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Molecular characterisation of meticillin-resistant Staphylococcus aureus isolates from two ceftobiprole Phase 3 complicated skin and skin-structure infection clinical trials
International Journal of Antimicrobial Agents 34 (2009) 166–168
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Short communication
Molecular characterisation of meticillin-resistant Staphylococcus aureus isolates from two ceftobiprole Phase 3 complicated skin and skin-structure infection clinical trials Todd A. Davies a,∗ , Wenchi Shang a , Karen M. Amsler a , Saralee Bajaksouzian b , Michael R. Jacobs b , Karen Bush a a b
Johnson & Johnson Pharmaceutical Research & Development L.L.C., Raritan, NJ 08869, USA Case Western Reserve University and University Hospitals Case Medical Center, Cleveland, OH, USA
a r t i c l e
i n f o
Article history: Received 15 January 2009 Accepted 9 February 2009 Keywords: MRSA Ceftobiprole SCCmec USA300 Panton–Valentine leukocidin Pulsed-field gel electrophoresis
a b s t r a c t Meticillin-resistant Staphylococcus aureus (MRSA) isolates from two worldwide ceftobiprole Phase 3 clinical trials for the treatment of complicated skin and skin-structure infections were characterised by clonality, staphylococcal cassette chromosome mec (SCCmec) type and the presence of Panton–Valentine leukocidin (PVL). PVL was predominantly found in US isolates (196/231 vs. 13/110 non-US isolates). SCCmec type IV was the most common (253/329) owing to the predominance of clone USA300 in isolates from the USA (197/226). In Europe, SCCmec type III was the most prevalent (30/74). Ceftobiprole minimum inhibitory concentrations (MICs) ranged from 0.25 g/mL to 4 g/mL, with MICs ≤ 2 g/mL for 99.7% of isolates, regardless of SCCmec or clone type. © 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Management of diseases caused by Staphylococcus aureus is problematic due to the increase of meticillin-resistant S. aureus (MRSA), its relatively rapid appearance in community-acquired illnesses and the reduced effectiveness of previously useful classes of antibiotics. Many virulent strains originating from the community have been identified, with the clonal spread of a small number of MRSA types, frequently associated with the Panton–Valentine leukocidin (PVL) cytotoxin genes that may act as a marker for other virulence factors [1–3]. Ceftobiprole, a broad-spectrum cephalosporin with activity against Gram-negative and Gram-positive pathogens including meticillin-resistant staphylococci [4,5], has recently been approved in Switzerland, Ukraine, and Canada for the treatment of complicated skin and skin-structure infections (cSSSIs), including those caused by MRSA. Two Phase 3 ceftobiprole clinical trials for the treatment of cSSSI have been completed, and the in vitro activity of ceftobiprole for the baseline pathogens has been reported [4,6,7]. In this study, MRSA isolates from these trials were characterised by detection of PVL genes, staphylococcal cassette chromosome mec
∗ Corresponding author. Present address: Johnson & Johnson Pharmaceutical Research & Development L.L.C., Room B215, 1000 Route 202 South, Raritan, NJ 08869, USA. Tel.: +1 908 704 3465; fax: +1 908 707 3501. E-mail address: [email protected] (T.A. Davies).
(SCCmec) typing and clonal relatedness by pulsed-field gel electrophoresis (PFGE). 2. Materials and methods The presence of mecA and the PVL genes lukS and lukF was determined for all S. aureus isolates by multiplex polymerase chain reaction (PCR) [6]. SCCmec typing of MRSA was performed by multiplex PCR [8]. PFGE was performed as described previously [9]. Control MRSA strains USA100–USA800 were purchased from the Network on Antimicrobial Resistance in S. aureus (NARSA) (Herndon, VA). Susceptibility to ceftobiprole and comparators was determined using Clinical and Laboratory Standards Institute methodology [10]. 3. Results and discussion There were 932 S. aureus isolates in the two clinical trials, of which 591 (63%) were meticillin-susceptible and 341 (37%) were MRSA. All MRSA isolates, based on oxacillin MICs, were mecApositive by PCR. Six isolates were oxacillin-susceptible yet were mecA-positive. These isolates expressed mecA based on identification of penicillin-binding protein 2a (PBP2a) using an agglutination test (MRSA-Screen Kit; Denka Seiken Co., Tokyo, Japan). Sequencing of mecA did not yield any notable mutations, thus the reason for this discrepancy was not due to PBP2a alterations.
0924-8579/$ – see front matter © 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2009.02.013
T.A. Davies et al. / International Journal of Antimicrobial Agents 34 (2009) 166–168
167
Table 1 Incidence and geographical distribution of Panton–Valentine leukocidin (PVL) genes and staphylococcal cassette chromosome mec (SCCmec) types with corresponding ceftobiprole minimum inhibitory concentrations (MICs) in meticillin-resistant Staphylococcus aureus from two Phase 3 complicated skin and skin-structure infection clinical trials. Phenotype/genotype
N (%)
Ceftobiprole MIC (g/mL) MIC50
MIC90
No. of isolates by region (%) Asia or othera
Range
Europeb
Latin Americac
USA
PVL PVL −ve PVL +ve
132 (38.7) 209 (61.3)
1 0.5
2 1
0.25–4 0.25–1
6 (50.0) 6 (50.0)
72 (92.3) 6 (7.7)
19 (95.0) 1 (5.0)
35 (15.2) 196 (84.8)
SCCmec type All types I II III IV (all) IVa IV IVcE V Other (all) III (5A) 4A Unknown
329 (100)d 6 (1.8) 20 (6.1) 34 (10.3) 253 (76.9) 208 (63.2) 24 (7.3) 21 (6.4) 4 (1.2) 12 (3.6) 4 (1.2) 3 (0.9) 5 (1.5)
0.5 – 1 2 0.5 0.5 0.5 1 – 1 – – –
2 – 2 2 1 1 1 1 – 2 – – –
0.25–4 1–2 0.25–2 0.5–2 0.5–2 0.5–2 0.5–1 0.5–1 0.5–1 0.5–4 2–4 0.5–1 0.5–1
10 (3.0) 0 1 (10.0) 3 (30.0) 2 (20.0) 0 1 (10.0) 1 (10.0) 4 (40.0) 0 0 0 0
74 (22.5) 2 (2.7) 1 (1.4) 30 (40.5) 33 (44.6) 10 (13.5) 5 (6.8) 18 (24.3) 0 8 (10.8) 4 (5.4) 0 4 (5.4)
19 (5.8) 4 (21.1) 3 (15.8) 0 12 (63.2) 10 (52.6) 0 2 (10.5) 0 0 0 0 0
226 (68.7) 0 15 (6.6) 1 (0.4) 206 (91.2) 188 (83.2) 18 (8.0) 0 0 4 (1.8) 0 3 (1.3) 1 (0.4)
MIC50/90 , MIC for 50% and 90% of the organisms, respectively; –, insufficient isolates to determine MIC50 or MIC90 . a Countries included India, Israel, South Africa, South Korea, Taiwan and Thailand. b Countries included Bulgaria, Czech Republic, Hungary, Latvia, Lithuania, Romania, Russia, Serbia and Montenegro, and Ukraine. c Countries included Argentina, Costa Rica and Mexico. d Of the 341 MRSA baseline isolates, 329 isolates were available for molecular characterisation.
The majority of MRSA isolates (61.3%) were PVL-positive, mostly due to the high prevalence (84.8%; 196/231) of PVL genes in isolates from the USA (Table 1). PVL genes were present in only 7.7% (6/78) of European MRSA and 5.0% (1/20) of Latin America isolates. Among countries from Asia and other parts of the world, PVL was present in 50.0% (6/12) of MRSA isolates. Of the 341 MRSA isolates, 329 were available for SCCmec typing and PFGE analysis. SCCmec type IVa was most prevalent in MRSA from the USA and Latin America (83.2% and 52.6%, respectively), whilst type III was the most prevalent in Europe (40.5%) (Table 1). SCCmec type V was only found in isolates from Asia, where small numbers of types II, III and IV were also identified. Twelve isolates from the USA or Europe were untypeable or had previously undescribed SCCmec types. The predominant USA MRSA clone was USA300 (n = 197), followed by USA100 (n = 10), USA400 (n = 2) and USA800 (n = 1) (Table 2). Isolates related to one of the USA clones were from the USA, except for one USA100 isolate from the Czech Republic and a single USA800 isolate from Argentina. The remaining 119 isolates had PFGE patterns unrelated to any of the USA clones; however,
a group of SCCmec type III isolates (n = 22) from Latvia were of the Brazilian/Hungarian clone lineage and were comprised of two PFGE types. Susceptibility of isolates from the various MRSA clones varied according to clonality, with ceftobiprole and trimethoprim/sulfamethoxazole (SXT) retaining high activity. Isolates belonging to any of the clonal groups had ceftobiprole MICs ≤ 2 g/mL; all but 11 isolates (of the Latvia-2 clone) were susceptible to SXT (Tables 1 and 2). In contrast, 92% of the clones were erythromycin-resistant and 53% were levofloxacin-resistant (Table 2). The USA100 and Latvia clones demonstrated lower susceptibilities to aminoglycosides and clindamycin compared with the other clones (Table 2). The presence of PVL genes has been noted as one of the markers for community-associated MRSA (CA-MRSA) [3]. In this study, 61.3% of MRSA isolates were PVL-positive. Results from tigecycline worldwide Phase 3 clinical trials were slightly lower, with 47% (18/38) of MRSA being PVL-positive [2]. The high incidence of PVL genes in MRSA from the ceftobiprole trial was driven by isolates from the USA, as 84.8% of US isolates were PVL-positive whilst only 11.8% of
Table 2 Phenotypic and genotypic characteristics of major meticillin-resistant Staphylococcus aureus (MRSA) clones from two Phase 3 complicated skin and skin-structure infection trials. Characteristic
SCCmec PVL +ve (%) Susceptibility (%) Aminoglycosidesa Clindamycinb Erythromycin Levofloxacin SXT Isolates with ceftobiprole MIC ≤ 2 g/mL (%)
MRSA clone USA100 (n = 10)
USA300 (n = 197)
USA400 (n = 2)
USA800 (n = 1)
Latvia-1 (n = 9)
Latvia-2 (n = 13)
II 10
IVa 94
IVa 100
IVa 100
III 0
III 0
50 20 0 0 100 100
97 96 9 54 100 100
100 100 0 100 100 100
100 100 100 100 100 100
0 11 0 0 100 100
15 85 0 0 15 100
SCCmec, staphylococcal cassette chromosome mec type; PVL, Panton–Valentine leukocidin; SXT, trimethoprim/sulfamethoxazole; MIC, minimum inhibitory concentration. a Gentamicin or tobramycin. b Isolates were not screened for inducible resistance to clindamycin.
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T.A. Davies et al. / International Journal of Antimicrobial Agents 34 (2009) 166–168
isolates from the rest of the world were PVL-positive, similar to a recent report showing the high prevalence of PVL-positive MRSA in the USA [1]. SCCmec type IV was the most common SCCmec type for all MRSA (76.9%), with an even higher prevalence in the USA (91.2%). SCCmec type IV, specifically SCCmec type IVa, is common in the USA and is associated with the widespread CA-MRSA clone USA300 [1–3,11]. In the current study, 96% (197/206) of the US MRSA isolates having SCCmec type IV or IVa were related to USA300. These isolates exhibited a susceptibility profile indicative of CA-MRSA, i.e. resistance to macrolides and -lactams, with the exception of ceftobiprole, but susceptibility to most other drug classes. MRSA having SCCmec type IV from Europe, Latin America and Asia were not related to USA300, consistent with other reports showing the low incidence of this clone in these regions [3,12,13]. SCCmec type III was the second most common SCCmec type, found primarily among European isolates (41%) and similar to other reports showing SCCmec types III and IV to be prevalent in parts of Europe [11–13]. Twenty MRSA had SCCmec type II; 15 of these were from the USA, with the majority (10/15; 67%) having PFGE patterns related to the predominant hospital-acquired MRSA USA100 clone [2,11]. Unlike the USA300 clones, the USA100 clones were resistant to many antibiotics including -lactams (other than ceftobiprole), macrolides, clindamycin, aminoglycosides and fluoroquinolones. Molecular characterisation of MRSA causing cSSSI from two ceftobiprole clinical trials showed that MRSA isolates from Europe and other parts of the world were more diverse and distinct than those from the USA, in large part due to the predominance (87%) of SCCmec type IV USA300 in the USA. Acknowledgments The authors would like to thank Anne Windau from Case Western Reserve University and University Hospitals Case Medical Center, OH, for technical contributions to the susceptibility and multiplex PCR data, and Markus Heep from Basilea Pharmaceutica for suggestions regarding the PVL testing paradigm. Funding: This work was funded by Johnson & Johnson Pharmaceutical Research & Development, L.L.C. Competing interests: TAD, WS, KMA and KB own shares in Johnson & Johnson and are employees of Johnson & Johnson Pharmaceutical Research & Development, L.L.C. MRJ has received research funding from Johnson & Johnson Pharmaceutical Research & Development, L.L.C.
Ethical approval: Not required. References [1] Jones RN, Nilius AM, Akinlade BK, Deshpande LM, Notario GF. Molecular characterization of Staphylococcus aureus isolates from a 2005 clinical trial of uncomplicated skin and skin structure infections. Antimicrob Agents Chemother 2007;51:3381–4. [2] McAleese F, Murphy E, Babinchak T, Singh G, Said-Salim B, Kreiswirth B, et al. Use of ribotyping to retrospectively identify methicillin-resistant Staphylococcus aureus isolates from phase 3 clinical trials for tigecycline that are genotypically related to community-associated isolates. Antimicrob Agents Chemother 2005;49:4521–9. [3] Vandenesch F, Naimi T, Enright MC, Lina G, Nimmo GR, Heffernan H, et al. Community-acquired methicillin-resistant Staphylococcus aureus carrying Panton–Valentine leukocidin genes: worldwide emergence. Emerg Infect Dis 2003;9:978–84. [4] Amsler KM, Davies TA, Shang W, Jacobs MR, Bush K. In vitro activity of ceftobiprole against pathogens from two phase 3 complicated skin and skin structure infection clinical trials. Antimicrob Agents Chemother 2008;52:3418– 23. [5] Hebeisen P, Heinze-Krauss I, Angehrn P, Hohl P, Page MGP, Then RL. In vitro and in vivo properties of Ro 63-9141, a novel broad-spectrum cephalosporin with activity against methicillin-resistant staphylococci. Antimicrob Agents Chemother 2001;45:825–36. [6] Noel G, Bush K, Bagchi P, Ianus J, Strauss R. A randomized, double-blind trial comparing ceftobiprole medocaril with vancomycin plus ceftazidime for the treatment of patients with complicated skin and skin-structure infections. Clin Infect Dis 2008;46:647–55. [7] Noel GJ, Strauss RS, Amsler K, Heep M, Pypstra R, Solomkin JS. Results of a double-blind, randomized trial of ceftobiprole treatment of complicated skin and skin structure infections caused by gram-positive bacteria. Antimicrob Agents Chemother 2008;52:37–44. [8] Zhang K, McClure J-A, Elsayed S, Louie T, Conly JM. Novel multiplex PCR assay for characterization and concomitant subtyping of staphylococcal cassette chromosome mec types I to V in methicillin-resistant Staphylococcus aureus. J Clin Microbiol 2005;43:5026–33. [9] Davies TA, Goldschmidt R, Pfleger S, Loeloff M, Bush K, Sahm DF, et al. Crossresistance, relatedness and allele analysis of fluoroquinolone-resistant United States clinical isolates of Streptococcus pneumoniae (1998–2000). J Antimicrob Chemother 2003;52:168–75. [10] Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard. 7th ed. Document M7-A7. Wayne, PA: CLSI; 2006. [11] McDougal LK, Steward CD, Killgore GE, Chaitram JM, McAllister SK, Tenover FC. Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database. J Clin Microbiol 2003;41:5113–20. [12] Cookson BD, Robinson DA, Monk AB, Murchan S, Deplano A, de Ryck R, et al. Evaluation of molecular typing methods in characterizing a European collection of epidemic methicillin-resistant Staphylococcus aureus strains: the HARMONY collection. J Clin Microbiol 2007;45:1830–7. [13] Deurenberg RH, Vink C, Oudhuis GJ, Mooij JE, Driessen C, Coppens G, et al. Different clonal complexes of methicillin-resistant Staphylococcus aureus are disseminated in the Euregio Meuse-Rhine region. Antimicrob Agents Chemother 2005;49:4263–71.
Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Short communication
Molecular characterisation of meticillin-resistant Staphylococcus aureus isolates from two ceftobiprole Phase 3 complicated skin and skin-structure infection clinical trials Todd A. Davies a,∗ , Wenchi Shang a , Karen M. Amsler a , Saralee Bajaksouzian b , Michael R. Jacobs b , Karen Bush a a b
Johnson & Johnson Pharmaceutical Research & Development L.L.C., Raritan, NJ 08869, USA Case Western Reserve University and University Hospitals Case Medical Center, Cleveland, OH, USA
a r t i c l e
i n f o
Article history: Received 15 January 2009 Accepted 9 February 2009 Keywords: MRSA Ceftobiprole SCCmec USA300 Panton–Valentine leukocidin Pulsed-field gel electrophoresis
a b s t r a c t Meticillin-resistant Staphylococcus aureus (MRSA) isolates from two worldwide ceftobiprole Phase 3 clinical trials for the treatment of complicated skin and skin-structure infections were characterised by clonality, staphylococcal cassette chromosome mec (SCCmec) type and the presence of Panton–Valentine leukocidin (PVL). PVL was predominantly found in US isolates (196/231 vs. 13/110 non-US isolates). SCCmec type IV was the most common (253/329) owing to the predominance of clone USA300 in isolates from the USA (197/226). In Europe, SCCmec type III was the most prevalent (30/74). Ceftobiprole minimum inhibitory concentrations (MICs) ranged from 0.25 g/mL to 4 g/mL, with MICs ≤ 2 g/mL for 99.7% of isolates, regardless of SCCmec or clone type. © 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction Management of diseases caused by Staphylococcus aureus is problematic due to the increase of meticillin-resistant S. aureus (MRSA), its relatively rapid appearance in community-acquired illnesses and the reduced effectiveness of previously useful classes of antibiotics. Many virulent strains originating from the community have been identified, with the clonal spread of a small number of MRSA types, frequently associated with the Panton–Valentine leukocidin (PVL) cytotoxin genes that may act as a marker for other virulence factors [1–3]. Ceftobiprole, a broad-spectrum cephalosporin with activity against Gram-negative and Gram-positive pathogens including meticillin-resistant staphylococci [4,5], has recently been approved in Switzerland, Ukraine, and Canada for the treatment of complicated skin and skin-structure infections (cSSSIs), including those caused by MRSA. Two Phase 3 ceftobiprole clinical trials for the treatment of cSSSI have been completed, and the in vitro activity of ceftobiprole for the baseline pathogens has been reported [4,6,7]. In this study, MRSA isolates from these trials were characterised by detection of PVL genes, staphylococcal cassette chromosome mec
∗ Corresponding author. Present address: Johnson & Johnson Pharmaceutical Research & Development L.L.C., Room B215, 1000 Route 202 South, Raritan, NJ 08869, USA. Tel.: +1 908 704 3465; fax: +1 908 707 3501. E-mail address: [email protected] (T.A. Davies).
(SCCmec) typing and clonal relatedness by pulsed-field gel electrophoresis (PFGE). 2. Materials and methods The presence of mecA and the PVL genes lukS and lukF was determined for all S. aureus isolates by multiplex polymerase chain reaction (PCR) [6]. SCCmec typing of MRSA was performed by multiplex PCR [8]. PFGE was performed as described previously [9]. Control MRSA strains USA100–USA800 were purchased from the Network on Antimicrobial Resistance in S. aureus (NARSA) (Herndon, VA). Susceptibility to ceftobiprole and comparators was determined using Clinical and Laboratory Standards Institute methodology [10]. 3. Results and discussion There were 932 S. aureus isolates in the two clinical trials, of which 591 (63%) were meticillin-susceptible and 341 (37%) were MRSA. All MRSA isolates, based on oxacillin MICs, were mecApositive by PCR. Six isolates were oxacillin-susceptible yet were mecA-positive. These isolates expressed mecA based on identification of penicillin-binding protein 2a (PBP2a) using an agglutination test (MRSA-Screen Kit; Denka Seiken Co., Tokyo, Japan). Sequencing of mecA did not yield any notable mutations, thus the reason for this discrepancy was not due to PBP2a alterations.
0924-8579/$ – see front matter © 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2009.02.013
T.A. Davies et al. / International Journal of Antimicrobial Agents 34 (2009) 166–168
167
Table 1 Incidence and geographical distribution of Panton–Valentine leukocidin (PVL) genes and staphylococcal cassette chromosome mec (SCCmec) types with corresponding ceftobiprole minimum inhibitory concentrations (MICs) in meticillin-resistant Staphylococcus aureus from two Phase 3 complicated skin and skin-structure infection clinical trials. Phenotype/genotype
N (%)
Ceftobiprole MIC (g/mL) MIC50
MIC90
No. of isolates by region (%) Asia or othera
Range
Europeb
Latin Americac
USA
PVL PVL −ve PVL +ve
132 (38.7) 209 (61.3)
1 0.5
2 1
0.25–4 0.25–1
6 (50.0) 6 (50.0)
72 (92.3) 6 (7.7)
19 (95.0) 1 (5.0)
35 (15.2) 196 (84.8)
SCCmec type All types I II III IV (all) IVa IV IVcE V Other (all) III (5A) 4A Unknown
329 (100)d 6 (1.8) 20 (6.1) 34 (10.3) 253 (76.9) 208 (63.2) 24 (7.3) 21 (6.4) 4 (1.2) 12 (3.6) 4 (1.2) 3 (0.9) 5 (1.5)
0.5 – 1 2 0.5 0.5 0.5 1 – 1 – – –
2 – 2 2 1 1 1 1 – 2 – – –
0.25–4 1–2 0.25–2 0.5–2 0.5–2 0.5–2 0.5–1 0.5–1 0.5–1 0.5–4 2–4 0.5–1 0.5–1
10 (3.0) 0 1 (10.0) 3 (30.0) 2 (20.0) 0 1 (10.0) 1 (10.0) 4 (40.0) 0 0 0 0
74 (22.5) 2 (2.7) 1 (1.4) 30 (40.5) 33 (44.6) 10 (13.5) 5 (6.8) 18 (24.3) 0 8 (10.8) 4 (5.4) 0 4 (5.4)
19 (5.8) 4 (21.1) 3 (15.8) 0 12 (63.2) 10 (52.6) 0 2 (10.5) 0 0 0 0 0
226 (68.7) 0 15 (6.6) 1 (0.4) 206 (91.2) 188 (83.2) 18 (8.0) 0 0 4 (1.8) 0 3 (1.3) 1 (0.4)
MIC50/90 , MIC for 50% and 90% of the organisms, respectively; –, insufficient isolates to determine MIC50 or MIC90 . a Countries included India, Israel, South Africa, South Korea, Taiwan and Thailand. b Countries included Bulgaria, Czech Republic, Hungary, Latvia, Lithuania, Romania, Russia, Serbia and Montenegro, and Ukraine. c Countries included Argentina, Costa Rica and Mexico. d Of the 341 MRSA baseline isolates, 329 isolates were available for molecular characterisation.
The majority of MRSA isolates (61.3%) were PVL-positive, mostly due to the high prevalence (84.8%; 196/231) of PVL genes in isolates from the USA (Table 1). PVL genes were present in only 7.7% (6/78) of European MRSA and 5.0% (1/20) of Latin America isolates. Among countries from Asia and other parts of the world, PVL was present in 50.0% (6/12) of MRSA isolates. Of the 341 MRSA isolates, 329 were available for SCCmec typing and PFGE analysis. SCCmec type IVa was most prevalent in MRSA from the USA and Latin America (83.2% and 52.6%, respectively), whilst type III was the most prevalent in Europe (40.5%) (Table 1). SCCmec type V was only found in isolates from Asia, where small numbers of types II, III and IV were also identified. Twelve isolates from the USA or Europe were untypeable or had previously undescribed SCCmec types. The predominant USA MRSA clone was USA300 (n = 197), followed by USA100 (n = 10), USA400 (n = 2) and USA800 (n = 1) (Table 2). Isolates related to one of the USA clones were from the USA, except for one USA100 isolate from the Czech Republic and a single USA800 isolate from Argentina. The remaining 119 isolates had PFGE patterns unrelated to any of the USA clones; however,
a group of SCCmec type III isolates (n = 22) from Latvia were of the Brazilian/Hungarian clone lineage and were comprised of two PFGE types. Susceptibility of isolates from the various MRSA clones varied according to clonality, with ceftobiprole and trimethoprim/sulfamethoxazole (SXT) retaining high activity. Isolates belonging to any of the clonal groups had ceftobiprole MICs ≤ 2 g/mL; all but 11 isolates (of the Latvia-2 clone) were susceptible to SXT (Tables 1 and 2). In contrast, 92% of the clones were erythromycin-resistant and 53% were levofloxacin-resistant (Table 2). The USA100 and Latvia clones demonstrated lower susceptibilities to aminoglycosides and clindamycin compared with the other clones (Table 2). The presence of PVL genes has been noted as one of the markers for community-associated MRSA (CA-MRSA) [3]. In this study, 61.3% of MRSA isolates were PVL-positive. Results from tigecycline worldwide Phase 3 clinical trials were slightly lower, with 47% (18/38) of MRSA being PVL-positive [2]. The high incidence of PVL genes in MRSA from the ceftobiprole trial was driven by isolates from the USA, as 84.8% of US isolates were PVL-positive whilst only 11.8% of
Table 2 Phenotypic and genotypic characteristics of major meticillin-resistant Staphylococcus aureus (MRSA) clones from two Phase 3 complicated skin and skin-structure infection trials. Characteristic
SCCmec PVL +ve (%) Susceptibility (%) Aminoglycosidesa Clindamycinb Erythromycin Levofloxacin SXT Isolates with ceftobiprole MIC ≤ 2 g/mL (%)
MRSA clone USA100 (n = 10)
USA300 (n = 197)
USA400 (n = 2)
USA800 (n = 1)
Latvia-1 (n = 9)
Latvia-2 (n = 13)
II 10
IVa 94
IVa 100
IVa 100
III 0
III 0
50 20 0 0 100 100
97 96 9 54 100 100
100 100 0 100 100 100
100 100 100 100 100 100
0 11 0 0 100 100
15 85 0 0 15 100
SCCmec, staphylococcal cassette chromosome mec type; PVL, Panton–Valentine leukocidin; SXT, trimethoprim/sulfamethoxazole; MIC, minimum inhibitory concentration. a Gentamicin or tobramycin. b Isolates were not screened for inducible resistance to clindamycin.
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isolates from the rest of the world were PVL-positive, similar to a recent report showing the high prevalence of PVL-positive MRSA in the USA [1]. SCCmec type IV was the most common SCCmec type for all MRSA (76.9%), with an even higher prevalence in the USA (91.2%). SCCmec type IV, specifically SCCmec type IVa, is common in the USA and is associated with the widespread CA-MRSA clone USA300 [1–3,11]. In the current study, 96% (197/206) of the US MRSA isolates having SCCmec type IV or IVa were related to USA300. These isolates exhibited a susceptibility profile indicative of CA-MRSA, i.e. resistance to macrolides and -lactams, with the exception of ceftobiprole, but susceptibility to most other drug classes. MRSA having SCCmec type IV from Europe, Latin America and Asia were not related to USA300, consistent with other reports showing the low incidence of this clone in these regions [3,12,13]. SCCmec type III was the second most common SCCmec type, found primarily among European isolates (41%) and similar to other reports showing SCCmec types III and IV to be prevalent in parts of Europe [11–13]. Twenty MRSA had SCCmec type II; 15 of these were from the USA, with the majority (10/15; 67%) having PFGE patterns related to the predominant hospital-acquired MRSA USA100 clone [2,11]. Unlike the USA300 clones, the USA100 clones were resistant to many antibiotics including -lactams (other than ceftobiprole), macrolides, clindamycin, aminoglycosides and fluoroquinolones. Molecular characterisation of MRSA causing cSSSI from two ceftobiprole clinical trials showed that MRSA isolates from Europe and other parts of the world were more diverse and distinct than those from the USA, in large part due to the predominance (87%) of SCCmec type IV USA300 in the USA. Acknowledgments The authors would like to thank Anne Windau from Case Western Reserve University and University Hospitals Case Medical Center, OH, for technical contributions to the susceptibility and multiplex PCR data, and Markus Heep from Basilea Pharmaceutica for suggestions regarding the PVL testing paradigm. Funding: This work was funded by Johnson & Johnson Pharmaceutical Research & Development, L.L.C. Competing interests: TAD, WS, KMA and KB own shares in Johnson & Johnson and are employees of Johnson & Johnson Pharmaceutical Research & Development, L.L.C. MRJ has received research funding from Johnson & Johnson Pharmaceutical Research & Development, L.L.C.
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