Use of Faropenem as an Indicator of Carbapenemase Activity

JCM Accepts, published online ahead of print on 10 April 2013 J. Clin. Microbiol. doi:10.1128/jcm.00720-13 Copyright 2013, American Society for Microbiology. All Rights Reserved. 1 2 Use of Faropenem as an Indicator of Carbapenemase Activity in the Enterobacteriaceae 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Kathryn M. Day, a,b Rachel Pike, c Trevor G. Winstanley, d Clare Lanyon, b Stephen P. Cummings, b Muhammad W. Raza, a Neil Woodford, c John D. Perry a,b# Microbiology Department, Freeman Hospital, Newcastle upon Tyne, NE7 7DN, UK a ; School of Life Sciences, University of Northumbria, Newcastle upon Tyne, NE1 8ST, UK b ; Antimicrobial Resistance and Healthcare Associated Infections Reference Unit (AMRHAI), HPA Microbiology Services - Colindale, London, NW9 5EQ, UK c ; Department of Laboratory Medicine, Northern General Hospital, Sheffield, S5 7AU, UK d # Corresponding author. Telephone: +44 191 2231226; Fax: +44 191 2231224; E-mail: john.perry@nuth.nhs.uk. Running title: Faropenem for detection of carbapenemases 18 19 20 21 22 23 Keywords: β-lactamases, susceptibility testing, antimicrobial resistance mechanisms, carbapenems. 1

24 ABSTRACT 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 The aim of this study was to determine the ability of a disc susceptibility test using faropenem (10-µg) to predict carbapenemase activity in Enterobacteriaceae. A collection of 166 isolates of carbapenemase-producing Enterobacteriaceae (CPE) and 82 isolates of Enterobacteriaceae that produced other β-lactamases was compiled from diverse sources. Disc susceptibility testing was performed using CLSI / EUCAST methodology with discs of faropenem (10-µg), temocillin (30-µg) and four carbapenems (each 10-µg). A further prospective evaluation of the faropenem disc susceptibility test was performed using 205 consecutive isolates referred to a UK reference laboratory in parallel with molecular methods for carbapenemase detection. Of 166 isolates of CPE, 99% showed growth up to the edge of a 10-µg faropenem disc compared with only 6% of other β-lactamase producers (sensitivity: 99%, specificity: 94%). A double zone around 10-µg faropenem discs was frequently associated with OXA-48 producers. Of the carbapenems, the most useful agent was imipenem, where a zone diameter of 23 mm as a predictor of carbapenemase activity had a sensitivity of 99% and a specificity of 85%. No zone of inhibition around a temocillin 30-µg disc was a consistent feature of strains producing OXA-48 carbapenemase. For 205 isolates of Enterobacteriaceae referred to a UK reference laboratory, growth up to a 10-µg faropenem disc correctly identified 84 of 86 carbapenemase producers (98% sensitivity) with a specificity of 87%. Disc susceptibility testing using faropenem (10-µg) is a simple, convenient and highly predictive screening test for carbapenemase-producing Enterobacteriaceae. 45 2

46 INTRODUCTION 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 Carbapenems are relied upon as one of the few effective options for treatment of Enterobacteriaceae that produce extended-spectrum and/or AmpC β-lactamases. However, their utility is becoming compromised by the increasing occurrence of Enterobacteriaceae that produce carbapenemases (1, 2). Clinical laboratories must have access to simple phenotypic screening tests for the accurate detection of carbapenemaseproducing Enterobacteriaceae (CPE). A number of tests have been devised for detection of carbapenemases (3-8), but these are performed only if there is sufficient suspicion based on the results of antimicrobial susceptibility testing. However, inferring carbapenemase activity from an antibiogram is not always straightforward because some isolates of CPE appear susceptible to carbapenems while isolates without carbapenemases may appear resistant (2). Faropenem medoxomil is an oral penem antibiotic with efficacy for the treatment of community-acquired infections caused by Enterobacteriaceae, including strains with AmpC or extended spectrum ß-lactamases (9). In the first part of this study, we evaluated the effectiveness of disc susceptibility testing with faropenem as a means of predicting carbapenemase activity and compared it with four carbapenems using a collection of 248 Enterobacteriaceae with defined β- lactamases. In a second evaluation we examined the utility of the faropenem disc susceptibility test alone to predict carbapenemase activity in 205 consecutive isolates referred to a UK reference laboratory. 3

69 MATERIALS AND METHODS 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 Bacterial isolates. A collection of 248 Enterobacteriaceae were obtained from diverse international sources and all possessed β-lactamases that had been defined at a molecular level by reference laboratories and/or recognized experts in the field. A total of 166 were carbapenemase producers including; 115 with NDM-1 (Citrobacter freundii n = 5, other Citrobacter spp. n = 6, Enterobacter cloacae n = 27, E. coli n = 52, Klebsiella pneumoniae n = 22, Kluyvera sp. n = 1, Providencia rettgeri n = 2); fourteen with IMP (K. pneumoniae n = 11, E. coli n = 2, Klebsiella oxytoca n = 1); fifteen with KPC (K. pneumoniae n = 12, E. coli n = 1, E. cloacae n = 1, K. oxytoca n = 1); thirteen with OXA-48 (K. pneumoniae n = 9, E. coli n = 2, E. cloacae n = 2) and nine with VIM (K. pneumoniae n = 5, E. coli n = 3, E. cloacae n = 1). Most of the carbapenemaseproducers, co-produced extended-spectrum ß-lactamase (ESBL) or AmpC β-lactamase (AmpC), but these are not documented here for the sake of clarity. Eighty-two isolates were non-carbapenemase producers, of which 60 produced ESBLs including; 25 isolates producing ten varieties of CTX-M (K. pneumoniae n = 14, E. coli n = 5, K. oxytoca n = 1, P. mirabilis n = 3, E. cloacae n = 1, E. aerogenes n = 1); 23 isolates producing nine varieties of SHV (K. pneumoniae n = 16, E. coli n = 2, K. oxytoca n = 1, E. cloacae n = 3, Salmonella sp. n = 1); and 12 isolates all producing distinct varieties of TEM (E. coli n = 7, K. pneumoniae n = 3, E. cloacae n = 1, Proteus mirabilis n = 1). Nineteen Enterobacteriaceae producing acquired AmpC enzymes included four with ACC-1 (K. pneumoniae n = 2, P. mirabilis n = 1, Salmonella livingstone n = 1), five with CMY (P. mirabilis n = 4, Salmonella sp. n = 1), five with DHA-1 (all K. pneumoniae), two with 4

92 93 94 FOX (K. pneumoniae n = 1, K. oxytoca n = 1); and three with LAT (E. coli n = 2, K. pneumoniae n = 1). Three K1 hyper-producing K. oxytoca were also included as well as a control isolate of E. coli without β-lactamases (NCTC 10418). 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 Disc susceptibility testing. Each of the 248 isolates was sub-cultured on to chromid CPS agar (biomérieux, Basingstoke, UK) and incubated for 18 h in air. Colonies were then suspended in sterile saline (0.85 %) to a density equivalent to 0.5 McFarland units using a densitometer (Densimat; biomérieux). A dipped swab was then used to inoculate a Mueller-Hinton agar plate (Oxoid, Basingstoke, UK). Faropenem powder was kindly supplied by biomérieux, La Balme-les Grottes, France. Ten milligrams of powder (100 % potency) was dissolved in 10 ml of sterile deionised water and 10 µl of this solution was added to blank paper discs to give a final load of 10-µg faropenem. One of these discs was added to each of the inoculated plates along with the following discs obtained from Oxoid: meropenem (10-µg), doripenem (10-µg), imipenem (10-µg), ertapenem (10-µg) and temocillin (30-µg). Culture plates were incubated for 18 h at 37 C in air and zone diameters were recorded. All tests were performed in duplicate on separate occasions. E. coli (NCTC 10418) was included with every batch of tests during the course of the study to ensure that reproducible results were obtained. MIC testing. For the four carbapenems listed above, powders of known potency were obtained from their respective manufacturers to determine MICs by agar dilution. Each was incorporated into Mueller-Hinton agar to produce a concentration range of 0.125 64 mg/liter for each agent. Faropenem was incorporated into Mueller-Hinton agar 5

115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 plates in identical fashion. All of the 248 test isolates were inoculated at 10 4 cfu/spot using a multipoint inoculator and plates were incubated for 18 h at 37 C in air before determination of MICs. Antimicrobial-free plates were inoculated in parallel and E. coli NCTC 10418 was inoculated on every batch of plates as a control. Further validation of disc susceptibility testing with isolates referred to a UK reference laboratory. During December 2012, 205 isolates of Enterobacteriaceae were referred to the HPA s AMRHAI Reference Unit from clinical laboratories across the UK. Isolates were typically referred for investigation of suspected carbapenemase activity or for investigation of an unusual susceptibility pattern. These isolates were subjected to MIC testing using a standard agar dilution methodology (10), with 30 different antimicrobials including 23 β-lactam antibiotics with or without β-lactamase inhibitors. Isolates that showed any evidence of possible carbapenemase activity were further evaluated using molecular assays targeting common carbapenemases (specifically, KPC and OXA-48 non-metallo-enzymes and IMP, NDM and VIM metallo-enzymes). All isolates were also subjected to susceptibility testing with a 10-µg faropenem disc only, using the method described above. E. coli (NCTC 10418) was included with every batch of tests. 6

138 RESULTS 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 Evaluation of disc susceptibility testing with a collection of 248 isolates of Enterobacteriaceae with defined β-lactamases. For some disc susceptibility tests a zone of inhibition was apparent with colonies growing inside the zone of inhibition. Unless stated otherwise, all zone diameters recorded here take account of the colonies growing inside the zones when measurements were made i.e. the zone of inhibition corresponds to a clear zone of inhibition without any colonies. The inhibition zone diameters (and MICs) obtained for four carbapenems against E. coli NCTC 10418 were all within acceptable limits of published values (11). The MIC of faropenem for this strain was 0.25 mg/liter and this corresponded to an inhibition zone diameter of 26 +/- 1 mm, which was consistently achieved in every batch of tests using CSLI / EUCAST methodology (11). The average zone diameters for faropenem and four carbapenems against β-lactamase producers are shown in Table 1. A total of 152/166 isolates of CPE (92%) showed confluent growth up to the edge of the 10-µg faropenem disc (i.e. zone diameter = 6 mm). These included 14/15 isolates that produced KPC and all isolates that produced IMP (n = 14), VIM (n = 9) or NDM-1 (n = 115) carbapenemase. Twelve of 13 isolates that produced OXA-48 and one isolate that produced KPC-3 generated a zone of inhibition to faropenem, but with large numbers of colonies inside the zone growing up to the edge of the disc. Only one isolate of CPE (an OXA-48 producer) had a clear zone of inhibition without colonies growing up to the disc when tested against faropenem 10-µg. 7

160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 In contrast, only 5/82 (6%) non-cpe isolates showed growth up to the edge of faropenem discs. All five were Klebsiella pneumoniae and included two ESBL producers (one with CTX-M-3 and one with SHV-11 plus CTX-M-15 plus porin loss) and three isolates with DHA-1 acquired AmpC β-lactamase. The four carbapenems generated mean zone diameters of 10.9 16.7 mm for CPE (Table 1). Figure 1 shows the sensitivity and specificity for all possible zone diameters as a predictor of carbapenemase activity where a zone diameter of X mm was used to infer the presence of a carbapenemase. These data show that a zone diameter of 6 mm (i.e. no inhibition) for faropenem predicted carbapenemase activity with a sensitivity of 99% and a specificity of 94%. The next most useful agent was imipenem, where a zone diameter of 23 mm as a predictor of carbapenemase activity had a sensitivity of 99% and a specificity of 85%. MICs of faropenem were 64 mg/liter for all isolates with VIM, IMP and KPC and 99% of isolates with NDM-1 carbapenemase. In contrast, 32 mg/liter was sufficient to inhibit the growth of all but four isolates of non-cpe (range 0.5 - > 64 mg/liter). MICs for isolates with OXA-48 enzyme ranged from 2 32 mg/liter. A faropenem MIC of 16 mg/liter was indicative of carbapenemase production with a sensitivity of 96% and a specificity of 91%. Fifty-seven isolates of CPE (34%) showed no zone of inhibition when tested against a 30-µg temocillin disc, including all producers (n = 13) of OXA-48 enzyme. One (1.2%) non-cpe isolate (an isolate of K. pneumoniae with CTX-M-3) produced no zone of inhibition to temocillin. Thus, high-level resistance to temocillin has high 8

183 184 185 specificity (99%), but low sensitivity (34%) for detection of carbapenemase producers and was a consistent feature of those producing OXA-48. Figure 2 illustrates the typical antibiogram of some carbapenemase producers and a carbapenemase-negative isolate. 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 Evaluation of faropenem discs vs. 205 isolates of Enterobacteriaceae referred to a UK reference laboratory. Of 205 isolates, 86 (42%) were confirmed as carbapenemase producers using molecular methods. A range of species were found with carbapenemases including K. pneumoniae (n = 50), K. oxytoca (n = 14), E. coli (n = 9), E. cloacae (n = 6), E. aerogenes (n = 2) Providencia stuartii (n = 2), P. rettgeri (n = 1), C. freundii (n = 1) and Kluyvera ascorbata (n = 1). Table 2 shows the enzyme types that were encountered and the presumptive phenotype of all other isolates for which there was no evidence of carbapenemase activity. The presumptive phenotype of noncarbapenemase producers was derived from analysis of the antibiogram. Table 2 also shows details of inhibition zones to faropenem 10-µg generated by all 205 isolates. A total of 79/86 isolates of CPE (92%) showed confluent growth up to the edge of a 10-µg faropenem disc (i.e. zone diameter = 6 mm). These included 32/34 isolates with KPC and all isolates with VIM (n = 27) or NDM-1 (n = 15) carbapenemase genes. Four isolates with OXA-48 and a single isolate with KPC produced a double zone of inhibition with colonies growing up to the faropenem disc. Only 2/86 isolates of CPE (one with OXA-48 and one with KPC) had a clear zone of inhibition (without colonies growing up to the disc) when tested against faropenem 10-µg. All isolates with OXA-48 enzyme, showed high-level resistance to temocillin in MIC tests (MIC 64 mg/liter). Only 16 of 9

206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 119 isolates (13%) with other resistance mechanisms were able to grow up to the edge of a 10-µg faropenem disc and ten of these showed evidence of AmpC production plus decreased permeability to β-lactam agents. For this second collection of Enterobacteriaceae, a zone diameter of 6 mm (i.e. no inhibition) for faropenem predicted carbapenemase activity with a sensitivity of 98% and a specificity of 87%. As before, E. coli (NCTC 10418) consistently produced an inhibition zone diameter of 26 +/- 1 mm when tested with a 10-µg faropenem disc in every batch of tests. DISCUSSION The spread of carbapenemases is currently the most pressing resistance issue in Gramnegative bacteria. Rapid detection of carbapenemase producers in clinical laboratories is essential for infection control, epidemiology and, more contentiously, as a guide for treatment, but this goal is confounded by the absence of simple phenotypic tests that can reliably detect all enzyme types in the range of species affected. We have shown that the inclusion of a faropenem 10-µg disc in routine susceptibility tests on Enterobacteriaceae may provide a very good early indication of isolates that produce a carbapenemase. This enables prompt confirmation by other phenotypic or genotypic tests. An advantage of using faropenem is that most isolates of CPE grow up to the disc, and consequently they are easily detected without measurement of inhibition zone diameters. The appearance of a double zone of inhibition with colonies growing up to the disc was characteristic of 16 of 21 isolates with OXA-48. 10

229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 A number of studies have examined disc susceptibility testing with carbapenems as a means of predicting carbapenemase activity, with particular focus on KPCcarbapenemase (12-15). Based on the available evidence, zone diameter cut-off values for detecting putative carbapenemase producers were proposed by a EUCAST subcommittee for detection of resistance mechanisms in December 2012 (16). They recommend that investigations of carbapenemase activity should be performed on Enterobacteriaceae with inhibition zone diameters of <23 mm for imipenem or <25 mm for ertapenem or meropenem. Applying these criteria to our collection of 248 Enterobacteriaceae resulted in a sensitivity and specificity of 99% and 69% for ertapenem, 98% and 88% for imipenem, 99% and 79% for meropenem, respectively. The faropenem disc test therefore showed an equivalent sensitivity (99%) and superior specificity (94%) than any of these recommended screening indicators of potential carbapenemase activity. When applied to 205 consecutive isolates referred to a UK reference laboratory, the test retained good sensitivity (98%) but lower specificity (87%). This lower specificity is perhaps not surprising as the reference laboratory often receives atypical isolates and the information accompanying 85 of these isolates mentioned an unusual antibiogram among the reasons for referring the isolate. Glupczynski et al. (1) noted that high-level resistance to temocillin (MIC 256 mg/liter) was a valuable and specific phenotypic tool for the presumptive detection of OXA-48 and this is supported by others (17, 18). The lack of any inhibition zone to temocillin 30-µg in this study was a highly specific marker for carbapenemase production - albeit with low sensitivity - and was a consistent feature of OXA-48 producers. 11

252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 Mushtaq et al. performed MIC testing of faropenem against a large collection of 847 cephalosporin-resistant Enterobacteriaceae (including 646 ESBL-producers) isolated from 16 UK laboratories and found that 95% were inhibited by 2 mg/liter (14). None of the isolates possessed carbapenemases and all isolates were inhibited by a faropenem concentration of 16 mg/liter. Of five isolates in this study with MICs of 16 mg/liter faropenem, all had a zone of inhibition to a faropenem 10-µg disc (range 8-13 mm). We conclude that inclusion of a faropenem 10-µg disc in routine susceptibility tests provides an excellent screening test for presumptive detection of Enterobacteriaceae that produce carbapenemases. It is possible that the local prevalence of particular β- lactamase producers may impact on the value of the test, for example 3/5 isolates that produced DHA-1 enzyme gave no zone of inhibition to faropenem and would be regarded as presumptive carbapenemase producers. It would therefore be very useful for this test to be validated with other collections of Enterobacteriaceae in different countries to further establish its utility. As faropenem is effectively hydrolyzed by carbapenemases it may also be a useful substrate for inclusion in a variety of other assay formats for rapid phenotypic detection of carbapenemases and differentiation from other types of β- lactamase (3, 4, 6, 7, 20). ACKNOWLEDGEMENTS The authors are most grateful to the following collaborators who generously shared isolates with characterized resistance mechanisms: Patrice Nordmann, Hôpital de Bicêtre, France; Gilles Zambardi, biomérieux, La Balme les Grottes, France; Enno Stürenburg, 12

275 276 277 278 Universitätsklinikum Hamburg-Eppendorf, Germany; and Jaroslav Hrabák, Plzen Charles University, Prague. This study was supported by internal funding only. The authors are grateful to biomérieux, La Balme les Grottes, France for the provision of faropenem powder and to Mast Laboratories, Bootle, UK, for providing sterile blank discs. 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 13

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388 389 390 391 392 393 394 395 396 Table 1. Mean inhibition zone diameters (mm) and ranges (mm) produced by six antimicrobials against Enterobacteriaceae (n = 248) with various β-lactamases. n Faropenem Ertapenem Imipenem Meropenem Doripenem Temocillin All CPE 166 6 (6-12) 10 (6-28) 17 (6-24) 14 (6-28) 15 (6-26) 10 (6-25) IMP 14 6 13 (6-21) 20 (17-23) 17 (9-23) 17 (11-22) 8 (6-15) KPC 15 6 10 (6-19) 13 (6-20) 13 (6-22) 13 (6-21) 13 (6-25) NDM-1 115 6 11 (6-22) 16 (6-24) 14 (6-22) 14 (6-22) 11 (6-25) OXA-48 13 6 (6-12) 10 (6-18) 19 (7-23) 17 (6-24) 18 (6-25) 6 VIM 9 6 16 (6-28) 16 (6-21) 17 (6-28) 16 (6-26) 7 (6-7) NON CPE 82 19 (6-30) 25 (6-36) 27 (16-36) 28 (12-36) 28 (13-35) 19 (6-28) ESBL 60 20 (6-29) 25 (6-36) 28 (16-33) 28 (12-36) 28 (14-35) 19 (6-28) K1 3 19 (15-26) 27 (24-32) 30 (24-36) 31 (29-32) 30 (30-31) 23 (15-28) AmpC 19 18 (6-30) 24 (6-34) 25 (18-30) 27 (15-33) 27 (13-34) 20 (10-28) 397 398 399 18

400 Table 2. Results of faropenem susceptibility testing against 205 consecutive isolates of Enterobacteriaceae referred to a UK reference laboratory. 401 No. of isolates among confirmed carbapenemase producers Carbapenemase Total No zone Double zone Inhibition Detected Sensitivity (%) KPC 34 32 1 1 33 97.1 NDM-1 15 15 0 0 15 100 NDM-1 & OXA-48 2 2 0 0 2 100 OXA-48 8 3 4 1 7 87.5 VIM 27 27 0 0 27 100 Total 86 79 5 2 84 98 Presumptive phenotype No. of isolates among carbapenemase non-producers Total No zone Double zone Inhibition False positives Average zone diameter (mm) AmpC 27 2 0 25 2 17.1 AmpC plus impermeability 29 8 2 19 10 11.1 ESBL 20 1 0 19 1 23.0 ESBL plus AmpC 3 0 0 3 0 15.8 ESBL plus AmpC plus impermeability 1 0 0 1 0 14.2 ESBL plus impermeability 18 0 0 18 0 17.9 K1 penicillinase 2 0 0 2 0 28.2 K1 penicillinase plus impermeability 2 0 0 2 0 11.6 Impermeability 3 1 0 2 1 10.5 Undefined 14 1 1 12 2 20.2 Total 119 13 3 103 16 17.0 402 403 404 19

405 406 407 408 Figure 1. Percentage sensitivity ( ) and specificity ( ) for zone diameters of value X, where X mm is used as a predictor of carbapenemase activity using 10-µg discs of: faropenem (FAR), ertapenem (ETP), imipenem (IPM), meropenem (MEM) and doripenem (DOR). 409 410 411 412 413 414 Downloaded from http://jcm.asm.org/ on April 4, 2019 by guest 20

415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 Figure 2. Susceptibility testing of Enterobacteriaceae against meropenem 10-µg (M), doripenem 10-µg (D), faropenem 10-µg (F), ertapenem 10-µg (E), temocillin 30-µg (T) and imipenem 10-µg (I). (1) E. coli with NDM-1; (2) E. coli with OXA-48 (note: colonies growing up to the faropenem disc); (3) K. pneumoniae with KPC-2 and (4) K. pneumoniae with CTX-M-15. 436 1 21

ETP % MEM FAR % 100 90 80 70 60 50 40 30 20 10 0 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 100 90 80 70 60 50 40 30 20 10 0 Zone diameter (mm) 100 90 80 70 60 50 40 30 20 10 0 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Zone diameter (mm) IPM % DOR % % 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 0 Zone diameter (mm) 100 90 80 70 60 50 40 30 20 10 0 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 100 90 80 70 60 50 40 30 20 10 Zone diameter (mm) 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Zone diameter (mm)

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