Charles Darwin University

Reduced in vitro activity of ceftaroline by Etest among clonal complex 239 methicillin-resistant Staphylococcus aureus clinical strains from Australia

Abbott, I. J.; Jenney, A. W. J.; Jeremiah, Cameron; MirÄeta, M.; Kandiah, J P; Holt, Deborah;Tong, Steven; Spelman, DenisPublished in:Antimicrobial Agents and Chemotherapy

DOI:10.1128/AAC.02015-15

Published: 01/12/2015

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Citation for published version (APA):Abbott, I. J., Jenney, A. W. J., Jeremiah, C., MirÄeta, M., Kandiah, J. P., Holt, D., ... Spelman, D. (2015).Reduced in vitro activity of ceftaroline by Etest among clonal complex 239 methicillin-resistant Staphylococcusaureus clinical strains from Australia. Antimicrobial Agents and Chemotherapy, 59(12), 7837-7841. DOI:10.1128/AAC.02015-15

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TITLE: Reduced In-Vitro Activity of Ceftaroline by Etest among CC239 Methicillin-1

Resistant Staphylococcus aureus (MRSA) Clinical Strains from Australia. 2

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RUNNING TITLE: Ceftaroline Resistance in MRSA 4

5 I. J. Abbott1#†, A. W. J. Jenney1, C. J. Jeremiah1‡, M. Mirčeta1, J. P. Kandiah2, D. C. Holt2, S. Y. 6 C. Tong2, and D. W. Spelman1 7

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1 Alfred Hospital, Melbourne, Victoria, Australia 9

2 Menzies School of Health Research, Charles Darwin University, Darwin, NT, Australia 10

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†Present address: Victorian Infectious Diseases Reference Laboratory, Victoria. 12

‡Present address: The Northern Hospital, Melbourne, Victoria, Australia 13

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# CORRESPONDING AUTHOR 15 Name: Dr I. J. Abbott 16 Email: iainabbott@gmail.com 17

AAC Accepted Manuscript Posted Online 21 September 2015Antimicrob. Agents Chemother. doi:10.1128/AAC.02015-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 18 421 methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates were tested for 19 ceftaroline susceptibility by Etest (bioMérieux). Multidrug resistant phenotype was 20 found in 40.9% and clonal complex 239 (CC239) in 33.5%. Ceftaroline non-21 susceptibility (MIC >1.0 µg/mL) was 16.9% overall. Non-susceptibility was significantly 22 higher in CC239 (41.1%, 58/141) and isolates with a multidrug resistant phenotype 23 (35.5%, 61/172) when compared to comparators (p<0.0001). Non-susceptibility of 24 common multidrug resistant MRSA clones limits the empirical use of ceftaroline for 25 these infections. 26

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BODY TEXT 27 Ceftaroline, an oxyimino-cephalosporin active against methicillin-resistant 28

Staphylococcus aureus (MRSA) due to enhanced affinity for Penicillin Binding Protein 29 (PBP) 2a, was approved for use in Australia in 2013 for the treatment of complicated 30 skin and soft-tissue infections (SSTI) and community-acquired bacterial pneumonia 31 (CABP), following approvals for the same indications in the U.S. in 2010 and Europe in 32 2012. S. aureus minimum inhibitory concentration (MIC) breakpoints have been set by 33 Clinical Laboratory Standards Institute (CLSI) (1) and European Committee on 34 Antimicrobial Susceptibility Testing (EUCAST) (2). Both report susceptibility MIC ≤1.0 35 μg/ml, although differ in resistance designation (CLSI, resistant MIC >4.0 μg/ml; 36 EUCAST, resistant MIC >1.0 μg/ml). Resistance appears to be associated with decreased 37 PBP2a binding affinity (3-7) and heteroresistance (8). Reports have largely 38 demonstrated minimal resistance, although geographical variation has been noted (4) 39 (Table 1). More recently, in hospital-associated MRSA (HA-MRSA) isolates from China, 40 ceftaroline non-susceptibility was 33.5% (84/251), most (95.2%) belonging to clonal 41 complex (CC) 8, with sequence type (ST) 239-III in the majority (9). Similarly, 42 ceftaroline non-susceptible MRSA isolates from Eastern Australia were all of the ST239-43 III clone (10). A German study also demonstrated increased ceftaroline MIC50/90 values 44 for isolates of clonal lineages ST228 and ST239 (7). 45

46 Non-duplicate, consecutive clinical MRSA isolates were tested from patients treated at 47 the Alfred Hospital, a tertiary-care metropolitan hospital in Melbourne, Australia, over 48 two time periods; July to December 2010 and August to November 2013. Ceftaroline 49 was not in use clinically before 2014. Isolates from 2010 and 2013 were identified using 50 Vitek 2 and Vitek MS (bioMérieux) respectively. Susceptibility testing was performed 51

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using the Vitek 2 Gram Positive Susceptibility Card (AST-P612) applying EUCAST 52 breakpoints. Multidrug resistant phenotype was defined as resistance to ≥3 non β-53 lactam antibiotics, including each of ciprofloxacin, erythromycin/clindamycin, 54 gentamicin, cotrimoxazole and tetracycline. All isolates were assessed for ceftaroline 55 and vancomycin susceptibility by Etest (bioMérieux). Isolates from 2010 were thawed 56 from frozen and sub-cultured twice before testing. Isolates from 2013 were collected 57 and tested in real time. Ceftaroline Etests were set up on Mueller Hinton Agar (Becton 58 Dickinson BBL, Ref: 211438), incubated and read as per manufacturer’s guidelines. 59 Ceftaroline non-susceptibility was defined as MIC >1.0 μg/ml. Confirmatory broth 60 microdilution (BMD) was not available. Typing was performed by a high-resolution 61 melting-based method, giving inferred CCs (11). 62 63 421 non-duplicate MRSA isolates were identified for testing. 270 isolates were from 64 2010, and 151 isolates from 2013. SSTI accounted for the majority (70.3%) of 65 specimens. Strains with a multidrug resistant phenotype made up 40.9% of the isolates, 66 although this proportion declined over the two time periods, from 47.0% (127/270) in 67 2010 to 29.8% (24/151) in 2013. CC239, the dominant HA-MRSA strain found in 68 Australia and often multidrug resistant (12), was the most commonly identified clonal 69 complex, accounting for 33.5% of all isolates. This proportion also fell between the two 70 time periods, from 41.5% (112/270) in 2010 to 19.2% (29/151) in 2013. CC22 and 71 CC45 were the next most commonly identified clonal complexes, found in 28.5% and 72 11.4% respectively. The proportion of CC22 isolates increased from 26.7% (72/270) in 73 2010 to 31.8% (48/151) in 2013. Among all MRSA isolates, vancomycin MIC50/90 was 74 1.5 μg/ml. Reduced vancomycin susceptibility (i.e. vancomycin MIC > 1.0 μg/ml) was 75 most common in isolates with a multidrug resistant phenotype (84.9%, 146/172) and 76

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CC239 isolates (83.7%, 118/141), compared with those with a non-multidrug resistant 77 phenotype (32.9%, 82/249), or another CC-type (39.3%, 110/280). 78 79 Overall, ceftaroline MIC50 and MIC90 were 0.75 μg/ml and 1.5 μg/ml, with a non-80 susceptibility rate of 16.9% (71/421). The majority of ceftaroline non-susceptible 81 isolates had a multidrug resistant phenotype (85.9%, 61/71), typed as CC239 (81.7%, 82 58/71) and had a vancomycin MIC > 1.0μg/ml (76.1%, 54/71). All ceftaroline non-83 susceptible isolates had MICs that were either 1.5 µg/mL or 2.0 µg/mL, representing the 84 upper tail of a unimodal distribution, resistant by EUCAST criteria and intermediate by 85 CLSI (Figure 1). Two-sample test of proportions demonstrated that ceftaroline non-86 susceptibility was significantly higher in CC239 MRSA isolates than in non-CC239 87 isolates (58/141, 41.1%; compared to 13/280, 4.6%: p<0.0001). Similarly, ceftaroline 88 non-susceptibility in multidrug resistant MRSA isolates was significantly higher than in 89 non-multidrug resistant isolates (61/172, 35.5%; compared to 10/249, 4.0%: 90 p<0.0001). 91 92 MIC gradient strip testing and disc diffusion remain the most common way for clinical 93 laboratories to test ceftaroline susceptibility in the absence of automated methods and 94 the impracticalities of BMD (13). Ceftaroline gradient strip tests have been reported to 95 underestimate (7), overestimate (14) and demonstrate reasonably good MIC correlation 96 with BMD (10). Livermore et al. report discrimination between MICs of 1.0 and 2.0 97 μg/mL by Etest, compared with agar dilution, was poor (15). Despite this, and 98 differences in clinical breakpoints set by CLSI (1) and EUCAST (2), an Etest ceftaroline 99 MIC of 1.0 μg/ml remains an important and conservative breakpoint for the laboratory 100 to report susceptibility. 101

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102 The pharmacokinetic and pharmacodynamic index that best correlates with ceftaroline 103 efficacy is the percentage of time during the dosing interval that free-drug 104 concentrations remain above the MIC of the infecting organism (f %T>MIC). In a S. aureus 105 neutropenic murine thigh model, the mean f %T>MIC required for bacterial stasis, 1-log10 106 and 2-log10 kill was 26 ±8, 33 ±9 and 45 ±13, respectively. Less killing was observed 107 with more widely spaced dosing intervals (12- and 24-h)(16). Suggestions that 108 susceptibility test interpretive criteria could be MIC ≤2.0 μg/ml (4, 17) are based on 109 achieving a f %T>MIC target of 26%, reflecting bacterial stasis, and would be considered 110 the minimum value required when treating uncomplicated infections in patients with an 111 intact immune system (18). At MIC of 2.0 μg/ml, standard dosing with 600mg twice 112 daily demonstrated lower target attainments aiming for 1- or 2-log10 kill (74.5% and 113 28.8% respectively, in simulated patients with normal renal function, applying f %T>MIC 114 of 36% and 51% for a 1- and 2-log10 kill, respectively) (17). Monte Carlo simulation with 115 ceftaroline administered 600 mg 8-hourly as a 2 hour infusion in patients with normal 116 renal function demonstrated a higher probability of treatment success (from 72% in 117 CABP and 79% in SSTI to ≥99% in both) and may represent a better option than 118 standard dosing, especially when targeting MRSA (19). In practice, off-label dosing has 119 been commonly reported for serious infections (bacteremia, endocarditis, MRSA 120 pneumonia) with good clinical outcomes, although such dosing risks higher toxicity 121 rates (20). 122 123 Ceftaroline resistance among MRSA isolates from Melbourne, Australia, especially 124 isolates with a multidrug resistant phenotype and the CC239 strain, is significant and 125 would preclude its empirical use prior to dedicated susceptibility testing. Empiric usage 126

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in other settings should be determined at an individual institutional level. Ceftaroline 127 non-susceptibility has been identified prior to the clinical use of the drug, and therefore 128 does not represent emergence of resistance. Despite the limitations of this study, which 129 might be biased towards hospital-specific clones, or represent the known laboratory 130 issues with gradient strip susceptibility test with the lack of confirmatory BMD testing, 131 our findings linking ceftaroline resistance to multidrug resistance phenotype and CC239 132 is supported by other recently published studies (4, 9, 10). Our results may also 133 represent changes in resistance over time, highlighting the importance of monitoring 134 MRSA molecular epidemiology in order to understand the underlying genetic 135 background to non-susceptibility and the dynamic relationship that is seen between the 136 pathogen and the drug. In regions where CC239 contributes to a large proportion of 137 MRSA, caution should be exercised in using ceftaroline for suspected or known MRSA 138 infections. Although the association of CC239 and multidrug resistant phenotype are 139 inherently linked, the clinical appreciation, at the time of culture result, that a multidrug 140 resistant MRSA isolate has a higher background rate of ceftaroline non-susceptibility, is 141 an important one. Further studies are required on the clinical efficacy and safety of 142 using more intensive ceftaroline dosing regimens in such settings. 143 144

ACKNOWLEDGMENTS 145 The authors acknowledge the laboratory assistance from the Microbiology Department, 146 Alfred Hospital. Funding received from Alfred Research Trusts Small Projects Grants. 147 SYCT is an Australian National Health and Medical Research Council Career 148 Development Fellow (1065736). 149

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Table 1. Published reports of ceftaroline susceptibility in MRSA isolates. 313 314 Isolates were tested for susceptibility to ceftaroline by reference broth microdilution 315 methods as described by CLSI M07-A9, unless otherwise indicated. 316

a Suceptibility testing by MIC Evaluator (MICE) strips (Oxoid) 317

b Study drug was ceftaroline-avibactam. 318

c Includes isolates from Chile of which 83.9% had an ceftaroline MIC of 2.0µg/ml. 319

d Suceptibility testing by Etest (bioMérieux) 320

e Broth microdilution methods as described by EUCAST 321

f Broth microdilution methods as described by BSAC 322 CA-MRSA, community-associated MRSA. 323 HA-MRSA, healthcare-associated MRSA. 324 SSTI, skin and soft tissue infection. 325 RESP, respiratory. 326

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Figure 1. Ceftaroline MIC distributions in relation to antibiotic susceptibility 327

phenotype and clonal complex (CC). 328

329 Dark shaded area represents ceftaroline non-susceptible MIC range. 330 nmMRSA, non-multidrug resistant MRSA phenotype. 331 mMRSA, multidrug resistant MRSA phenotype. 332 CC239, clonal complex 239 strain. 333 CC22, clonal complex 22 strain. 334

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Table 1. Published reports of ceftaroline susceptibility in MRSA isolates. Time

period Region No. isolates Source MIC50 MIC90 % suscep. Ref1997-2008 Australia 103 Blood 0.5 1.0 100% (14)1.0 1.0 100%a 2008 U.S. 2,254 SSTI 1.0 1.0 94.8% (21)Europe 734 1.0 2.0 82.6% 2008-09 U.S. 215 RESP 1.0 1.0 94.4% (22)Europe 60 1.0 2.0 81.7% 2008-10 U.S. 4,453 Various 1.0 1.0 96.1% (23)2008-11 U.S. 9,875 Various 0.5 1.0 97.1% (24)2009 U.S. 2,247 Various 0.5 1.0 98.0% (25)2009 Canada 74 CA-MRSA Various 0.5 0.5 100% (26)151 HA-MRSA 1.0 1.0 100% 2009-11 U.S. 532 RESP 0.5 1.0 96.2% (27)2009-11 U.S. 1,492 SSTI 0.5 1.0 99.1% (28)2009-13 U.S. 12,514 Various 0.5 1.0 97.6% (29)2009-13 U.S. 2013 Blood 0.5 1.0 95.4% (30)2010 U.S. 1,072 Various 0.5 1.0 98.4% (31)2010 Europe 331 SSTI 1.0 2.0 88.8% (32)2010 Sth Africa + Asia Pacific 211 SSTI & RESP 1.0 2.0 80.6% (33)2010-11b U.S. 2,143 Various 0.5 1.0 96.8% (34)2010-12b U.S. 4,333 SSTI 0.5 1.0 98.9% (35)2011 Latin America 409c Various 1.0 2.0 61.6% (36)2011 Australia 713 Various 0.75 1.0 98.2%d (12)2011 U.S. 2,093 Various 0.5 1.0 98.9% (37)2011 China 251 HA-MRSA SSTI 1.0 2.0 66.5% (9)2012 Global 4324 Various 1.0 2.0 85.2% (4)2012 Germany 133 Various 1.0 1.5 63.9%e (7)0.5 0.75 96.2%d 2012-13 U.K. 531 SSTI 1.0 1.0 95.3%f (38)2012-13 France 67 SSTI 0.5 1.0 97.0% (39)2013 Australia 100 Various 0.5 2.0 85.0% (10)1.0 2.0 85.0%d 2013 U.S. 2642 SSTI 1.0 1.0 98.8% (40)2014 India 50 Various 0.5 1.0 96.0%d (41)Isolates were tested for susceptibility to ceftaroline by reference broth microdilution methods as described by CLSI M07-A9, unless otherwise indicated.

a Suceptibility testing by MIC Evaluator (MICE) strips (Oxoid) b Study drug was ceftaroline-avibactam.

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c Includes isolates from Chile of which 83.9% had an ceftaroline MIC of 2.0µg/ml. d Suceptibility testing by Etest (bioMérieux) e Broth microdilution methods as described by EUCAST f Broth microdilution methods as described by BSAC CA-MRSA, community-associated MRSA. HA-MRSA, healthcare-associated MRSA. SSTI, skin and soft tissue infection. RESP, respiratory.