Synergy of beta-lactams with vancomycin against methicillin-resistant. Staphylococcus aureus: correlation of the disk diffusion and the

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1 JCM Accepted Manuscript Posted Online 16 December 2015 J. Clin. Microbiol. doi: /jcm Copyright 2015, American Society for Microbiology. All Rights Reserved Synergy of beta-lactams with vancomycin against methicillin-resistant Staphylococcus aureus: correlation of the disk diffusion and the checkerboard methods 4 Cheng Len Sy 1,Tsi-Shu Huang 2, Chii Shiang Chen 2, Yao-Shen Chen 1,3, Hung-Chin Tsai 1,3, 5 Shue-Renn Wann 1, Kuan-Sheng Wu 1,3, Jui-Kuang Chen 1, Susan Shin-Jung Lee 1,3#, 6 Yung-Ching Liu Division of Infectious Diseases, Department of Internal Medicine, Kaohsiung 8 Veterans General Hospital, Kaohsiung, Taiwan 9 2. Division of Microbiology, Department of Pathology and Laboratory Medicine, 10 Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan National Yang-Ming University, Taipei, Taiwan Division of Infectious Diseases, Shuang-Ho Hospital, Taipei, Taiwan; Taipei 13 Medical University, Taipei, Taiwan Running Title: Synergy of beta-lactams and vancomycin 16 #Address correspondence to: Susan Shin-Jung Lee, ssjlee28@yahoo.com.tw 17 *Present Address: Division of Infectious Diseases, Department of Medicine,

2 18 Kaohsiung Veterans General Hospital,386, Ta-chung 1 st Rd, Kaohsiung 813, Taiwan

3 19 Abstract 20 Modified disk diffusion (MDD) and checkerboard tests were employed to 21 assess synergy of combinations of vancomycin and β-lactam antibiotics for 59 clinical 22 isolates of methicillin resistant Staphylococcus aureus (MRSA) and mu50 23 (ATCC700699). Bacterial inocula equivalent to McFarland 0.5 and 2.0 were 24 inoculated on agar plates containing 0, 0.5, 1 and 2 μg/ml of vancomycin. Oxacillin, 25 cefazolin and cefoxitin impregnated disks were applied to the surface and the zones of 26 inhibition were measured at 24h. The CLSI-recommended checkerboard method was 27 used as a reference to detect synergy. The minimum inhibitory concentrations (MIC) 28 for vancomycin were determined using the Etest method, broth microdilution and the 29 Vitek2 automated system. 30 Synergy was observed with the checkerboard method in 51-60% of the isolates 31 when vancomycin was combined with any β-lactam. The fractional inhibitory 32 concentration indices were significantly lower in MRSA isolates with higher 33 vancomycin MICs combinations (p<0.05). 34 The overall agreement between the MDD methods and the checkerboard to 35 detect synergy in MRSA isolates with bacterial inocula equivalent to McFarland 0.5

4 36 were 33% and 62.5% for oxacillin, 45.1% and 52.4% for cefazolin and 43.1% and % for cefoxitin when combined with 0.5 and 2 μg/ml of vancomycin, 38 respectively. Based on our study, the simple MDD method is not recommended as a 39 replacement of the checkerboard method to detect synergy. However, it may serve 40 as an initial screening method for detection of potential synergy when it is not feasible 41 to perform other labor-intensive synergy tests.

5 42 Introduction 43 Vancomycin and other glycopeptide antibiotics are currently the standard 44 treatment for methicillin-resistant Staphylococcus aureus (MRSA) (1). The 45 emergence of MRSA with reduced vancomycin susceptibility (SA-RVS) (2) raises 46 concerns about the reliability of vancomycin for treatment of MRSA. The occurrence 47 of resistance during treatment with daptomycin has raised similar concerns about its 48 effectiveness for life-threatening MRSA infections (3). Low serum levels and the 49 bacteriostatic activity of tigecycline against MRSA limit its use for the treatment of 50 MRSA bacteremia (4). 51 The combination of vancomycin and β-lactam antibiotics offers a potential 52 option for management of MRSA infections (5). This concept is based on several 53 in-vitro and animal studies that demonstrated a synergistic effect of 54 vancomycin-β-lactam combinations against MRSA (6-11). 55 The current study was designed to determine the utility of a modified disk 56 diffusion test (8, 12) to assess synergy and antagonism for combinations of 57 vancomycin and β-lactam antibiotics among clinical isolates of MRSA. The CLSI 58 checkerboard method was used as the reference for the determination of synergy. We

6 59 determined the vancomycin minimal inhibitory concentration (MIC) breakpoint 60 where synergy is most commonly observed in MRSA isolates

7 61 Materials and Methods: 62 Bacterial isolates 63 Fifty-nine unique clinical isolates of MRSA with a vancomycin minimum 64 inhibitory concentrations (MIC) 0.5μg/mL were obtained from patients at the 65 Kaohsiung Veterans General Hospital in Taiwan during 2008 to All isolates and 66 S. aureus ATCC (Mu50) were identified as S. aureus by the tube coagulase 67 method and the VITEK 2 system (BioMerieux, France). Mu50 is an MRSA isolate 68 with reduced vancomycin susceptibility that was isolated in Japan in All 69 isolates tested positive for meca by PCR as previously described (13). 70 Susceptibility tests 71 Methicillin resistance was determined using 30 μg cefoxitin and 1μg oxacillin 72 disks according the disk diffusion method recommended by the Clinical and 73 Laboratory Standards Institute (CLSI) (14). The MIC to oxacillin and vancomycin 74 were also determined by CLSI-recommended broth microdilution, the VITEK 2 75 system and the Etest method (AB, Biodisk, Solna, Sweden). In the Etest method, 76 colonies were suspended in trypticase soy broth to obtain inoculum equivalent of 77 McFarland 0.5. For uniformity, 200μL of bacterial inoculum was streaked evenly

8 78 onto a 90mm agar plate and allowed to dry. Oxacillin and vancomycin Etest strips 79 were applied on the inoculated agar and incubated at 35 C for 24 hours. Interpretation 80 was done according to the manufacturer s instructions. Staphylococcus aureus 81 ATCC and ATCC served as controls for MIC testing and disk diffusion 82 studies, respectively Modified Disk Diffusion Method of Synergy Testing 85 Synergy between vancomycin and β-lactams was determined by a MDD 86 method (8) using inocula with turbidities equivalent to 0.5 and 2.0 of the McFarland 87 standard. The agar plates were defined to have low, intermediate and high 88 vancomycin concentration when they contain 0.5, 1 and 2μg/mL of vancomycin, 89 respectively. Two hundred microliters of the bacterial suspension were inoculated on 90 brain heart infusion agar (BHIA) (CMP, Taiwan) containing 0, 0.5, 1 and 2μg/mL of 91 vancomycin. Antimicrobial disks containing oxacillin (1μg), cefazolin (30μg), and 92 cefoxitin (30μg) were placed on the agar plates. The zones of inhibition were 93 measured at 24 hours incubation at 37 o C. The definition of synergy was modified 94 from a previous study utilizing a MDD method for testing for synergy and antagonism

9 95 (8). Synergy was defined as an increase in the zone of inhibition by > 2 mm around 96 the antimicrobial disk in the vancomycin-containing agar compared to the drug free 97 agar. Indifference was defined a difference of +/- 2mm in the zone of inhibition of 98 the antimicrobial disk in the vancomycin-containing agar plates. Antagonism was 99 defined as a decrease of >2mm in the zone of inhibition around the antimicrobial disk 100 in the vancomycin-containing agar. 101 Broth Microdilution Checkerboard Method 102 Checkerboard synergy testing was performed by the microdilution method as 103 previously described (15). Vancomycin was tested from concentrations of 0.06 to μg/ml. Vancomycin was combined with oxacillin in concentrations of 1 to μg/ml in 60 isolates of MRSA. Fifty-one isolates were tested using checkerboard 106 synergy by combining vancomycin with cefazolin and cefoxitin in concentrations of to 1024μg/ml. Microtiter plates were incubated at 37 C and were read at 24 h. The 108 fractional inhibitory concentration (FIC) index was calculated using the first 109 non-turbid well in each row and column (16). An FIC index of 0.5 was defined as 110 synergy, an FIC index of >0.5 to 4.0 was defined as additive or indifferent, and an 111 FIC index of >4.0 was defined as antagonistic (17).

10 112 Statistical analysis 113 Descriptive statistics were expressed as frequency, percentage, mean and 114 standard deviation. Means and standard deviations were calculated for continuous 115 variables and percentages were calculated for categorical variables. Linear 116 regression analysis was used to determine the correlation of the FIC indices and 117 vancomycin MIC using the Etest, broth microdilution and automated Vitek 2method. 118 A p-value less than 0.05 was considered significant. Analyses were performed using 119 SPSS version 20.0 (IBM, Armonk, NY, USA) and Stata release 10 (STATA Corp. 120 College Station TX, USA).

11 121 Results 122 Demonstration of Synergy for Vancomycin and β-lactams by the Disk Diffusion 123 Method 124 Sixty percent of the isolates of MRSA failed to grow on the plates with 2μg/mL 125 when its concentration was equivalent to a 0.5 McFarland standard. Fifty percent of 126 the isolates grew when the inoculum was increased to McFarland 2.0. The number 127 of isolates with demonstrable synergy increased when the vancomycin concentration 128 increased from 0.5 to 2μg/mL (Table 1). The increase in synergistic activity was seen 129 in both inoculum concentrations of 0.5 and 2.0 McFarland standard. Antagonism 130 was observed in % of isolates in the presence of a low vancomycin 131 concentration and all β-lactam antibiotics. Antagonism was present in only 1 isolate 132 (1.67%) when oxacillin was combined with an intermediate concentration of 133 vancomycin. There was no antagonism when intermediate concentration of 134 vancomycin was combined with cefazolin and cefoxitin. Antagonism was not seen 135 when a high vancomycin concentration was combined with any β-lactam antibiotics. 136 Demonstration of Synergy for Vancomycin and β-lactams by the Checkerboard 137 Method

12 138 Synergy was seen in varying proportions of the MRSA isolates when 139 vancomycin was combined with either oxacillin, cefoxitin or cefazolin (Table 1). 140 Fifty-one to sixty percent of the isolates showed synergy when vancomycin was 141 combined with oxacillin, cefazolin or cefoxitin. The combination resulted in additive 142 activity against the rest of the isolates. No antagonism was observed for the 143 combinations against any test isolate. 144 The mean FIC indices for combinations of vancomycin with oxacillin, cefazolin 145 or cefoxitin were significantly inversely correlated to the vancomycin MIC of the 146 isolates using linear regression analysis, except for cefoxitin using Vitek 2 for 147 vancomycin MIC (Figure 1). The rate of synergy observed in combinations of 148 vancomycin with oxacillin, cefoxitin and cefazolin differs with the 3 methods of 149 determining of vancomycin MIC (Table 2). Synergy was seen more frequently in 150 oxacillin-vancomycin combination when isolates had a vancomycin MIC of >1μg/mL 151 using the Vitek2 and BMD method. Synergy was more frequent when vancomycin 152 was combined with cefoxitin or cefazolin in higher vancomycin MIC. The current 153 study was not able to determine the vancomycin breakpoint where frequency of

13 154 synergy is statistically significant when cefazolin and cefoxitin was combined with 155 vancomycin. 156 There was poor agreement between the checkerboard and the modified disk 157 diffusion methods to detect synergy irrespective of the concentration of vancomycin 158 in the disk diffusion plates and the inoculum size used. Overall agreement ranged 159 from 33.3% to 60.0% with poor kappa values (0.15 to -0.24). The overall agreement 160 between the MDD methods and the checkerboard to detect synergy in MRSA isolates 161 with bacterial inocula equivalent to McFarland 0.5 were 33% and 62.5% for oxacillin, % and 52.4% for cefazolin, and 43.1% and 52.4% for cefoxitin when combined 163 with 0.5 and 2 μg/ml of vancomycin, respectively. Sensitivity to detect synergy 164 with oxacillin by the MDD method at low inoculums (McFarland 0.5) increased from % to 83.3% when vancomycin concentrations was increased from 0.5 μg/ml to μg/ml, cefazolin increased by 46.2% to 100%, and cefoxitin, 53.6% to 100.0%

14 171 Discussion 172 The disk diffusion test has been previously shown to be a valid method to 173 detect synergy for combinations of vancomycin and β-lactam antibiotics against 174 staphylococci with reduced susceptibility to vancomycin (8) and to detect synergistic 175 activities of triazoles with caspofungin against Candida glabrata (12). It is more rapid, 176 less expensive and labor intensive than the more commonly used time-kill or 177 checkerboard method. However, our study shows that the MDD method correlated 178 poorly with the checkerboard method for testing for synergy in combinations of 179 vancomycin and β-lactams in MRSA isolates. The definition of synergy using the 180 MDD is not known. Currently, there is no current consensus on the optimum 181 method for determining zone diameter breakpoints (18). Climo et al defined synergy 182 as an enhancement in the zone of inhibition surrounding the antibiotic disk on 183 vancomycin agar compared to the vancomycin-free agar (8). A numerical definition 184 of enhancement was not specified in their study. Many factors affect the zone of 185 inhibition during disk diffusion studies. These include the variations in the culture 186 media, the preparation and handling of inoculum suspensions, inoculated plates and 187 antimicrobial disks, the duration of incubation of plates and reading of zones (19). A

15 188 natural variation of 1 to 2.4mm has been described in previous studies (19, 20). Our 189 definition of synergy included a consideration of these natural variations. 190 A low concentration of β-lactam antibiotic has been associated with an 191 antagonistic effect when combined with vancomycin (6, 10). Antagonism was 192 observed in the MDD only in the presence of a low to intermediate concentration of 193 vancomycin. The mechanisms responsible for synergy and antagonism are not well 194 defined. It has been proposed to be due a reduction of β-lactam resistance in MRSA 195 when the MIC for glycopeptides increases or due to simultaneous inhibition of 196 different stages of cell wall synthesis (21, 22) 197 The activities of combinations of β-lactams and vancomycin were enhanced in 198 MRSA isolates with higher MICs. Our findings are consistent with those of Climo 199 et al. who showed that the synergistic activity of vancomycin and oxacillin was 200 directly related to the vancomycin resistance of the isolate (8). 201 In conclusion, combinations of vancomycin with β-lactams appear to have a 202 synergistic activity in MRSA, particularly in isolates with a higher vancomycin MIC. 203 Synergy was observed in the oxacillin-vancomycin combination when isolates had a 204 vancomycin MIC of > 1μg/mL using the Vitek2 and BMD method. The current

16 205 findings failed to support the concept that MDD can be a replacement for the 206 checkerboard method to test for synergy. However, it may provide a rapid screening 207 test for clinical microbiology laboratories that do not have the resources to perform 208 the more labor-intensive, time-kill, checkerboard methods and population analyses to 209 detect potential synergy and antagonism for combinations of vancomycin with 210 β-lactam antibiotics. Further studies with larger sample sizes are needed to 211 determine the utility and validity of this method and to define the optimal zone of 212 inhibition that may correlate to the more complex standard synergy testing.

17 213 Acknowledgement: This study was funded by a grant from the Medical Foundation 214 in Memory of Dr. Deh-Lin Cheng, Taiwan ( ). The authors wish to 215 express their appreciation to Calvin M. Kunin for his critical review of the 216 manuscript

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20 264 beta-lactam antibiotics. Antimicrob Agents Chemother 50: Haraga I, Nomura S, Nagayama A The effects of vancomycin and 266 beta-lactam antibiotics on vancomycin-resistant Staphylococcus aureus. N 267 Engl J Med 341: Rochon-Edouard S, Pestel-Caron M, Lemeland JF, Caron F In 269 vitro synergistic effects of double and triple combinations of beta-lactams, 270 vancomycin, and netilmicin against methicillin-resistant Staphylococcus 271 aureus strains. Antimicrob Agents Chemother 44: Kiraz N, Dag I, Yamac M, Kiremitci A, Kasifoglu N, Oz Y Synergistic activities of three triazoles with caspofungin against Candida 274 glabrata isolates determined by time-kill, Etest, and disk diffusion 275 methods. Antimicrob Agents Chemother 54: Tiwari HK, Sen MR Emergence of vancomycin resistant 277 Staphylococcus aureus (VRSA) from a tertiary care hospital from northern 278 part of India. BMC Infect Dis 6: Wayne P Performance standards for antimicrobial susceptibility 280 testing; twenty-third informational supplement, document M100-S23 ed.

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24 1 Table 1. Frequency of a synergy for combinations of vancomycin and oxacillin, cefoxitin or cefazolin for 60 isolates of methicillin-resistant 2 Staphylococcus aureus by a modified disk diffusion and the checkerboard method Drug Combination Inoculum Modified Disk Diffusion Method Checkerboard Method Concentration (Mc Farland b Low vancomycin c Intermediate vancomycin d High vancomycin Numerator / Total no.(%) Standard) Numerator / Total no. (%) Numerator / Total no. (%) Numerator / Total no. (%) Vancomycin + Oxacillin /60 (16.7) 28/60 (46.7) 21/24(87.5) e 36/60(60.0) 2.0 5/60(8.3) 14/60 (23.3) 25/30(83.3) Vancomycin +Cefoxitin /60 (58.3) 59/60 (98.3) 24/24(100) e 28/51(54.9) /60(33.3) 47/60 (78.3) 30/30(100) Vancomycin + Cefazolin /60 (45) 58/60 (96.7) 24/24 (100) e 26/51(51.0) /60(21.7) 48/60 (80.0) 30/30(100)

25 3 a Modified disk diffusion method: Synergy is defined as an increase in the zone of inhibition by >2mm around the antimicrobial disk in the 4 vancomycin containing agar compared to the drug free agar. 5 Checkerboard method: Synergy is defined as a fractional inhibitory concentration index of b Vancomycin concentration in agar is 0.5 μg/ml 7 c Vancomycin concentration in agar is 1 μg/ml 8 d Vancomycin concentration in agar is 2 μg/ml 9 e No growth was seen in 6 of the 30 BHIA plates high vancomycin concentration when the inoculum was equivalent to a McFarland 0.5 standard

26 10 11 Table 2. Frequency of synergy using the checkerboard method for VSSA and VISA isolates using different methods of determination of vancomycin MIC. %Synergy %Synergy %Synergy %Synergy %Synergy %Synergy Combination Vitek VAN MIC>1 Vitek VAN MIC<=1 BMD VAN MIC>1 BMD VAN MIC<=1 Etest VAN MIC>2 Etest VAN MIC<=2 Vancomycin + Oxacillin n = 60 Vancomycin +Cefoxitin n = 51 Vancomycin + Cefazolin n = 51 15/17(88.2) 21/43(48.8) 11/11(100.0) 25/49(51.0) 7/9(77.8) 29/51(56.9) 10/16(62.5) 18/35(51.4) 8/11(72.7) 20/40(50.0) 7/8(87.5) 21/43(48.8) 10/16(62.5) 16/35(45.7) 8/11(72.7) 18/40(45.0) 7/8(87.5) 19/43(44.2) Note: a BMD, broth microdilution method bvan, vancomycin