This is the accepted version of the review article: Rivera, Alba; Viñado, Belén; Benito, Natividad; [et al.]. «Recommendations of the Spanish Antibiogram Committee (COESANT) for in vitro susceptibility testing of antimicrobial agents by disk diffusion». Enfermedades infecciosas y microbiología clínica, Vol. 41, Issue 9 (November 2023), p. 571-576. DOI 10.1016/j.eimc.2022.04.015 This version is available at https://ddd.uab.cat/record/285155 under the terms of the license ref. EIMC-D-22-00168 REVISADO 1 Recommendations of the Spanish Antibiogram Committee (COESANT) for in 2 vitro susceptibility testing of antimicrobial agents by disk diffusion 3 4 Abstract 5 Disk diffusion is a well standardized method that provides reliable categorical results to guide 6 antimicrobial therapy in numerous types of infections. Based on the guidelines of the European 7 Committee on Antimicrobial Susceptibility Testing (EUCAST), which are widely implemented in 8 Spain, the Spanish Antibiogram Committee (COESANT) has drawn up recommendations for 9 antimicrobial selection by the disk diffusion technique, including selective reporting and its use 10 for the detection of resistance mechanisms. Factors affecting disk diffusion results, along with 11 advantages and shortcomings of the method, are also discussed. 12 13 Introduction 14 Disk diffusion is a conventional phenotypic method for antimicrobial susceptibility testing that 15 combines simplicity and cost-effectiveness. Even though automated antimicrobial systems are 16 now widely employed in many clinical microbiology laboratories, the disk diffusion method is 17 still in use, as it provides reliable qualitative results to guide antimicrobial therapy in numerous 18 types of infections. Rigorous compliance with the methodology, the use of high-quality reagents 19 (culture media and antibiotic-containing disks) together with an adequate quality control allow 20 accurate and reproducible results to be obtained.1–3 21 22 The method is based on the diffusion of a predefined amount of a given antimicrobial agent 23 contained in a paper disk or tablet, which is placed on the surface of an agar plate previously 24 inoculated with a standardized inoculum of the microorganism. As the drug diffuses, it creates 25 a gradient of concentration, leading to the formation of inhibition zones where the 26 concentration is sufficient to inhibit the growth of the inoculated microorganism. The inhibition- 27 zone diameters are classified into susceptibility categories according to regularly updated clinical 28 breakpoints.4,5 Several factors influence the zone diameters, which can be related to the drug 29 (disk content, diffusion rate, activity against the tested isolate), agar (depth, composition), 30 incubation conditions (temperature, duration, atmosphere) or microorganism (growth rate, 31 inoculum density). Disk diffusion methodology guidelines are provided by the CLSI (Clinical and 32 Laboratory Standards Institute) and EUCAST (European Committee on Antimicrobial 33 Susceptibility Testing), with some differences regarding the testing media for fastidious 34 organisms and the disk concentration for certain antimicrobial agents.6,7 Fortunately, CLSI and 35 EUCAST have created a joint disk-diffusion working group to develop standardized 36 recommendations for disk content selection, which will henceforth be applied to new 37 antimicrobial agents.8,9 38 39 For each bacterium-drug combination, clinical breakpoints for interpreting zone diameter are 40 commonly set by selecting an optimal disk content and evaluating the correlation between the 41 minimum inhibitory concentration (MIC) values obtained by reference methods and the 42 inhibition zone diameters.10,11 In 2019, EUCAST revised interpretative clinical categories, 43 redefining category I (susceptible, increased exposure) and establishing arbitrary "off-scale" 44 breakpoints for some species/agents corresponding to a zone diameter of S ≥ 50 mm, which 45 indicates that the microorganism may require a high exposure to the drug. 46 47 A relevant advantage of the disk diffusion technique is its flexibility in the choice of 48 antimicrobials as opposed to commercial systems in which both the antimicrobials and their 49 concentrations are chosen by the manufacturer. Thus, disk panels can be readily modified 50 according to the specific needs of each center. Moreover, the incorporation of new 51 antimicrobials into disk diffusion antibiograms is easier and less time-consuming compared with 52 commercial panels, which also tend to be costly.12 In contrast, disk diffusion is considered the 53 least expensive of all susceptibility methods. Another benefit is that growth visualization allows 54 the detection of inoculum adequacy, mixed cultures, heteroresistance and interactions between 55 antibiotics as synergy or antagonism effects, which is highly useful for the phenotypic detection 56 of resistance mechanisms. Moreover, interpretive reading of the antibiogram and education can 57 be an advantage of disk diffusion due to its easy adaptability. 58 59 One of the main reasons against the use of the disk diffusion method is that it does not provide 60 MIC values, which are used in pharmacokinetic/pharmacodynamic (PK/PD) models for 61 personalized antibiotic therapy, especially in critically ill patients.13 However, as occurs in disk 62 diffusion, MIC determination is inherently variable, influenced by biological factors (strain-to- 63 strain differences) and the type of assay (accepted variation of one doubling dilution), and 64 therefore MIC values cannot be considered as absolute.14 65 Although the qualitative results obtained by disk diffusion are considered adequate for most 66 infections, choosing the most suitable antibiotic treatment regimen for some types of infections 67 (e.g., endocarditis) or microorganisms (e.g., carbapenemase-producing Enterobacterales) 68 requires establishing MIC values.15 MIC determination can also be useful for the confirmation of 69 unexpected results, when the disk diffusion result falls within the newly defined EUCAST area of 70 technical uncertainty (ATU) or in cases where disk diffusion is unreliable (e.g. vancomycin and 71 staphylococci). 72 73 Another shortcoming is that although the method has been validated against the most common 74 bacteria, as stated by the international committees EUCAST and CLSI, it is not yet well 75 standardized against some fastidious and/or slow-growing microbes. It is worth noting that 76 ongoing EUCAST research on setting new disk breakpoints has led to the development of a new 77 disk diffusion method for rapidly growing anaerobic bacteria, and the breakpoints have been 78 published in the 2022 EUCAST document.5,16 79 80 Furthermore, the disk diffusion method is labor-intensive, not fully automated, and the 81 interpretation of the inhibition zone diameters is subject to inter-observer variation. The 82 implementation of equipment that allows automatic reading and interpretation of inhibition 83 zone diameters, such as Sirscan (i2a, Montpellier, France), ADAGIO (Bio-Rad, Marnes-la- 84 Coquette, France), or BIOMIC (Giles Scientific Inc., Santa Barbara, USA), has gone some way to 85 address these problems.17–19 This equipment incorporates an expert system to improve the 86 quality of interpretation and allows the storage and further management of data. Disk diffusion 87 could also benefit from automatization of the entire process, including inoculum preparation, 88 streaking of media plates, incubation, and reading, as demonstrated by Cherkaoui et al. using 89 the WASPLabTM system (Copan, Brescia, Italy).20 A study analyzing the most frequently 90 encountered pathogens in blood cultures has shown that this device could also allow early 91 reading (6-12 h).21 92 93 A recently introduced improvement of the disk diffusion method has been the standardization 94 of rapid susceptibility testing for bloodstream infections with the aim of shortening the 95 turnaround time. The EUCAST rapid method performed directly from positive blood culture 96 bottles provides reliable results within 4–8 h and so far has been validated for seven 97 pathogens.22 98 99 In 2020, the Spanish Antibiogram Committee (COESANT) and the Study Group on Mechanisms 100 of Action and Resistance to Antimicrobial Agents (GEMARA) from the Spanish Society of 101 Infectious Diseases and Clinical Microbiology (SEIMC) published recommendations for the 102 inclusion of antimicrobials and the selection of concentration ranges in automated systems 103 according to the clinical breakpoints and the epidemiological breakpoints (ECOFF) defined by 104 EUCAST.23 105 Similarly, the objective here is to provide recommendations on the antimicrobials to be studied 106 using the diffusion disk technique and suggestions for selective reporting. The usefulness of the 107 method for the detection of resistance mechanisms is also outlined. 108 109 General recommendations for antimicrobial susceptibility testing using the disk diffusion 110 method 111 The lists of antimicrobial agents to be tested and reported are shown in supplementary tables 112 S1-S11. Antimicrobials have been divided into five categories (A to E) along with 113 recommendations of testing and selective reporting (Table 1).23 These recommendations should 114 be adapted to each individual center according to the institutional formulary and local antibiotic 115 stewardship programs. Antimicrobial selection criteria are based on microbiological, clinical and 116 PK/PD data, as stated in the previous COESANT document.23 It should be noted that some 117 antimicrobial agents (category E) are used to detect resistance mechanisms or as surrogate 118 markers to extrapolate results for other agents and should not be reported. According to 119 EUCAST guidelines, the list of disk diffusion screening tests that can be used for these purposes 120 is included in Table 2.24 121 122 Susceptibility testing by the disk diffusion method is challenging for some drugs, such as colistin 123 and daptomycin, due to their molecular size and physico-chemical traits, and the results for 124 some anaerobes, Neisseria and Helicobacter are unreliable, in which case an MIC method should 125 be used (Table 3).15 There are also some drug-organism combinations for which both disk and 126 MIC EUCAST breakpoints are lacking. In these cases, EUCAST recommends using the PK/PD non- 127 species-related breakpoints only available as MIC values.25 EUCAST also recommends referring 128 to the epidemiologic cutoff (ECOFF) values to determine whether the MIC against the targeted 129 isolate is consistent with the wild type MIC distribution against the species. There may still be 130 cases in which the use of breakpoints defined by other organizations such as the Clinical and 131 Laboratory Standards Institute (CSLI), the Societe Francaise de Microbiologie (SFM) or the U.S. 132 Food and Drug Administration (FDA) could be an alternative when EUCAST breakpoints are 133 unavailable. In these cases, methodological recommendations of each organization regarding 134 disk content and testing media must be taken into account. 135 Additional recommendations for the detection of resistance mechanisms in the primary panel 136 by the disk diffusion method and other peculiarities of different bacterial groups are outlined 137 below. Suggested disk diffusion panels for Enterobaterales, Enterobacterales from urinary tract 138 infections (UTI), Pseudomonas spp., Staphylococcus spp., Streptococcus spp./Enterococcus spp., 139 Enterococcus spp. from UTI, Haemophilus influenzae/Haemophilus parainfluenzae and 140 Campylobacter jejuni/Campylobacter coli as well as the recommended media are shown in 141 Figure 1. 142 143 Enterobacterales 144 The analysis of antimicrobial resistance patterns and the observation of antibiotic interactions 145 is very useful for the detection of resistance mechanisms. Disk diffusion constitutes a suitable 146 technique for the detection of several beta-lactamases of clinical and epidemiological 147 importance, such as extended spectrum beta-lactamases (ESBLs), AmpC and carbapenemases. 148 Phenotypic confirmation by diffusion techniques is mainly based on the use of beta-lactamase 149 inhibitors and indicator beta-lactam drugs. One of the recommended methods for ESBL 150 detection is the double-disk synergy test, which can be easily incorporated into the primary disk 151 diffusion antibiogram. This method involves placing an amoxicillin-clavulanic acid (10 µg) disk at 152 a distance of 30 mm (center to center, the distance provided by several types of disk-dispensers) 153 from disks containing third and fourth generation cephalosporins (30 µg) used as beta-lactam 154 indicators. The distance may be reduced to 15-20 mm according to the inhibition zone 155 diameters. ESBL production is demonstrated if the inhibition zone of any of the indicators is 156 extended by the action of clavulanic acid.26,27 157 158 Phenotypic confirmation of ESBL can also be performed by the combination disk test using 159 cephalosporin disks (30 µg) with and without clavulanic acid (10 µg). An increase in the inhibition 160 zone diameter of ≥5mm for cephalosporin with clavulanic acid compared to cephalosporin alone 161 is considered a positive result. It is worth noting that the cephalosporin disk content initially 162 recommended by EUCAST for the detection of ESBLs (cefotaxime 30 µg and ceftazidime 30 µg) 163 differed from that used for standard susceptibility testing (cefotaxime 5 µg and ceftazidime 10 164 µg). In 2019, the EUCAST technical guidance on the use of the combination disk test to confirm 165 ESBLs in Enterobacterales was modified in favor of the concentration used in standard 166 susceptibility testing.28 Nevertheless, the recommendation for disk content in the double-disk 167 synergy technique currently remains unchanged. Detection of ESBLs in isolates co-producing 168 AmpC beta-lactamases may be challenging due to resistance to clavulanic acid, which can 169 mitigate the synergistic effect. Several approaches could be used to improve ESBL detection in 170 these cases, including the use of AmpC-stable fourth-generation cephalosporins such as 171 cefepime or incorporating cloxacillin (200-250 mg/L), an inhibitor of AmpC enzymes, into the 172 medium. 173 Similarly, the double-disk synergy test or the combination disk test can be performed to detect 174 AmpC beta-lactamases, using disks with AmpC inhibitors such as cloxacillin (500-750 µg) or 175 boronic acid (400-600 µg) and third generation cephalosporins as indicators. It should be noted 176 that boronic acid inhibitors are not specific for AmpC enzymes and also affect class A beta- 177 lactamases. Inducible AmpC beta-lactamase production can also be detected in a conventional 178 disk diffusion assay by the appearance of a flattening inhibition zone between beta-lactams 179 (such as third generation cephalosporins, aztreonam or piperacillin-tazobactam) and beta- 180 lactam inducers (such as imipenem, cefoxitin or amoxicillin-clavulanate). Additionally, the 181 appearance of scattered colonies near the edge of the inhibition zone of cefoxitin, cefotaxime, 182 ceftazidime and aztreonam has been described as a useful phenotypic indicator to differentiate 183 between plasmidic and chromosomal AmpC.29 184 185 Disk diffusion-based tests can also be performed to detect carbapenemase-producing 186 Enterobacterales. The most commonly used method is the combination disk test based on the 187 potentiation of the action of meropenem or imipenem in the presence of specific inhibitors of 188 each class of carbapenemases (boronic acid for class A carbapenemases, and dipicolinic acid or 189 EDTA for class B carbapenemases), in which the inhibition zones of the carbapenem with and 190 without the inhibitor are compared.26,27,30 Commercial kits are available that include cloxacillin 191 to differentiate between AmpC hyperproduction plus porin loss and carbapenemase- 192 production. For OXA-48-type carbapenemases, the only disk-based phenotypic marker is 193 temocillin resistance (zone diameter <11 mm), although it lacks specificity. The combination disk 194 method is not included in the primary disk diffusion panel, and is used for positively screened 195 isolates according to the zone diameter cut-off values for carbapenemase-producing 196 Enterobacterales. Therefore, the main drawback of this method is that it requires overnight 197 incubation and has been replaced by other techniques that provide faster results such as 198 biochemical tests, lateral flow immunoassays or molecular assays.27,30 199 200 Non-fermentative Gram-negative bacilli 201 For non-fermenting Gram-negative bacilli other than Pseudomonas spp., Acinetobacter spp., 202 Achromobacter xylosoxidans, Stenotrophomonas maltophilia and Burkholderia pseudomallei, 203 disk diffusion and MIC EUCAST breakpoints are unavailable. In these cases, it is recommended 204 to perform a MIC-based technique and interpret the results according to the EUCAST PK/PD non- 205 species-related breakpoints.25 206 207 As for Enterobacterales, disk-based assays using cloxacillin as an inhibitor and ceftazidime as an 208 indicator could be employed to detect AmpC beta-lactamase overproduction in P. aeruginosa. 209 The double-disk synergy test or the combination disk using EDTA or dipicolinic acid and 210 carbapenems can also be useful for the detection of class B carbapenemases in P. aeruginosa 211 and other non-fermentative Gram-negative bacilli. 212 213 Staphylococcus spp., Streptococcus spp. and Enterococcus spp. 214 The appearance (sharp or fuzzy) of the inhibition zone edges of certain antimicrobial agents can 215 provide information on resistance mechanisms. For Staphylococcus aureus, isolates with 216 penicillin zone diameters in the susceptible range and sharp zone edges should be considered 217 beta-lactamase producers. Cefoxitin disk diffusion results reliably predicts methicillin resistance 218 in staphylococci, except in Staphylococcus pseudintermedius, Staphylococcus schleiferi and 219 Staphylococcus coagulans. Conventional disk diffusion tests can also detect inducible resistance 220 to clindamycin in Staphylococcus and Streptococcus species, manifested by the appearance of a 221 flattening inhibition zone adjacent to an erythromycin disk (D-zone effect). 222 223 Disk diffusion is unreliable for glycopeptide susceptibility testing among staphylococci as it 224 cannot distinguish between wild type isolates and those with non-vanA-mediated glycopeptide 225 resistance. For enterococci, fuzzy zone edges and colonies within the inhibition zone are highly 226 suggestive of glycopeptide resistance and should be investigated further with an MIC method. 227 228 Additional uses of diffusion testing 229 The gradient strip diffusion method combines the principles of dilution and diffusion and allows 230 the MIC to be determined directly. It shares several similarities with the disk diffusion technique 231 regarding inoculum preparation, culture media and incubation conditions, as well as procedural 232 simplicity and versatility in the choice of antimicrobials. The gradient diffusion method generally 233 produces results that match those obtained with standardized dilution methods and has the 234 advantage of providing a more accurate MIC value, due to the use of a higher number of 235 dilutions than the conventional double series. However, it is not considered a reference 236 technique and the degree of concordance with reference dilution methods may vary for some 237 microorganism-drug combinations. 238 The gradient diffusion method has also been used to detect resistance mechanisms. Double- 239 sided strips containing cephalosporins or carbapenems with and without inhibitors have been 240 developed for the detection of ESBLs, AmpC betalactamases or metallo-beta-lactamases. A 241 method for the screening of heteroresistant vancomycin-intermediate Staphylococcus aureus 242 has also been described. 243 244 Concluding remarks 245 Disk diffusion remains a reliable method for the antimicrobial susceptibility testing of most 246 bacterial pathogens. Unlike commercial automated microdilution systems, the disk diffusion 247 method combines flexibility in the choice of antimicrobials and low cost. It also allows the 248 recognition of phenotypic traits, including inducible and synergistic effects, that are highly useful 249 in detecting certain resistance mechanisms. Although a labor-intensive method, this drawback 250 could be partially resolved by the incorporation of instrumentation for reading zone diameters. 251 Currently, disk diffusion is not well standardized for some bacterial groups; however, the test is 252 being updated according to ongoing research and new disk breakpoints are being set, as 253 reflected in EUCAST and CLSI publications. 254 255 Conflict of interest 256 The authors declare they have no conflict of interest. 257 258 Table 1. Categories used for the inclusion and reporting of antimicrobial agents in susceptibility 259 testing by disk diffusion.23 260 Categories Definitions A Antimicrobials that must be routinely studied and reported. They are relevant for both clinical purpose and for the process of interpretive reading of the antibiogram. B Antimicrobials that must be routinely studied but selectively reported. They are useful for the process of interpretive reading of the antibiogram and should be selectively reported according to the type of patient, type of infection or the inferred resistance mechanism. C Antimicrobials that should be selectively studied and reported according to the type of patient, type of infection or to the inferred resistance mechanism. D Antimicrobials that are recommended to be routinely studied and reported in urine isolates. E Antimicrobials that should be studied but not reported. They are useful for the detection of antimicrobial resistance mechanisms, application of an expert rule or as surrogate markers of the susceptibility testing result of other antimicrobials. 261 262 263 264 Table 2. Antimicrobial agents (category E) used to detect resistance mechanisms or as 265 surrogates for the results of other agents and screening tests.24 Antimicrobial Microorganisms and screening tests Cefoxitin 30 µg Staphylococci, to exclude methicillin resistance except in S. pseudintermedius, S. schleiferi and S. coagulans Oxacillin 1 µg Streptococcus pneumoniae, to exclude all mechanisms of beta-lactam resistance. To exclude methicillin resistance in S. pseudintermedius, S. schleiferi and S. coagulans Benzylpenicillin 1U Haemophilus influenzae and viridans group streptococci, to exclude all mechanisms of beta-lactam resistance Norfloxacin 10 µg Gram-positive bacteria (staphylococci, pneumococci, enterococci, aerococci) to exclude fluoroquinolone resistance Pefloxacin 5 µg Salmonella enterica and Vibrio spp., to exclude fluoroquinolone resistance Nalidixic acid 30 µg H. influenzae, Moraxella catarrhalis and Pasteurella multocida, to exclude fluoroquinolone resistance 266 267 268 Table 3. Current antibiotic-organism combinations without disk diffusion breakpoints that 269 require MIC determination. (Adapted from ref. 15). 270 Antibiotic Group of bacteria Fosfomycin Enterobacterales except E. coli, Staphylococcus spp. Ciprofloxacin Salmonella spp. Colistin All Gram-negative bacilli Tigecycline Enterobacterales except E. coli Beta-lactams Penicillin non-susceptible Streptococcus pneumoniae Glycopeptides Staphylococcus spp. Daptomycin All Gram-positive Lipoglycopeptides All Gram-positive All antibiotics Some anaerobes, Neisseria spp., Helicobacter pylori 271 272 273 Figure 1. Panel distribution of antimicrobial agents to be tested by disk diffusion against 274 Enterobacterales (A), Enterobacterales from urinary tract infections (UTI) (B), Pseudomonas spp. 275 (C), Staphylococcus spp. (D), Streptococcus spp./Enterococcus spp. (E), Enterococcus spp. from 276 UTI (F), Haemophilus influenzae/Haemophilus parainfluenzae (G), and Campylobacter 277 jejuni/Campylobacter coli (H). Figures refer to square (A-E) or round (F-H) plates. Recommended 278 media: Mueller-Hinton agar for Enterobacterales, Pseudomonas spp., Staphylococcus spp., 279 Enterococcus spp.; Mueller-Hinton agar + 5% defibrinated horse blood and 20 mg/L β-NAD (MH- 280 F) for Streptococcus spp., Haemophilus influenzae/Haemophilus parainfluenzae and 281 Campylobacter jejuni/Campylobacter coli. 282 283 A B C 284 AMP PIT AMI GEN AMP PIT AMI GEN 285 PIP CTZ AMS MER 286 287 CXI CTA CUR ERT FOS CTA CUR ERT CEP PIT AZT IMI 288 289 CTZ AMC AZT CIP CTZ AMC AZT CIP GEN TOB AMI CIP 290 291 292 CTV CEP MER TRS NIT CEP MER TRS CTT MIN FOS TRS 293 294 295 D E 296 297 BEN AMP OXA LIN 298 BEN OXA VAN TEI 299 300 ERY CLI LIN TET ERY CLI TET TEI 301 302 RIF GEN LEV TRS VAN CTA STR GEN 303 304 305 FUS MUP TOB CXI LEV TRS RIF 306 307 308 309 F G H 310 311 AMP IMI GEN TRS 312 AMP VAN 313 NAL AMC ERY CIP FOS 314 BEN 315 AMC TET 316 NIT LEV CTA CUR 317 AZI 318 319 320 321 AMC Amoxicillin-clavulanate, AMI Amikacin, AMP Ampicillin, AMS Ampicillin- sulbactam, AZI Azithromycin, 322 AZT Aztreonam, BEN Benzylpenicillin, CEP Cefepime, CIP Ciprofloxacin, CLI Clindamycin, CTA Cefotaxime, 323 CTT Ceftolozane-tazobactam, CTV Ceftazidime- avibactam, CTZ Ceftazidime, CUR Cefuroxime, CXI 324 Cefoxitin, ERT Ertapenem, ERY Erythromycin, FOS Fosfomycin, FUS Fusidic acid, GEN Gentamicin, IMI 325 Imipenem, LEV Levofloxacin, LIN Linezolid, MER Meropenem, MIN Minocycline, MUP Mupirocin, NAL 326 Nalidixic acid, NIT Nitrofurantoin, OXA Oxacillin, PIP Piperacillin, PIT Piperacillin-tazobactam, RIF 327 Rifampicin, STR Streptomycin, TEI Teicoplanin, TET Tetracycline, TOB Tobramycin, TRS Trimethoprim- 328 sulfamethoxazole, VAN Vancomycin. 329 330 Bibliography 331 1. 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