1:100 151 isolate = S. aureus resistant to oxacillin Oxacillin salt agar (4%NaCl, 6μg 6 Oxacillin) 37 o C, 24 h. Fox OX (NCCLS : National Committee of Clinical Laboratory Standards, 2004) 21
MRSA? TSA (working stocks) PBP2a MRSA latex agglutination test (PBP2a) (Denka Seiken, Japan) PBP2a + PBP2a + = 147 isolates PBP2a - = 4 isolates (C30, C34, 37 and C39) 22
Approach (ii) Antibiotic test TSA TSB (37 o C, 24 h) TSB (Mc Farland No.0.5) Disk diffusion method MHA 37 o C, 16-18 18 h. Trimethoprim-sulfamethoxazole(sxt) 25μg, Oxacillin (ox) 1μg, Erythromycin (E) 15μg,Vancomycin (Va) 30μg, Fosfomycin (FM)30μg, Fusidic acid (FD) 10μg, Chloramphenical (C) 30μg, Rifampicin (RD) 2μg, Linezolid (LZ) 30μg, Clindamycin(DA) 2μg. 23
1. Trimethoprim-sulfamethoxazole(sxt sulfamethoxazole(sxt) ) 25μg 2. Oxacillin (ox) 1μg 1 3. Erythromycin (E) 15μg 4. Vancomycin (Va)) 30μg 5. Fosfomycin (FM)30μg 6. Fusidic acid (FD) 10μg 7. Chloramphenical (C) 30μg 8. Rifampicin (RD) 2μg2 9. Linezolid (LZ) 30μg 10. Clindamycin(DA) ) 2μg. 2 R <10 10 <13 < 9 <11 29 <12 29 - <15 I S 11-15 15 >16 11-12 12 13 14-17 17 >18 10-11 11 >12 12-14 14 >15-30 13-17 17 >18-30 - 21 16-19 19 >20 (NCCLS : National Committee of Clinical Laboratory Standards, 2004) 24
Antibiogram of MRSA Sensitivity No. Antibiotics Antibiotic patterns 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 Vancomycin S S S S S S S S S S S S S S S S S 2 Linezolidin S S S S S S S S S S S S S S S S S 3 Fusidic acid R S R S S S S S S S S S S S S S S 4 Chloramplinical S S S S S S S R S S I S S R S I I 5 Fosfomycin R R S R S R S S S S S S S R S S S 6 Rifampicin R R R S R S S R R S S S S R S S S 7 Clindamycin R R R R R S R S S S R R R I I R I 8 Cotrimoxazole R R R R R R R R R R S S S R R R R 9 Erythromycin R R R R R R R R R R R R S R R R R year 2005 0 4 0 10 8 1 11 0 0 2 1 2 1 1 1 5 1 2006 3 20 2 42 14 1 16 1 1 3 0 0 0 0 0 0 0 Total. 151 isolates % 3 2 24 15.9 2 1.3 52 34.4 22 14.6 2 1.3 27 17.9 1 0.7 1 0.7 5 3.3 1 0.7 2 1.3 1 0.7 1 0.7 1 0.7 5 3.3 1 0.7 17 Patterns.
Oxacillin Erythromycin Cotrimoxazole 160 140 120 100 80 60 40 20 0 Antibiotic sensitivity Chloramplinical Fosfomycin Rifampicin Clindamycin Antibiotic S R I Linezolidin Fusidic acid Vancomycin Number of isolate(s)
Approach (ii) MRSA typing PFGE PFGE (Pulsed-Field Gel Electrophoresis, Gold standard) - PFGE pattern (Cluster) Multiplex PCR - SCCmec typing 27
Pulsed-Field Gel Electrophoresis (PFGE) In particular, PFGE resolved extremely large DNA for the first time, raising the upper size limit of DNA separation in agarose from 30-50 kb to well over 10 Mb (10,000 kb). 28
PFGE Cell suspension abs ~1.8 24 hour culture Lyse cells and wash plugs Make agarose plugs 29
Slice 2mm piece of plug Digestion DNA by SmaI. + Gel loading buffer (Ambion) Load slices onto comb Pour gel and remove comb 30
CHEF (Contour-clamped homogeneous electric field ) (Chu, et al., 1986, 1990) PFGE marker 31
CHEF
33
The Analysis Process: Normalization 34
35 PFGE 100 98 96 94 92 90 88 86 84 82 80 78 76 74 72 70 68 66 64 62 60 58 56 54 52 50 48 46 44 42 40 PFGE G@FFPLUS@A1-20@002 G@FFPLUS@A1-20@004 G@FFPLUS@A1-20@009 G@FFPLUS@A1-20@010 G@FFPLUS@A11-20@006 G@FFPLUS@A11-20@007 G@FFPLUS@A11-20@009 G@FFPLUS@A11-20@010 G@FFPLUS@A21-33@002 G@FFPLUS@A48-A60@009 G@FFPLUS@A48-A60@014 G@FFPLUS@B1-B13@005 G@FFPLUS@B1-B13@007 G@FFPLUS@B1-B13@008 G@FFPLUS@B1-B13@011 G@FFPLUS@C1-C13@012 G@FFPLUS@B40-B52@003 G@FFPLUS@A11-20@008 G@FFPLUS@C14-C26@003 G@FFPLUS@B1-B13@004 G@FFPLUS@B14-B26@004 G@FFPLUS@A1-20@003 G@FFPLUS@A21-33@011 G@FFPLUS@A48-A60@012 G@FFPLUS@A48-A60@006 G@FFPLUS@A48-A60@007 G@FFPLUS@A48-A60@008 G@FFPLUS@B14-B26@002 G@FFPLUS@C40-C43-I,V-EPI-A3'@004 G@FFPLUS@B14-B26@003 G@FFPLUS@A34-47@011 G@FFPLUS@A48-A60@002 G@FFPLUS@A48-A60@003 G@FFPLUS@A48-A60@010 G@FFPLUS@A48-A60@013 G@FFPLUS@B1-B13@003 G@FFPLUS@C14-C26@004 G@FFPLUS@C14-C26@010 G@FFPLUS@C14-C26@012 G@FFPLUS@B40-B52@002 G@FFPLUS@C1-C13@011 G@FFPLUS@A34-47@008 G@FFPLUS@A48-A60@005 G@FFPLUS@A1-20@011 G@FFPLUS@B1-B13@013 G@FFPLUS@C14-C26@009 G@FFPLUS@C14-C26@008 G@FFPLUS@A11-20@004 G@FFPLUS@A34-47@007 G@FFPLUS@A11-20@002 G@FFPLUS@C14-C26@011 G@FFPLUS@A11-20@003 G@FFPLUS@A34-47@012 G@FFPLUS@A34-47@013 G@FFPLUS@B1-B13@002 G@FFPLUS@B1-B13@006 G@FFPLUS@B40-B52@005 G@FFPLUS@A34-47@014 G@FFPLUS@B40-B52@012 G@FFPLUS@B40-B52@006 G@FFPLUS@B40-B52@009 G@FFPLUS@A1-20@005 G@FFPLUS@C14-C26@013 G@FFPLUS@A1-20@007 G@FFPLUS@A1-20@008 G@FFPLUS@A11-20@005 G@FFPLUS@A11-20@011 G@FFPLUS@A48-A60@004 G@FFPLUS@B1-B13@009 G@FFPLUS@B14-B26@005 G@FFPLUS@B14-B26@006 G@FFPLUS@B14-B26@010 G@FFPLUS@A21-33@006 G@FFPLUS@B27-B39@009 G@FFPLUS@B14-B26@013 G@FFPLUS@B40-B52@004 G@FFPLUS@A1-20@006 G@FFPLUS@C14-C26@005 G@FFPLUS@B1-B13@014 G@FFPLUS@B27-B39@002 G@FFPLUS@B14-B26@011 G@FFPLUS@C14-C26@002 G@FFPLUS@B27-B39@012 G@FFPLUS@B27-B39@014 G@FFPLUS@B14-B26@014 G@FFPLUS@B27-B39@006 G@FFPLUS@B14-B26@008 G@FFPLUS@B27-B39@011 G@FFPLUS@B14-B26@009 G@FFPLUS@B14-B26@012 G@FFPLUS@B27-B39@003 G@FFPLUS@B27-B39@005 G@FFPLUS@B27-B39@010 G@FFPLUS@A21-33@005 G@FFPLUS@B27-B39@004 G@FFPLUS@A21-33@007 G@FFPLUS@A21-33@009 G@FFPLUS@A21-33@008 G@FFPLUS@A21-33@003 G@FFPLUS@A21-33@004 G@FFPLUS@A21-33@013 G@FFPLUS@A21-33@010 G@FFPLUS@A21-33@012 G@FFPLUS@B27-B39@007 G@FFPLUS@A34-47@002 G@FFPLUS@A48-A60@011 G@FFPLUS@C1-C13@010 G@FFPLUS@B40-B52@013 G@FFPLUS@C1-C13@002 G@FFPLUS@B27-B39@013 G@FFPLUS@C14-C26@006 G@FFPLUS@A34-47@009 G@FFPLUS@C1-C13@004 G@FFPLUS@B40-B52@010 G@FFPLUS@C1-C13@005 G@FFPLUS@C27-C39@009 G@FFPLUS@C1-C13@008 G@FFPLUS@C27-C39@005 G@FFPLUS@C27-C39@006 G@FFPLUS@C27-C39@007 G@FFPLUS@C27-C39@011 G@FFPLUS@C27-C39@012 G@FFPLUS@C27-C39@014 G@FFPLUS@B1-B13@012 G@FFPLUS@B40-B52@008 G@FFPLUS@A34-47@003 G@FFPLUS@C1-C13@013 G@FFPLUS@B40-B52@007 G@FFPLUS@C27-C39@013 G@FFPLUS@C27-C39@010 G@FFPLUS@C1-C13@006 G@FFPLUS@B14-B26@007 G@FFPLUS@B27-B39@008 G@FFPLUS@C14-C26@014 G@FFPLUS@A34-47@004 G@FFPLUS@A34-47@010 G@FFPLUS@A21-33@014 G@FFPLUS@A34-47@005 G@FFPLUS@C1-C13@009 G@FFPLUS@C27-C39@008 G@FFPLUS@B1-B13@010 G@FFPLUS@B40-B52@011 G@FFPLUS@C1-C13@014 G@FFPLUS@C1-C13@007 G@FFPLUS@C27-C39@003 G@FFPLUS@A34-47@006 G@FFPLUS@A1-20@001 G@FFPLUS@A1-20@012 G@FFPLUS@A11-20@001 G@FFPLUS@A11-20@012 G@FFPLUS@A21-33@001 G@FFPLUS@A21-33@015 G@FFPLUS@A34-47@001 G@FFPLUS@A34-47@015 G@FFPLUS@A48-A60@001 G@FFPLUS@A48-A60@015 G@FFPLUS@B1-B13@001 G@FFPLUS@B1-B13@015 G@FFPLUS@B14-B26@001 G@FFPLUS@B14-B26@015 G@FFPLUS@B27-B39@001 G@FFPLUS@B27-B39@015 G@FFPLUS@B40-B52@001 G@FFPLUS@B40-B52@014 G@FFPLUS@C1-C13@001 G@FFPLUS@C1-C13@015 G@FFPLUS@C14-C26@001 G@FFPLUS@C14-C26@015 G@FFPLUS@C27-C39@001 G@FFPLUS@C27-C39@015 G@FFPLUS@C40-C43-I,V-EPI-A3'@001 G@FFPLUS@C40-C43-I,V-EPI-A3'@006 A1 A3 A8 A9 A15 A16 A18 A19 A21 A55 A60 B4 B6 B7 B10 C11 B42 A17 C15 B3 B16 A2 A30 A58 A52 A53 A54 B14 C42 B15 A44 A48 A49 A56 A59 B2 C16 C22 C24 B41 C10 A41 A51 A10 B12 C21 C20 A13 A40 A11 C23 A12 A45 A46 B1 B5 B44 A47 B51 B45 B48 A4 C25 A6 A7 A14 A20 A50 B8 B17 B18 B22 A25 B34 B25 B43 A5 C17 B13 B27 B23 C14 B37 B39 B26 B31 B20 B36 B21 B24 B28 B30 B35 A24 B29 A26 A28 A27 A22 A23 A32 A29 A31 B32 A34 A57 C9 B52 C1 B38 C18 A42 C3 B49 C4 C34 C7 C30 C31 C32 C36 C37 C39 B11 B47 A35 C12 B46 C38 C35 C5 B19 B33 C26 A37 A43 A33 A38 C8 C33 B9 B50 C13 C6 C28 A39 Maker Maker 39.91% Cluster A 53.50% Cluster B 66.89% Cluster C 72.76% Cluster D Cluster A = 1 isolate ( C39) Cluster B = 1 isolate ( C28) Cluster C = 1 isolate ( C6) Cluster D = 148 isolates
Detection of SCCmec types by M-PCR. M Sccmec typing (I-V) References strains. (Oliveira and Lencastre 2002) Sccmec type I Original strains NCTC10442 II N315 III 85/2082 IVa IVc V JCSC4744 JCSC4788 WIS, JCSC3624 36
DNA extraction. TSA 5 colony Suspend in 100 μl Tris-EDTA buffer + l μ1 Achromopeptidase Incubate 55 C C for 10 min. Centrifugation at 13,200 rpm for 5 min, 4 o C DNA template, -20 o C 37
M-PCR : Sccmec typing (I-V) (Oliveira and Lencastre 2002)
(Oliveira and Lencastre 2002) M-PCR : Sccmec typing (I-V) 39
III IIIA IS431, pt181 (Oliveira and Lencastre 2002) 40
bp. 1000 500 400 300 200 100 I II III IVa IVc V
bp. 1000 500 300 200 100 III IIIA III 91 isolates (60.26%) IIIA 49 isolates (33.11%) Non-type able 7 isolates (4.63%) meca-nagative (3 isolates) = 151 isolates Nontypeable. (Only meca) 42
meca-nagative (3 isolates) C 34, C37, C39 meca+ PBP2a - C 34, C37, C39, C30 43
A42 DCS + meci+ meca Sccmec type IIa or III1.1.4? Chongtrakool et al. 2006 B38 B49 44
S. aureus Number of isolate(s) 60 50 40 30 20 10 0 MRSA isolate 151 isolates MRSA Antibiotic Patterns. 17 Patterns. Total 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Antibiotic patterns Antibiogram of MRSA Sensitivity Pattern 4 and 7 were most found with frequency of 34.4 9%.and 17.9%, respectively. Total PBP2a + = 147 isolates Summary PBP2a - = 4 isolates (C30, C34, 37 and C39) PFGE Cluster A = 1 isolate ( C39) Cluster B = 1 isolate ( C28) Cluster C = 1 isolate ( C6) Cluster D = 148 isolates M-PCR for Sccmec typing (I-V) Type III = 91 isolates (60.26 % ) Type IIIA = 50 isolates (33.11% ) Non-type able = 10 isolates (6.62%) 45
Future Work meca- positive C30 meca- negatives (3 isolates) C 34, C37, C39 PBP2a - Beta-lactamase test All data Analysis 46
Thank you. 47