Evaluation of surrogate disk tests for the detection of ciprofloxacin and 1 levofloxacin resistance in clinical isolates of Salmonella enterica 2 3 Eszter Deak1,2, Robert Skov3, Janet A. Hindler1 and Romney M. Humphries1* 4 1. Pathology and Laboratory Medicine, University of California, Los Angeles CA 5 2. Santa Clara Valley Medical Center, Santa Clara CA 6 3. Robert Skov, Statens Serum Institut, Copenhagen, Denmark 7 8 *corresponding author: Romney M. Humphries, rhumphries@mednet.ucla.edu 9

JCM Accepted Manuscript Posted Online 19 August 2015J. Clin. Microbiol. doi:10.1128/JCM.01393-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Abstract 10 11 Detection of fluoroquinolone resistance in Salmonella enterica has become 12 increasingly difficult due to evolving resistance mechanisms to this antimicrobial 13 class in this organism. We evaluated two quinolone and 5 fluoroquinolone disks for 14 their ability to act as a surrogate agent for the detection of fluoroquinolone 15 resistance in a collection of 136 S. enterica isolates, including 111 with intermediate 16 or resistant ciprofloxacin MICs mediated by a variety of resistance mechanisms. 17 Ciprofloxacin, ofloxacin, and pefloxacin disks detected all isolates resistant to 18 ciprofloxacin (0% very major error) and yielded false resistance (major error) in 8, 19 4 and 12% of susceptible isolates, respectively. Ciprofloxacin and pefloxacin 20 provided a more clear differentiation of susceptible and resistant isolates. 21

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22 Introduction 23 Fluoroquinolones are an important class of antimicrobials for the treatment 24 of typhoid fever (1). However, defining an optimal method for the detection of 25 fluoroquinolone resistance in Salmonella enterica that is practical for laboratories in 26 developing countries, where typhoid is endemic, has proven difficult. The Clinical 27 and Laboratories Standards Institute (CLSI) has published several revisions to the 28 M100 document recommendations for the detection of fluoroquinolone resistance 29 in Salmonella over the past three years. These changes were driven by; 1) the 30 recognition that several emerging fluoroquinolone resistance mechanisms are not 31 detected by traditional phenotypic methods; 2) re-evaluation of fluoroquinolone 32 pharmacokinetics and pharmacodynamics; and 3) several excellent reports 33 documenting fluoroquinolone treatment failures in patients infected with an isolate 34 with low-level fluoroquinolone resistance (2). 35 The most common mechanism of fluoroquinolone resistance in clinical 36 isolates of S. enterica is mutation to the quinolone resistance determining regions 37 (QRDR) of the gyrA gene. This mutation is associated with ciprofloxacin MICs above 38 those found in wild-type strains (MIC >0.06 μg/ml) and nalidixic acid resistance 39 (MIC ≥32 μg/mL). Less frequently, fluoroquinolone resistance can occur following 40 mutation to the QRDR of gyrB or the topoisomerase genes, parC and parE, or by 41 acquisition of plasmid-mediated quinolone resistance genes, such as qnr or aac-6’-42 Ib-cr (2). Resistance via these latter pathways confers elevated MICs, but not 43

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resistance, to nalidixic acid, which was recommended as an indicator of 44 fluoroquinolone resistance for S. enterica by the CLSI prior to 2012. 45 In order to resolve this testing issue, the CLSI revised the ciprofloxacin, 46 levofloxacin and ofloxacin MIC breakpoints for Salmonella, to better reflect the wild-47 type distribution of Salmonella MICs to these antimicrobials (Table 1) (3). The 48 European Committee on Antimicrobial Susceptibility Testing (EUCAST) 49 independently published Salmonella-specific MIC breakpoints for ciprofloxacin and 50 the U.S. Food and Drug Administration (FDA) revised the ciprofloxacin MIC 51 breakpoint to match those of the CLSI, but for S. enterica ser. Typhi alone (Table 1). 52 CLSI established Salmonella disk diffusion breakpoints for ciprofloxacin (3), but 53 levofloxacin and ofloxacin disk breakpoints have not yet been established, although 54 these have been proposed by Sjolund-Karlsson and colleagues (4, 5). 55 Several laboratories both in developing countries, where enteric fever is 56 endemic and fluoroquinolone resistance is prevalent but MIC testing is infrequently 57 performed, and developed countries where this disease is less common, have noted 58 difficulties in interpreting ciprofloxacin disk results (2). In order to address this 59 concern, both EUCAST and CLSI now recommend as an option the use of a 5 μg 60 pefloxacin disk test as a surrogate marker by which to detect fluoroquinolone 61 resistance in Salmonella (Table 1). Studies performed in Europe and the U.S., and 62 presented to the CLSI Microbiology Subcommittee on Antimicrobial Susceptibility 63 Testing in July 2014, demonstrated pefloxacin zone diameters of ≥24 mm to be an 64 excellent indicator of wild-type isolates (i.e. those without fluoroquinolone 65 resistance mechanisms). This report summarizes the studies performed in the U.S. 66

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to identify pefloxacin as a surrogate agent for the detection of fluoroquinolone 67 resistance in S. enterica. Performance of 5 other quinolone/fluoroquinolone and 68 nalidixic acid disks were also evaluated for their abilities to detect fluoroquinolone 69 resistance in Salmonella spp. 70 71 Methods 72

Isolates 73 A collection of 136 S. enterica, including 29 S. enterica ser. Typhi, 2 S. enterica 74 ser. Paratyphi A, 1 S. enterica ser. Paratyphi B, and 104 S. enterica of non-typhoid 75 serovars were included in these studies (Table 2). Fluoroquinolone resistance 76 mechanisms in these isolates were pre-determined by molecular methods, using 77 PCR for the specific targets (Skov et al, companion article). Twenty-four isolates had 78 no resistance genes, 37 isolates harbored a qnr gene, 1 an aac(6’)-lb-cr gene, and 45 79 had mutations in the QRDR region of the gyrA topoisomerase gene. An additional 29 80 isolates were not characterized for resistance mechanism, but were S. enterica ser. 81 Typhi with the ciprofloxacin-intermediate, nalidixic-acid resistant phenotype that is 82 typically associated with mutation to the QRDR. Twenty-five isolates were 83 ciprofloxacin susceptible (18%), 91 (65%) were ciprofloxacin intermediate and 20 84 (18%) were ciprofloxacin resistant (5) (Figure 1). The isolates with ciprofloxacin-85 intermediate MICs are the same isolates evaluated in the companion article by Skov 86 and colleagues. 87 Susceptibility Testing 88

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Disk diffusion was performed according to CLSI standards (3). Briefly, a 89 suspension of the test organism equivalent to a 0.5 McFarland was prepared in 90 saline from 3-5 well-isolated colonies of each isolate grown overnight on a Blood 91 Agar plate (BBL, BD, Sparks MD). Using a swab, the organism was inoculated onto a 92 Mueller Hinton Agar II plate (BBL) and disks were applied. Disks tested include 5 93 fluoroquinolones: ciprofloxacin (BD, 5 μg), enoxacin (BD, 10 μg), levofloxacin (BD, 5 94 μg), norfloxacin (BD, 10 μg) and ofloxacin (BD, 5 μg), and two quinolones: nalidixic 95 acid (BD, 30 μg) and pefloxacin (Oxoid, ThermoFisher, Reinach, Switzerland, 5 μg). 96 Plates were incubated for 18 hr at 35°C, and zones were read visually using 97 reflected light, by two independent readers. If discrete colonies were present within 98 the zone of inhibition, testing was repeated. If colonies within the zone were 99 observed a second time, the colony-free zone was measured. Using the same 100 inoculum suspension, isolates were tested by broth microdilution for ciprofloxacin 101 and levofloxacin MICs, on panels prepared in-house according to CLSI standards 102 using cation-adjusted Mueller Hinton broth (Difco, BD). Ciprofloxacin and 103 levofloxacin concentrations spanned a range from 0.008 μg/ml – 16 μg/ml. Quality 104 control was assessed using Escherichia coli ATCC 25922, Pseudomonas aeruginosa 105 ATCC 27853, Staphylococcus aureus ATCC 29213, and Enterococcus faecalis ATCC 106 29212 (MIC only). The quality control results for all MIC and disk diffusion tests 107 were within acceptable quality control ranges (3). 108 109 The precision of pefloxacin disk results across three manufacturers of disk 110 was evaluated, using 72 isolates with ciprofloxacin MICs in the intermediate range, 111

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as these isolates are the most problematic for laboratory testing (2). Disk diffusion 112 was performed as described above, and 5 μg pefloxacin disks from three 113 manufacturers (BD, MAST, Merseyside UK, and Oxoid) were placed on the same BBL 114 MHA plate. Plates were incubated for 18 hr and read as described above. 115 Data Analysis 116 Disk diffusion results were evaluated to determine if any of the 7 disks tested 117 could serve as a surrogate agent for the detection of fluoroquinolone resistance in S. 118

enterica. For the purpose of this study, fluoroquinolone resistance was defined as 119 not-susceptible to ciprofloxacin (MIC ≥0.12 μg/ml), using the current CLSI 120 breakpoints for Salmonella (3). The definition for the CLSI intermediate category 121 implies clinical efficacy when higher than normal dosage of a drug can be used (3), 122 however, isolates with intermediate MICs (0.12 – 0.5 μg/ml) were lumped together 123 with those demonstrating resistant MICs because there are no data to indicate 124 ciprofloxacin monotherapy is efficacious against isolates with ciprofloxacin MICs in 125 the intermediate range (0.12 to 0.5 μg/ml), regardless of dose (2). Therefore, the 126 breakpoint for each of the 7 “surrogate” agents was defined as the zone diameter 127 value that most reliably differentiated Salmonella isolates that were susceptible vs. 128 not-susceptible to ciprofloxacin, based on the assumption that ciprofloxacin MICs 129 determined by BMD could reliably differentiate fluoroquinolone susceptible from 130 fluoroquinolone not-susceptible isolates. Performance of 3 disks (ciprofloxacin, 131 nalidixic acid and pefloxacin) to act as surrogate agents for levofloxacin MIC was 132 also evaluated, in the same manner. Very major errors (VME, i.e. number of false 133 susceptible isolates / total number of ciprofloxacin not-susceptible isolates), and 134

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major errors (ME, i.e. number of false not-susceptible / total number of 135 ciprofloxacin susceptible isolates) were calculated. Overall categorical agreement 136 (susceptible or not-susceptible) was also calculated. 137 In addition, VME, ME and minor errors (mE, i.e. one result is intermediate 138 and the other is either susceptible or resistant) were calculated in the traditional 139 manner, using the current Enterobacteriaceae susceptible, intermediate and 140 resistant breakpoints for each disk surrogate agent, with the exception of ofloxacin 141 and levofloxacin, where proposed Salmonella disk diffusion breakpoints were used 142 (4), and pefloxacin, for which no intermediate breakpoint exists. 143 144 Results 145

Evaluation of a surrogate disk based on ciprofloxacin MIC results 146 Scattergrams for each of the 7 disks tested, as compared to ciprofloxacin 147 MICs, are presented in the Supplemental Figure. VME and ME calculations, using a 148 susceptible vs. not-susceptible definition as described in the methods section, are 149 presented in Table 3. No VME were noted for ciprofloxacin (Table 3), and 8% ME 150 were observed. It should be noted that only 25 ciprofloxacin-susceptible isolates 151 were tested and that 8% ME represents 2 isolates. One ME was for an isolate with a 152 QRDR mutation but ciprofloxacin MIC of 0.06 μg/ml and zone of 27 mm. This is the 153 only isolate in the study where a ciprofloxacin-susceptible MIC was found in an 154 isolate with a fluoroquinolone resistance mechanism. The second ME was for an 155 isolate with no resistance mechanism and ciprofloxacin MIC of 0.03 μg/ml and zone 156 of 30 mm. 157

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When evaluated in the traditional manner to include intermediate 158 breakpoints, 0 VME, 0 ME, and 11.0% mE (15 isolates) were observed between 159 ciprofloxacin disk and ciprofloxacin MIC results. The majority of these mEs (n=11) 160 were in isolates with a ciprofloxacin MIC of 1.0 μg/ml and ciprofloxacin disk 161 diffusion zones in the intermediate range (21-30 mm, Supplemental Figure 1). 162 The ofloxacin and levofloxacin disks were evaluated using proposed not- 163 susceptible disk breakpoints of ≤24 mm and ≤27 mm (4), respectively. No VME and 164 one ME (4.0%) were noted for both ofloxacin and levofloxacin (Table 3). Both ME 165 were for the same isolate with a ciprofloxacin MIC of 0.03 μg/ml and ofloxacin zone 166 of 24 mm and levofloxacin zone of 27 mm; this isolate had no resistance mechanism. 167 When ofloxacin and levofloxacin disk results were evaluated against ciprofloxacin 168 MIC to include the intermediate category, 11.0% and 11.8% mEs were noted, 169 respectively (Table 4). As was the case for ciprofloxacin, the majority of these mE 170 were for isolates that were intermediate by the ofloxacin or levofloxacin disk result 171 but a ciprofloxacin resistant MIC of 1.0μg/ml (Supplemental Figures 3 and 4). 172 Enoxacin and norfloxacin disks demonstrated 53.1% and 90.9% VME, 173 respectively, when evaluated using CLSI Enterobacteriaceae susceptible vs. not 174 susceptible breakpoints for these antimicrobials; no ME were noted (Table 3). If 175 susceptible, intermediate and resistant breakpoints were considered, the number of 176 VME was 25% and 50%, respectively (Table 4). However, a Salmonella-specific not-177 susceptible breakpoint could be assigned to enoxacin and norfloxacin as ≤23 and 178 ≤27 mm, respectively. These Salmonella-specific breakpoints result in VME rates of 179

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0.0% (enoxacin) and 0.9%, (norfloxacin) and ME rates of 4.0% and 8.0% for these 180 two surrogate disks (Table 3). 181 By the current CLSI resistant breakpoints, nalidixic acid testing does not 182 detect all mechanisms of fluoroquinolone resistance in S. enterica (3). However, we 183 interpreted nalidixic acid disk results as susceptible vs. not-susceptible (vs 184 ciprofloxacin MIC) to determine if this approach would improve performance of 185 nalidixic acid as a surrogate agent for detection of fluoroquinolone resistance. When 186 data were analyzed in this manner, 1.8% (n=2) VME and 20% (n=5) ME were noted 187 (Table 3). Both VME were in isolates with qnr resistance mechanisms, and 188 ciprofloxacin MICs of 0.12 – 0.5 μg/ml. In contrast, by the current CLSI nalidixic acid 189 susceptible, intermediate and resistant disk breakpoints 66.2% mE were observed 190 (Table 4). Of these mE, 84/90 (78%) were in isolates with a ciprofloxacin 191 intermediate MIC and a nalidixic acid-resistant zone which is the phenotype of the 192 QRDR fluoroquinolone resistance mechanism, although 29 of the 90 mE were in 193 isolates with plasmid-mediated quinolone resistance mechanisms (not shown). For 194 several isolates with ciprofloxacin intermediate MICs, discrete colonies within the 195 nalidixic acid zone of inhibition were observed, but these did not impede reading of 196 nalidixic acid zones, as even when these colonies were ignored, zone measurements 197 were interpreted as resistant (Figure 2a). 198 Pefloxacin disk results, interpreted by CLSI and EUCAST susceptible vs. not 199 susceptible breakpoints for Salmonella, yielded no VME and 12% (n=3) ME, as 200 compared to ciprofloxacin MICs. (Tables 3 and 4) One of the ME was in the isolate 201 with a QRDR mutation and ciprofloxacin susceptible MIC of 0.06 μg/ml and zone of 202

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21 mm. Similar to what was seen with nalidixic acid, for 47% of isolates with 203 ciprofloxacin MICs that were not susceptible, discrete colonies were observed 204 within the pefloxacin zone of inhibition (Figure 2b). We did not observe colonies 205 within the zone of inhibition for any of the fluoroquinolones evaluated, for this 206 collection of isolates. 207 When applying susceptible vs. not susceptible breakpoints, performance of 208 four of the seven surrogate disks (ciprofloxacin, pefloxacin, ofloxacin, and 209 levofloxacin) was equivalent (Table 3). In order to determine if one disk would 210 better separate ciprofloxacin susceptible from not-susceptible isolates (based on 211 ciprofloxacin MICs), the percentage of isolates with zone sizes near the proposed 212 breakpoint for each agent, arbitrarily chosen as 3 mm on either side of the 213 breakpoint, was calculated (Shown in Supplemental Figures). Twenty-six percent of 214 isolates included in the study had a zone size within 3 mm of the ofloxacin 215 breakpoint (zone sizes of 22 to 27 mm), and 30% within 3 mm of the levofloxacin 216 breakpoint (25 to 30 mm). In contrast, 16.9% of isolates had zone sizes within 3 mm 217 of the ciprofloxacin breakpoint (28 to 33 mm) and only 15.4% of isolates had a zone 218 size within 3 mm of the pefloxacin (21 to 26 mm). 219 220 Evaluation of a surrogate disk based on levofloxacin MIC results 221 No Salmonella levofloxacin disk breakpoints have been formally established, 222 and yet levofloxacin is commonly used in many U.S. hospitals as the formulary 223 fluoroquinolone. Performance of the proposed levofloxacin disk breakpoints for S. 224

enterica is reported elsewhere (4, 5). We therefore assessed the performance of 225

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ciprofloxacin, nalidixic acid and pefloxacin disks as potential surrogate agents for 226 the detection of levofloxacin susceptible vs. not-susceptible MICs. Ofloxacin, 227 enoxacin and norfloxacin were not included in this analysis due to poor 228 performance with ciprofloxacin. Categorical agreement between the ciprofloxacin 229 disk and levofloxacin MIC was 97.8%. One VME was noted (0.9% of 112 not-230 susceptible isolates, Table 5), for a S. enterica ser. Typhi with a ciprofloxacin 231 susceptible disk zone of 31 mm but a levofloxacin intermediate MIC of 0.25 µg/mL. 232 This isolate had a ciprofloxacin susceptible MIC of 0.06 μg/mL and a mutation in the 233 QRDR. Two ME were noted (8.7%) in isolates with levofloxacin MICs of 0.12 μg/mL 234 but ciprofloxacin disk zones of 28 and 30 mm. A categorical agreement of 95.6% 235 was found between nalidixic acid disks and levofloxacin MICs which included 3 VME 236 (2.7% of 112 not-susceptible isolates) and 3 ME (13% of 24 susceptible isolates) 237 (Table 5). Two of the VME were for isolates with qnr and levofloxacin MICs of 0.25 238 μg/ml; the third was for a S. enterica ser. Typhi with no fluoroquinolone resistance 239 mechanism detected and ciprofloxacin MIC of 0.06 μg/ml but levofloxacin MIC of 240 0.25 μg/ml. Categorical agreement was 100% for pefloxacin disk as compared to 241 levofloxacin MIC (Table 5). 242 Performance of pefloxacin disks from three manufacturers 243 As pefloxacin disks are not available in the United States and have not been 244 evaluated by CLSI prior to inclusion in M100-S25 for detection of Salmonella 245 fluoroquinolone resistance, we evaluated the performance of pefloxacin from three 246 manufacturers using a subset of 72 isolates. While BD and MAST disks yielded 247 equivalent zones, zone sizes obtained by Oxoid disks were on average 1.9 mm 248

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(standard deviation, 0.96) and 1.8 mm (standard deviation 0.66) larger than zones 249 determined using BD and MAST disks (p<0.0001). However, this difference did not 250 change the susceptible vs. not-susceptible interpretation for any of the 72 isolates 251 tested. 252 253 Discussion 254 This study evaluated the performance of 7 surrogate disks for the detection 255 of fluoroquinolone resistance in S. enterica. We demonstrated excellent performance 256 for ciprofloxacin and pefloxacin disks at differentiating ciprofloxacin-susceptible vs. 257 not-susceptible S. enterica. ME rates were higher than the typically accepted <3%, 258 when testing a random selection of organisms, however it should be noted that ME 259 rates are inflated in the present study due to the inclusion of few (n=25) susceptible 260 isolates. Levofloxacin and ofloxacin disks also performed well, with few VME and 261 ME. However, many isolates had zone sizes near the breakpoint for these two agents 262 (Supplemental Figures), which was of concern because disk diffusion is generally 263 accepted as precise to only ± 2 mm. This variability is related to disk lot and 264 manufacturer, media lot and manufacturer, incubation temperature and time, and 265 reader proficiency, among other factors. A major limitation of the present study is 266 that only one manufacturer of MHA (BD) was used. To this point, studies performed 267 at the EUCAST Development Laboratory, Växjö, Sweden and Staten Serum Institute 268 for EUCAST during their evaluation of pefloxacin disks, noted a much larger 269 proportion of isolates with zones within 1 to 3 mm of the ciprofloxacin breakpoint 270 when using three manufacturers of MHA (R. Skov, companion article). As a result, 271

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EUCAST does not recommend ciprofloxacin as a surrogate agent for the detection of 272 fluoroquinolone resistance in S. enterica. We also evaluated enoxacin and 273 norfloxacin as potential surrogate agents, but these did not perform well, with an 274 unacceptable number of VME (Table 3). 275 It is important to note that no surrogate agent will detect all fluoroquinolone 276 resistance mechanisms. For example, isolates that acquire aac-6’-lb-cr as the sole 277 fluoroquinolone resistance determinant will test susceptible to pefloxacin, nalidixic 278 acid, ofloxacin and levofloxacin, among others (6). This is because aac-6’-lb-cr 279 acetylates the amino nitrogen on the R7 piperazinyl substituent found on some, but 280 not all, quinolones, including ciprofloxacin, norfloxacin and enoxacin (6, 7). This 281 resistance mechanism remains rare and may be coupled with other fluoroquinolone 282 resistance mechanisms, as was the case for the 1 isolate tested in the present study 283 (R. Skov, unpublished observations). Similarly, we observed a S. enterica ser. Typhi 284 isolate with documented mutation to the QRDR that tested repeatedly susceptible to 285 ciprofloxacin by broth microdilution. Nalidixic acid did not detect qnr mutations in 286 5.4% of the 37 isolates with this resistance mechanism studied herein. 287 Both nalidixic acid and pefloxacin are currently included in the CLSI M100-288 S25 document as surrogate agents for the detection of fluoroquinolone resistance in 289 Salmonella spp. Laboratories need not test both agents but rather evaluate whether 290 surrogate disks should be used (i.e. cannot perform ciprofloxacin MIC), and which 291 surrogate disk best meets their needs. Laboratories that have noted difficulties in 292 reading ciprofloxacin disks and are in countries where pefloxacin disks are available 293 may opt to use pefloxacin disks as a surrogate agent for the detection of 294

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ciprofloxacin resistance. We noted several colonies within the zone of inhibition for 295 both pefloxacin and nalidixic acid; these should not be ignored, as these isolates all 296 harbored ciprofloxacin-intermediate MICs. The most common resistance 297 mechanism for typhoidal Salmonella remains QRDR mutation to gyrA, which is 298 readily detected by nalidixic acid disk testing (2). As such, laboratories in countries 299 where typhoid is endemic and that have noted success with nalidixic acid may opt to 300 continue to test with this agent. If this approach is taken, interpretation of nalidixic 301 acid disk zones as susceptible vs. not-susceptible (rather than susceptible, 302 intermediate and resistant) would be recommended, because some isolates will 303 yield ciprofloxacin MICs in the intermediate range and nalidixic acid resistant zones, 304 the classic phenotype of isolates with QRDR mutation (2) (Table 3 and Table 4). 305 However, laboratories that choose to continue testing with nalidixic acid must be 306 cognizant this disk will over-call resistance, as demonstrated by the 20% ME rate 307 (n=5 isolates). Furthermore, laboratories testing non-typhoidal Salmonella isolates 308 wherein plasmid mediated quinolone resistance is more common must be aware 309 that nalidixic acid will not detect such resistance mechanisms. 310 Laboratories in the U.S. and other developed countries may consider 311 performing an MIC test for ciprofloxacin or levofloxacin (depending on the agent to 312 be prescribed). However, no commercial MIC susceptibility test panels produced in 313 the U.S. contain ciprofloxacin or levofloxacin concentrations low enough to allow 314 use of the current CLSI susceptible breakpoints. We recently demonstrated that 315 Etest performs well compared to BMD at differentiating ciprofloxacin- and 316 levofloxacin-susceptible vs. not-susceptible isolates (5). However, U.S. laboratories 317

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that desire to use Etest for S. enterica with the current CLSI Salmonella breakpoints 318 would need to perform a verification study prior to implementation for clinical 319 testing, as this product is not FDA-cleared for use with these CLSI breakpoints. Since 320 S. enterica that require susceptibility testing (e.g. typhoid serovars or isolates from 321 extra-intestinal specimens) are infrequently encountered in most U.S. clinical 322 laboratories, investing the resources to perform such studies is likely not cost-323 effective. Pefloxacin disks are not available in the U.S., and are unlikely to become 324 available in the near future. Pefloxacin is not FDA-cleared for therapeutic use, which 325 precludes sale of pefloxacin disks without appropriate clinical trial data supporting 326 the performance of this test. Ciprofloxacin disk test may be a suitable alternative for 327 differentiating ciprofloxacin and levofloxacin susceptible vs non-susceptible isolates 328 of S. enterica for U.S. laboratories, if MIC tests are not available. If a laboratory opts 329 for the use a surrogate drug (Table 3), the results should be reported as susceptible 330 or resistant to ciprofloxacin or levofloxacin on the patient report. Results from the 331 surrogate agent tested (other than ciprofloxacin or levofloxacin) should not be 332 reported, as this could imply that the surrogate might be used for therapy. Reporting 333 results of surrogate agents is not recommended for other surrogate agents (eg: 334 cefoxitin for detection of mecA-mediated resistance in Staphylococcus aureus (2)). 335

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References 336 337 1. World Health Organization. 2003. Background document: The diagnosis, 338 treatment and prevention of typhoid fever. In Communicable Diseases 339 Surveillance and Response Vaccines and Biologicals. World Health 340 Organization, Geneva, Switzerland. 341 2. Humphries RM, Fang FC, Aarestrup FM, Hindler JA. 2012. In vitro 342 susceptibility testing of fluoroquinolone activity against Salmonella: recent 343 changes to CLSI standards. Clinical Infectious Diseases 55:1107-1113. 344 3. CLSI. 2014. Performance Standards for Antimicrobial Susceptibility Testing; 345 Twenty-Fourth Informational Supplement, M100 S24. Clinical Laboratory 346 and Standards Institute, Wayne, PA. 347 4. Sjolund-Karlsson M, Howie RL, Crump JA, Whichard JM. 2014. 348 Fluoroquinolone susceptibility testing of Salmonella enterica: detection of 349 acquired resistance and selection of zone diameter breakpoints for 350 levofloxacin and ofloxacin. Journal of Clinical Microbiology 52:877-884. 351 5. Deak E, Hindler JA, Skov R, Sjolund-Karlsson M, Sokovic A, Humphries 352

RM. 2015. Performance of Etest and Disk Diffusion for Detection of 353 Ciprofloxacin and Levofloxacin Resistance in Salmonella enterica. Journal of 354 Clinical Microbiology 53:298-301. 355 6. Robicsek A, Strahilevitz J, Jacoby GA, Macielag M, Abbanat D, Park CH, 356 Bush K, Hooper DC. 2006. Fluoroquinolone-modifying enzyme: a new 357 adaptation of a common aminoglycoside acetyltransferase. Nature Medicine 358 12:83-88. 359 7. Domagala JM. 1994. Structure-activity and structure-side-effect 360 relationships for the quinolone antibacterials. Journal of Antimicrobial 361 Chemotherapy. 33: 685-706. 362 363 364 365

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Figure Legends 366 367 Figure 1. Distribution of ciprofloxacin MICs among S. enterica isolates studied. 368 Shaded box indicates isolates with MICs in the intermediate (0.12-0.5 μg/ml) range 369 by M100 S25 CLSI breakpoints. 370 371 Figure 2. Representative disk diffusion result for nalidixic acid (panel A) and 372 pefloxacin (panel B) demonstrating colonies within the zone of inhibition. 373 374 375

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1

Table 1. Current CLSI, EUCAST and FDA fluoroquinolone breakpoints for Salmonella spp. Antimicrobial Disk Diffusion (mm) MIC (μg/ml) S I R S I R Ciprofloxacin CLSI ≥31 21-30 ≤20 ≤0.06 0.12-0.5 ≥1.0 EUCAST -1 - - ≤0.06 - >0.06 FDA (S. Typhi only) ≥31 21-30 ≤20 ≤0.06 0.12-0.5 ≥1.0 Levofloxacin2 CLSI - - - ≤0.12 0.25-1.0 ≥2.0 Ofloxacin3 CLSI - - - ≤0.12 0.25-1.0 ≥2.0 Surrogate / Screening Agents For Detection of Fluoroquinolone Resistance Nalidixic Acid CLSI ≥19 14-18 ≤13 ≤16 - ≥32 Pefloxacin CLSI ≥24 - ≤23 - - - EUCAST ≥24 - <24 - - - 1Use 5 μg pefloxacin disk to detect resistance in Salmonella spp. 2 Disk zones proposed by Karlsson (mm): ≥28 susceptible; 19-27 intermediate; ≤18 resistant (4) 3 Disk zones proposed by Karlsson (mm): ≥25 susceptible; 16-24 intermediate; ≤15 resistant (4)

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Table 2. S. enterica isolates included in this study No. of isolates (% typhoidal) Resistance mechanism Ciprofloxacin Nalidixic Acid Range MIC50 MIC90 Range MIC50 MIC901 (0) aac(6’)-lb-cr 1.0 1.0 1.0 32 32 32 37 (0) qnr 0.12 – 1.0 0.5 1.0 4.0-32 32 32 45 (0) QRDR mutation 0.06 – 0.5 0.25 0.25 >128 >128 >128 29 (90) Not characterized 0.12 – 16 0.5 16 128 - >128 >128 >128 24 (25) None ≤0.008 – 0.06 0.015 0.03 2 – 16 4 16

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Table 3. Performance of surrogate disks for the differentiation of ciprofloxacin S vs. ciprofloxacin not susceptible MICs in Salmonella spp.1 Surrogate Agent and not-susceptible breakpoint2 VME (%) ME (%) CA (%) Ciprofloxacin ≤30mm 0.0 8.0 98.5 Levofloxacin ≤27 mm3 0.0 4.0 99.3 Ofloxacin ≤24 mm3 0.0 4.0 99.3 Nalidixic Acid ≤18 mm 1.8 20.0 94.9 Enoxacin ≤17 mm ≤23 mm4 53.1 0.0 0.0 4.0 56.6 99.3 Norfloxacin ≤16 mm ≤27 mm4 90.9 0.9 0.0 8.0 25.7 97.8 Pefloxacin ≤23 mm 0.0 12.0 97.8 1111 isolates ciprofloxacin intermediate or resistant; 25 isolates ciprofloxacin susceptible 2Current CLSI not-susceptible disk breakpoints for Enterobacteriaceae (nalidixic acid, enoxacin, norfloxacin) or Salmonella (ciprofloxacin) were used 3 Intermediate (corresponds to not-susceptible) breakpoint proposed by Karlsson and colleagues (4) 4 Alternative not-susceptible breakpoint, proposed in this study VME, very major errors; ME, major errors; CA, categorical agreement

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Table 4. Performance of surrogate disks as compared to ciprofloxacin susceptible, intermediate, and resistant MICs in Salmonella spp.1 Surrogate Disk VME (%) ME (%) mE (%) CA (%) Ciprofloxacin 0.0 0.0 11.0 89.0 Levofloxacin 0.0 0.0 11.8 88.2 Ofloxacin 0.0 0.0 11.0 89.0 Nalidixic Acid 0.0 4.0 66.2 33.1 Enoxacin 25.0 0.0 47.8 48.5 Norfloxacin 50.0 0.0 72.1 20.6 Pefloxacin 0.0 12.0 - 97.8 120 isolates ciprofloxacin resistant; 91 isolates ciprofloxacin intermediate; 25 isolates ciprofloxacin susceptible VME, very major errors; ME, major errors; mE, minor errors; CA, categorical agreement on N

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Table 5. Performance of surrogate disks for the differentiation of levofloxacin S vs. levofloxacin not susceptible MICs in Salmonella spp.1 Surrogate Disk VME (%) ME (%) CA (%) Ciprofloxacin 0.9 8.7 97.8 Nalidixic Acid 2.7 13.0 95.6 Pefloxacin 0.0 0.0 100 120 isolates levofloxacin resistant; 92 isolates levofloxacin intermediate; 24 isolates levofloxacin susceptible VME, very major errors; ME, major errors; CA, categorical agreement

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