Microbiological and Genotypic Analysis of Methicillin-Resistant ACCEPTED. 1. Department of Medicine, New York Medical College, Valhalla, NY

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1 AAC Accepts, published online ahead of print on 7 July 2008 Antimicrob. Agents Chemother. doi: /aac Copyright 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. AAC Version 2 Microbiological and Genotypic Analysis of Methicillin-Resistant Staphylococcus aureus (MRSA) Bacteremia Carlo McCalla MD, 1 Davida S. Smyth PhD, 2 D. Ashley Robinson PhD, 2 Judith Steenbergen PhD, 3 Steven A. Luperchio PhD, 3 Pamela A. Moise PharmD, 3 Vance G. Fowler Jr MD, 4 and George Sakoulas MD 1 1. Department of Medicine, New York Medical College, Valhalla, NY 2. Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 3. Cubist Pharmaceuticals, Lexington, MA 4. Division of Infectious Disease, Duke University Medical Center, Durham, North Carolina Address Correspondence to: George Sakoulas, MD Division of Infectious Diseases New York Medical College Munger 245 Valhalla, NY Phone: (914) Fax: (914) george_sakoulas@nymc.edu 1

2 Abstract In a recent landmark MRSA bacteremia trial, vancomycin MIC s were > 1 µg/ml for only16% and accessory gene regulator (agr) function by delta hemolysin was absent or reduced in only 28.1% of the isolates. This clinical study did not capture a population of MRSA predictive of vancomycin treatment failure. 2

3 Methicillin-resistant Staphylococcus aureus (MRSA) with higher vancomycin MICs (> 1 µg/ml), reduced vancomycin bactericidal activity in vitro (5,13), the accessory gene regulator (agr) group II genotype (9), and attenuated agr function (1), have been associated with reduced vancomycin efficacy in bacteremia. Clinical studies targeting bacteremia by MRSA with these properties would be important to define the practical role of newer antimicrobials. We evaluated the properties of MRSA from a recent study demonstrating non-inferiority of daptomycin compared to standard therapy in cases of S. aureus bacteremia and endocarditis (2). Eighty-nine baseline MRSA isolates from the previously published study comparing standard therapy (vancomycin plus initial 4-days of low-dose gentamicin) to daptomycin in MRSA bacteremia and endocarditis were analyzed (87 of 89 were analyzed genotypically) (2). Vancomycin susceptibility testing was confirmed by CLSI broth microdilution methods. Susceptibility to other antimicrobials was performed by Cubist Pharmaceuticals as part of the prior study (2). Function of agr was determined semi-quantitatively using delta hemolysin activity as previously described (12). Genotype and SCCmec type was determined by PCR methods previously published (3,6,11). spa X-repeat polymorphisms were determined by nucleotide sequencing as described previously (7,11). spa types were compared with results from previous studies (11) and with the Ridom spa server (4) to provisionally assign isolates to MLST-defined clonal complexes (CCs). CC1 equaled spa types 131, 950; CC5 equaled spa types 2, 12, 23, 47, 65, 203, 302, 385, 402, 437; CC8 equaled spa types 1, 4, 7, 363, 954; CC30 equaled spa types 16, 19; CC45 equaled spa types 15, 756, 951, 953; CC59 spa equaled types 17, 206.Note that 4 new spa types are included above as follows: 950 (UJJJFFKBPE), 951 (XE3BMBKB), 953 (XE3BBKB), 954 (YHGCO). Clinical outcomes were defined as described in the initial published study (2). 3

4 Vancomycin MIC s for the 89 isolates were: 0.25 µg/ml (n=1, 1%), 0.5 µg/ml (n=74, 83%), and 1 µg/ml (n=14, 16%). There was 100% concordance (defined as within 1 dilution tube) between MIC s performed for the purposes of this study and the Cubist Central Microbiology Laboratory for the original clinical study. Semi-qualitative assessment of agr function demonstrated that 28% of isolates had a reduced or absent delta hemolysin phenotype. The distribution of agr function between the two treatment arms is shown in Table 1. MRSA genotyping results between the two treatment arms are shown in Table 1. Interestingly, over half of the isolates were SCCmec type IV, which is associated with community-acquired MRSA. This result was investigated further by analyzing the susceptibility data of other antibiotics and stratifying the results by SCCmec type. MRSA with SCCmec type IV were more likely to be susceptible to clindamycin and levofloxacin and less likely to be susceptible to tetracycline when compared to strains with SCCmec type II. MRSA with SCCmec II were 7.7% agr group I, 89.7% agr II, and 2.6% agr III whereas MRSA with SCCmec IV were 87.2% agr I, 4.3% agr II, and 8.5% agr III. This association is consistent with prior data (10). Clinical response to therapy with either daptomycin or vancomycin evaluated by SCCmec type, agr genotype, and agr function, is listed in Table 2. Currently the epidemiology of MRSA is in flux, with health-care associated MRSA marked by increases in vancomycin MIC s and the agr group II genotype that have been linked with inferior vancomycin efficacy on the one hand, and community-associated MRSA with lower vancomycin MICs but linked with rapidly invasive necrotizing infections and a high severity of illness on the other (10,16), with boundaries between these becoming less discriminatory. The MRSA from this study was heavily-weighted (84%) towards organisms with vancomycin MIC of <1 µg/ml, leaving too small a subset with higher vancomycin MICs to 4

5 evaluate. However, this observation brought to light the fact that the study comparing the efficacy of daptomycin to vancomycin plus an initial 4-days of gentamicin did not capture the organisms required to address the pressing problem of optimal antimicrobial therapy for MRSA bacteremia caused by organisms with vancomycin of 2 µg/ml. Only 25 of 89 (28%) of the isolates had significantly reduced or absent delta hemolysin activity, in contrast to prior evaluations of MRSA bacteremia isolates where 75% of 81 isolates had this phenotype (14). This finding, in addition to the results of the vancomycin MIC values, suggested that communityacquired MRSA represented a significant proportion of this group of isolates. Such strains represent an increasing cause of S. aureus bacteremia and endocarditis. In this trial, 44 of 87 (51%) of MRSA belonged to agr group I and 38 of 87 (44%) belonged to agr group II, in contrast to prior data showing that greater than 60% of MRSA bacteremia isolates collected from multiple centers around the United States belonged to agr group II (9). Extrapolation from spa typing revealed that 33 (38%) of the isolates belonged to clonal complex 8, the genetic background of the well-recognized USA300 community MRSA pulsotype (14). Consistent with this was the finding that 87% of agr group I strains harboured SCCmec type IV, another feature of community MRSA, while 90% of agr group II strains harboured SCC type mec II. Community-acquired SCCmec type IV MRSA as causes of hospital-acquired infections increased from 17% in 1999 to 56% in 2003 at one center, suggesting that this epidemiological shift in MRSA had been well under way during the enrollment period of this trial. (8) The shift in MRSA epidemiology towards community-acquired clones may render the evaluation of infections caused by strains with microbiological features that are currently predictive of vancomycin treatment failure (agr group II genotype, reduced agr function, 5

6 vancomycin MIC > 1 µg/ml) more difficult. Such investigations would need to enrich their samples for isolates with higher vancomycin MICs or enroll a higher number of patients in order capture subsets large enough to get statistically significant data, which may be prohibitively expensive and lengthy. It is important to appreciate that features predictive of treatment failure are dynamic and therefore may change temporally and geographically with shifts in molecular epidemiology and differences in antibiotic selection pressure over time and in different hospital centers. If the population studied in this trial is representative of the current predominant S. aureus strains, additional studies are warranted to determine the microbiological features associated with treatment failure in bacteremia caused by community acquired S. aureus. In this study, our attempts to show superiority of daptomycin over vancomycin for MRSA bacteremia caused by organisms within specific the agr group genotype subsets did not achieve statistical significance. In summary, MRSA with SCCmec type IV have emerged to become significantly represented in MRSA bacteremia, resulting in the trial comparing daptomycin with vancomycin plus gentamicin not capturing isolates with vancomycin MIC s of 2 mg/l, recently associated with higher mortality when vancomycin was used empirically for bacteremia (15). Additional studies would be needed to define optimal therapy in cases of MRSA bacteremia where the vancomycin MIC value is > 1 mg/l, the genotype is agr group II, and agr function is reduced or absent. Given the poor performance of vancomycin in MRSA bacteremia in such cases, the need for the timely completion of such investigations to keep up with the rapid evolution of MRSA is of utmost importance. Until this happens, clinicians are encouraged to quantitate vancomycin MIC s in their local hospitals and for individual patients in serious MRSA infections. 6

7 Acknowledgements/Funding This study was funded through a research grant from Cubist Pharmaceuticals (Lexington, MA). DAR is supported in part by grants from the American Heart Association and the National Institute of General Medical Sciences (GM080602). Authors, JNS, SAL, and PAM are employed by Cubist Pharmaceuticals. Author GS has received research funding from Cubist and Pfizer Pharmaceuticals and honoraria from Cubist, Pfizer, and Wyeth Pharmaceuticals. 7

8 Table 1: Genotypes and agr function distribution of MRSA between treatment arms Daptomycin Comparator Delta hemolysin (n = 45) (n = 44) p-value (24.4%) 014 (31.8%) (75.6%) 30 (68.2%) 0.49 agr type 1 20 (44.4%) 24 (54.5%) (46.7%) 17 (38.6%) (04.4%) 03 (06.8%) 0.68 ND. 02 (04.4%) 00 (00.0%) 0.49 SCCmec type I 1 (2.2%) 0 (0.0%) >0.99 II 19 (42.2%) 20 (45.5%) 0.83 IV 23 (51.1%) 24 (54.5%) 0.83 ND 02 (04.4%) 00 (00.0%) 0.49 Predicted clonal complex* 1 01 (02.2%) 02 (04.5%) (46.7%) 17 (38.6%) (28.9%) 20 (45.5%) (02.2%) 01 (02.3%) >0.99 8

9 45 05 (11.1%) 03 (06.8%) (04.4%) 01 (02.3%) >0.99 n.d. 02 (04.4%) 00 (00.0%) 0.49 *See text for spa types used to predict CCs ** p-values determined with a 2-sided Fisher s Exact Test 9

10 Table 2: Summary of IEAC Success Rates at Test-of-Cure stratified by agr function (delta hemolysin score), SCCmec type, and agr genotype a Delta Hemolysin Score Daptomycin Comparator (n = 45) (n = 44) p-value /11 (45.5%) 04/14 (28.6%) to 4 15/34 (44.1%) 10/30 (33.3%) 0.45 SCCmec type I II 0/1 (0.0%) 8/19 (42.1%) - 5/20 (25.0%) > IV 11/23 (47.8%) 9/24 (37.5%) 0.56 a. 2 SCCmec types were undetermined agr type 1 10/20 (50.0%) 9/24 (37.5%) /21 (38.1%) 3/17 (17.6%) /02 (50.0%) 2/03 (66.7%) >0.99 a Excludes the two isolates that did not have their SCCmec type determined ** p-values determined with a 2-sided Fisher s Exact Test 10

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13 13. Sakoulas, G., P.A. Moise, J.J. Schentag, A. Forrest, R.C. Moellering, Jr, and G.M. Eliopoulos Relationship of MIC and bactericidal activity to efficacy of vancomycin for the treatment of methicillin-resistant Staphylococcus aureus bacteremia. J. Clin. Microbiol. 42: Sakoulas, G The accessory gene regulator (agr) in methicillin-resistant Staphylococcus aureus: role in virulence and reduced susceptibility to glycopeptide antibiotics. Drug Discovery Today: Disease Mechanisms 3: Soriano, A., F. Marco, J.A. Martinez, E. Pisos E, M. Almela, V.P. Dimova, D. Alamo, M. Ortega, J. Lopez, and J. Mensa Influence of vancomycin minimum inhibitory concentration on the treatment of methicillin-resistant Staphyloccoccus aureus bacteremia. Clin. Infect. Dis., E-published Dec Tsuji, B.T., M.J. Rybak, C.M. Cheung, M. Amjad, and G.W. Kaatz Community- and health care-associated methicillin-resistant Staphylococcus aureus: a comparison of molecular epidemiology and antimicrobial activities of various agents. Diagn. Microbiol. Infect. Dis. 58:

Hong-Kai Wang 1, Chun-Yen Huang 1 and Yhu-Chering Huang 1,2*

Hong-Kai Wang 1, Chun-Yen Huang 1 and Yhu-Chering Huang 1,2* Wang et al. BMC Infectious Diseases (2017) 17:470 DOI 10.1186/s12879-017-2560-0 RESEARCH ARTICLE Open Access Clinical features and molecular characteristics of childhood communityassociated methicillin-resistant