Article Figures & Data
Figures
- FIG. 1.
C-MRSA strain shows heterogeneous phenotypic expression of methicillin resistance. Analysis of resistant subpopulations of the C-MRSA strain MW2, the related MSSA strain 476, and strain Mu3, with heterogeneous resistance to vancomycin, was performed with oxacillin (A), ceftriaxone (B), and imipenem (C) as described previously (13). Ceftriaxone was the antibiotic used in vain to treat the patient infected with MW2 (3). MW2 is an American Midwest MRSA strain representing the major C-MRSA (see the text). Strain 476 is an MSSA strain sharing its MLST allotype with MW2 (see Table 1). Mu3 is a representative H-MRSA strain with heterogeneous resistance to vancomycin (13). It is noted that MW2 contains subpopulations resistant to each of the three beta-lactam antibiotics.
- FIG. 2.
Illustrative representation of various types of SCCmec. SCCmec type is defined by the combination of the type of ccr gene complex and the class of mec gene complex. Type I SCCmec is defined by the combination of type 1 ccr and class B mec (IS1272-ΔmecR1-mecA); type II is defined by type 2 ccr and class A mec (mecI-mecR1-mecA); type III is defined by type 3 ccr and class A mec; and type IV is defined by type 2 ccr and class B mec. Type IV SCCmec is further classified into subtypes (type IVa and type IVb) based on the sequence difference in the J1 region of SCCmec (J stands for “junkyard”). Positions of primers used in this study to identify and type SCCmec are shown (see Table 2 for the nucleotide sequence of each primer). The allelic class of mec gene complex is determined by PCR detection of the presence or absence of IS1272, mecI, and mecR1 in two domains (PB, penicillin-binding domain; and MS, membrane-spanning domain), mecA, and IS431mec. PCR amplification was performed using 2.5 U of Ex Taq (Takara Shuzo Co., Ltd., Kyoto, Japan) in 50 μl of reaction mixture. Thermal cycling was set at 30 cycles (30 s for denaturation at 94°C, 1 min for annealing at 50°C, and 2 min for elongation at 72°C) using the Gene Amp PCR system 9600 (Perkin-Elmer, Wellesley, Mass.). For the detection of mecA-IS431mec, a long-range PCR method was used, set at 10 cycles (15 s for denaturation at 94°C, 30 s for annealing at 50°C, and 8 min for elongation at 68°C) followed by 20 cycles (15 s for denaturation at 94°C, 30 s for annealing at 50°C, and 12 min for elongation at 68°C). Note that this study identified a new type of SCCmec for three C-MRSA strains that carried the class C2 mec gene complex (21). The sequencing of the entire SCCmec is now ongoing.
Tables
- TABLE 1.
Genotyping and antibiogram of tested MRSA strains
Categorya Sourceb Strain name Coagulase isotype MIC (mg/liter) ofc: SCCmec typed PFGE pattern MLST luk-PVg Doubling time (min)h Reference(s) ERY KAN TOB GEN TET NOR OXA CZX IMP ST, allelic profilee CCf C MN, US C1998000370 7 0.5 8 0.5 0.5 32 1 4 512 0.25 IVa J4 1, 1-1-1-1-1-1-1 1 + 27.89 3, 24 C ND, US C1999000529 7 0.5 4 0.5 0.25 0.5 1 8 >512 0.5 IVa J4 1, 1-1-1-1-1-1-1 1 + 26.38 3, 24 C MN, US C1999000193 7 0.5 8 0.5 0.5 0.5 1 4 >512 0.25 IVa J3 1, 1-1-1-1-1-1-1 1 + 26.73 3, 24 C ND, US MW2 7 0.5 4 0.25 0.25 0.5 1 8 >512 0.5 IVa J4 1, 1-1-1-1-1-1-1 1 + 28.67 2, 3, 24 C MN, US C2001001201 7 >512 8 0.25 0.25 0.5 1 8 >512 0.5 IVa J4 1, 1-1-1-1-1-1-1 1 + 27.4 3, 24 C MN, US C2001000101 7 0.5 8 0.5 0.5 0.5 1 4 >512 0.25 IVa J4 1, 1-1-1-1-1-1-1 1 + 27.25 3, 24 C MN, US C200100818 7 >512 8 0.5 0.25 0.5 1 16 >512 0.25 IVa J5 1, 1-1-1-1-1-1-1 1 + 27.72 3, 24 C Woo, AU A80 3355 4 0.5 4 0.25 0.25 0.5 2 16 >512 0.125 IVa H1 30, 2-2-2-2-6-3-2 30 + 26.22 25 C Woo, AU A82 3549 4 0.5 2 0.25 0.25 0.5 2 16 >512 0.125 IVa H3 30, 2-2-2-2-6-3-2 30 + 28.09 25 C Woo, AU A83 0528 4 0.5 4 0.25 0.25 0.5 2 16 >512 ≤0.063 IVa H1 30, 2-2-2-2-6-3-2 30 + 27.08 25 C Woo, AU B82 6559 4 0.5 2 0.25 0.25 0.5 2 16 512 ≤0.063 IVa H1 30, 2-2-2-2-6-3-2 30 + 27.57 25 C Woo, AU D82 1552 4 0.5 2 0.25 0.25 0.5 2 16 >512 0.125 IVa H1 30, 2-2-2-2-6-3-2 30 + 61.03 25 C Woo, AU E80 2537 4 0.5 4 0.25 0.25 0.5 2 8 >512 ≤0.063 IVa H2 30, 2-2-2-2-6-3-2 30 + 26.97 25 C Woo, AU F81 0539 4 0.5 4 0.25 0.25 0.5 2 16 >512 ≤0.063 IVa H1 30, 2-2-2-2-6-3-2 30 + 26.44 25 C Woo, AU 180 2552 4 0.5 2 0.25 0.25 0.5 2 16 512 0.125 IVa H3 30, 2-2-2-2-6-3-2 30 + 26.86 25 C Per, AUi M9N 3 >512 4 0.25 0.25 0.5 2 16 512 0.125 IVa I1 78, 22-1-14-23-12-53-31 298 − 26.75 26 C Per, AUi M33T 3 >512 4 0.25 0.25 0.5 2 16 512 0.125 IVa I2 78, 22-1-14-23-12-53-31 298 − 26.44 26 C Per, AUi W1S 1 0.5 2 0.5 0.25 0.5 0.5 2 32 0.063 New E2 45, 10-14-8-6-10-3-2 45 − 27.74 26 C Per, AUi C7N 7 >512 4 0.5 0.5 0.5 2 16 512 0.25 IVa J6 1, 1-1-1-1-1-1-1 1 − 30.26 26 C TN, USj 00215 7 0.25 4 0.5 0.25 0.5 0.5 64 >512 2 IVa E1 45, 10-14-8-6-10-3-2 45 − 27.71 28 C MS, USk 01083 3 64 >512 0.5 0.5 32 16 8 512 0.125 IVa A3 8, 3-3-1-1-4-4-3 8 + 27.78 4 C MS, USk 01093 5 16 512 0.5 0.5 0.5 2 16 >512 0.125 IVa F 72, 1-4-1-8-4-4-3 8 − 25.94 4 C MS, USk 01102 3 0.25 4 0.5 0.5 0.5 1 8 >512 0.25 IVb A2 8, 3-3-1-1-4-4-3 8 + 28.29 4 N Ade, AU 81 0342 3 0.25 4 0.5 0.5 0.5 8 2 16 ≤0.063 New A1 8, 3-3-1-1-4-4-3 8 − 26.86 This study N Ade, AU 91 2572 7 0.5 4 0.25 0.25 0.5 1 4 >512 0.25 IVa J7 1, 1-1-1-1-1-1-1 1 − 27.15 This study N Ade, AU 91 2619 7 0.5 8 0.5 0.5 0.5 2 64 >512 2 IVa J7 76, 1-63-1-1-1-1-1 1 − 27.47 This study N Ade, AU WCH379 7 0.5 2 0.25 0.5 0.5 1 16 >512 4 IVa J8 1, 1-1-1-1-1-1-1 1 − 33.31 This study N Ade, AU 91 2574 7 0.25 1 0.25 0.25 0.5 128 8 256 ≤0.063 New G 22, 7-6-1-5-8-8-6 22 − 29.52 This study N Ade, AU SAP260 2 >512 4 0.5 0.25 0.5 0.5 4 >512 0.25 IVa B 73, 1-4-27-4-12-1-10 5 − 26.64 This study N Per, AU 81 0937 7 0.5 2 0.25 0.5 0.5 1 4 >512 0.5 IVa J1 1, 1-1-1-1-1-1-1 1 − 25.29 This study N Per, AU 91 2125 7 0.5 4 0.25 0.5 0.5 0.5 16 >512 0.5 IVa J2 1, 1-1-1-1-1-1-1 1 − 30.25 This study N Per, AU 91 1703 3 0.25 2 0.125 0.25 8 1 16 512 0.125 IVa A2 8, 3-3-1-1-4-4-3 8 − 32.0 This study N Bri, AU 81 1238 7 0.5 2 0.25 0.5 0.5 1 8 >512 0.5 IVa J1 1, 1-1-1-1-1-1-1 1 − 26.06 This study N Bri, AU 91 2666 7 0.5 2 0.5 0.25 0.5 0.5 2 512 0.125 IVa J2 1, 1-1-1-1-1-1-1 1 − 26.65 This study N Dar, AU SAP411 6 ≤0.125 4 0.25 0.5 32 1 4 128 0.125 IVa N 75, 36-3-43-34-39-52-49 S − 27.68 This study H Ade, AU 81 0508 4 >512 256 256 0.5 0.25 512 512 >512 128 II M2 36, 2-2-2-2-3-3-2 30 − 32.22 This study H Ade, AU 91 2231 4 >512 2 0.25 0.5 32 32 128 >512 16 III K4 239, 2-3-1-1-4-4-3 8 − 35.8 This study H Bri, AU 91 1573 4 >512 128 64 8 16 1 64 >512 4 III K2 239, 2-3-1-1-4-4-3 8 − 41.82 This study H Bri, AU 91 1575 4 >512 512 16 32 1 32 256 >512 32 III K6 239, 2-3-1-1-4-4-3 8 − 50.46 This study H Bri, AU 91 2145 4 >512 512 16 32 32 32 64 >512 16 III K1 239, 2-3-1-1-4-4-3 8 − 50.64 This study H Dar, AU SAP344 4 >512 4 0.25 0.5 32 32 256 >512 32 III K3 239, 2-3-1-1-4-4-3 8 − 34.51 This study H Per, AU 91 2118 4 >512 512 16 32 32 256 512 >512 64 III L 239, 2-3-1-1-4-4-3 8 − 38.45 This study H UK NCTC10442 3 0.125 1 0.125 0.5 128 1 256 >512 16 I D 250, 3-3-1-1-4-4-16 8 − 36.44 19 H JP N315 2 >512 >512 512 0.5 0.125 2 16 16 1 II C 5, 1-4-1-4-12-1-10 5 − 34.28 19 H NZ 85/2082 4 512 >512 8 64 128 2 32 >512 0.5 III K5 239, 2-3-1-1-4-4-3 8 − 43.53 19 H UK Strain 252l 4 >512 128 128 0.25 0.5 >512 512 >512 64 II M1 36, 2-2-2-2-3-3-2 30 − 29.79 20, —m H UK MSSA476 7 0.5 4 0.5 0.5 1 1 0.5 8 ≤0.063 J1 1, 1-1-1-1-1-1-1 1 − 27.66 —m ↵a C, C-MRSA; N, NORSA; H, H-MRSA.
↵b MN, Minnesota; US, United States; ND, North Dakota; Woo, Wooloongabba, Australia, AU, Australia; Per, Perth, Australia; TN, Tennessee; MS, Mississippi; Ade, Adelaide, Australia; Bri, Brisbane, Australia; Dar, Darwin, Australia; UK, United Kingdom; JP, Japan; NZ, New Zealand.
↵c MICs were determined by the NCCLS-based plate dilution method. Antibiotics: ERY, erythromycin; KAN, kanamycin; TOB, tobramycin; GEN, gentamicin; TET, tetracycline; NOR, norfloxacin; OXA, oxacillin; CZX, ceftizoxime; IMP, imipenem. Values in bold signify resistance to these antibiotics.
↵d New, new type of SCCmec possessing class C2 mec gene complex (see Fig. 2).
↵e ST, sequence type.
↵f Clonal complex, based on BURST (based upon related sequence types). S, singleton (not assigned to any clonal complex).
↵g luk-PV genes encode Panton-Valentin leucocidin proteins.
↵h Doubling time during exponential growth phase (optical density of 0.05 to ∼1.0 at 660 nm) measured by using TN-2612 (Advantec Toyo Kaisha, Ltd., Tokyo, Japan) as previously described (8).
↵i Isolated from aborigine.
↵j Isolated from food-poisoning strain.
↵k Isolated from state prison.
↵l E-MRSA16.
- TABLE 2.
Primer sets used for identifying SCCmec
Primer (previous name) for detection of: Nucleotide sequence Expected size of product (gene[s] reactive to the primer) Reference ccr gene complexa βc (β2) 5′-ATTGCCTTGATAATAGCCITCT-3′ (all types of ccrB) 19 αc 5′-ATCTATTTCAAAAATGAACCA-3′ 560 bp, βc (all types of ccrA) This study α1 (α2) 5′-AACCTATATCATCAATCAGTACGT-3′ 700 bp, βc (type 1 ccrA) 19 α2 (α3) 5′-TAAAGGCATCAATGCACAAACACT-3′ 1 kbp, βc (type 2 ccrA) 19 α3 (α4) 5′-AGCTCAAAAGCAAGCAATAGAAT-3′ 1.6 kbp, βc (type 3 ccrA) 19 mec gene complex (all types) mecR1 (MS domain) mcR4 5′-GTCGTTCATTAAGATATGACG-3′ (mecR1—MS domain) This study mcR3 5′-GTCTCCACGTTAATTCCATT-3′ 310 bp, mcR4 (mecR1—MS domain) 21 mecA mA1 5′-TGCTATCCACCCTCAAACAGG-3′ (mecA) 15 mA2 5′-AACGTTGTAACCACCCCAAGA-3′ 200 bp, mA1 (mecA) 15 mecA -IS431mec IS2 (iS-2) 5′-TGAGGTTATTCAGATATTTCGATGT-3′ 4 kbp, mA1 (IS431mec) 21 Class C mec gene complex mA2 5′-AACGTTGTAACCACCCCAAGA-3′ <3 kbp, IS2 (mecA) 21 Class B mec gene complex IS5 5′-AACGCCACTCATAACATATGGAA-3′ (IS1272) This study mA6 5′-TATACCAAACCCGACAAC-3′ 2 kbp, IS5 (mecA) 21 Class A mec gene complex mecI mI4 5′-CAAGTGAATTGAAACCGCCT-3′ (mecI) This study mI3 5′-CAAAAGGACTGGACTGGAGTCCAAA-3′ 180 bp, mI4 (mecI) This study mecR1 (PB domain) mcR2 5′-CGCTCAGAAATTTGTTGTGC-3′ (mecR1—PB domain) 21 mcR5 5′-CAGGGAATGAAAATTATTGGA-3′ 320 bp, mcR2 (mecR1—PB domain) This study SCCmec subtype IVa 4a1 5′-TTTGAATGCCCTCCATGAATAAAAT-3′ (J1 region of IVa) This study 4a2 5′-AGAAAAGATAGAAGTTCGAAAGA-3′ 450 bp, 4a1 (J1 region of IVa) This study SCCmec subtype IVb 4b1 5′-AGTACATTTTATCTTTGCGTA-3′ (J1 region of IVb) This study 4b2 5′-AGTCATCTTCAATATCGAGAAAGTA-3′ 1 kbp, 4b1 (J1 region of IVb) This study ↵a ccr type is determined by PCR using primer βc (the common primer for three types of ccrB) and either one of the three types of ccrA, α1 (ccrA1), α2 (ccrA2), and α3 (ccrA3). This typing actually reflects the allotype of ccrA.
