This is the author’s version of the review article: Cantón, Rafael; Oliver, Antonio; Alós, Juan Ignacio; [et al.]. «Recommendations of the Spanish Antibiogram Committee (COESANT) for selecting antimicrobial agents and concentrations for in vitro susceptibility studies using automated systems». Enfermedades infecciosas y microbiología clínica, Vol. 38, Issue 4 (April 2020), p. 182-187. 6 pàg. DOI 10.1016/j.eimc.2019.01.017 This version is available at https://ddd.uab.cat/record/285180 under the terms of the license *Primera página (incluye autores, filiación y correspondencia) 1 Recommendations of the Spanish Antibiogram Committee 2 (COESANT) for selecting antimicrobial agents and concentrations for 3 in vitro susceptibility studies using automated systems 4 a,b 5 Rafael Cantón , Antonio Oliverb,c, Juan Ignacio Alósd, Natividad de Benitoe, Germán Boub,f, 6 José Camposb,g, Jorge Calvob,h, Andrés Canuti, Javier Castilloj, Emilia Cercenadok, M. Ángeles 7 Domínguezb,l, Felipe Fernández-Cuencab,m, Jesús Guineak. Nieves Larrosab,n, Josefina Liñaresb,l, 8 Lorena López-Cererob,m, Antonio López-Navaso, Francesc Marcob,p, Beatriz Mirelisq, Miguel 9 Ángel Moreno-Romor, María Isabel Morosinia,b, Ferran Navarroq, Jesús Oteob,g, Álvaro 10 Pascualb,m, Emilio Pérez-Tralleros, María Pérez-Vázquezb,g, Alex Sorianot, Carmen Torresu, 11 Jordi Vilab,p and Luis Martínez-Martínezb,w. 12 a 13 Servicio de Microbiología. Hospital Universtario Ramón y Cajal and Instituto Ramón y Cajal 14 de Investigación Sanitaria (IRYCIS). Madrid. Spain; bRed Española de Patología Infecciosa 15 (REIPI). Instituto de Salud Carlos III. Madrid. Spain; cServicio de Microbiología. Hospital 16 Universitario Son Espases. Instituto de Investigación Sanitaria Illes Balears (IdISBa). Palma de 17 Mallorca. Spain; dServicio de Microbiología, Hospital Universitario de Getafe, Madrid. Spain; e 18 Unidad de Enfermedades Infecciosas. Hospital de la Santa Creu i Sant Pau. Instituto de 19 Investigación Biomédica Sant Pau (IIB Sant Pau). Universitat Autònoma.Barcelona. Spain; f 20 Servicio de Microbiología-INIBIC, Complejo Hospitalario Universitario A Coruña, A Coruña. 21 Spain; gLaboratorio de Referencia e Investigación en Resistencia a Antibióticos e Infecciones 22 Relacionadas con la Asistencia Sanitaria, Centro Nacional de Microbiología, Instituto de Salud 23 Carlos III, Majadahonda, Madrid. España. Spain; hServicio de Microbiología. Hospital 24 Universitario Marqués de Valdecilla and Instituto de Investigación Marqués de Valdecilla 25 (IDIVAL). Santander. Spain; iServicio de Microbiología, Hospital Universitario de Álava. 26 Vitoria. Spain; jServicio de Microbiología. Hospital Clínico Universitario. Zaragoza. Spain; k 27 Servicio de Microbiología y Enfermedades Infecciosas. Hospital General Universitario 28 Gregorio Mrañón. Madrid. Spain; lServicio de Microbiología. Hospital Universitario de 29 Bellvitge. Barcelona. Spain; mServicio de Microbiología. Hospital Virgen Macarena. Sevilla; 30 n Servicio de Microbiología. Hospital Universitario Vall d’Hebron. Barcelona. Spain;oAgencia 31 Española de Medicamentos y Productos Sanitarios (AEMPS). Madrid. Spain; pDepartamento de 32 Microbiología, Centro de Diagnóstico Biomédico (CDB), Hospital Clínic, Universidad de 33 Barcelona, and ISGlobal, Barcelona, Spain; qServicio de Microbiología. Hospital de la Santa 34 Creu i Sant Pau. Barcelona. Spain; rDepartamento de Sanidad Animal, Facultad de Veterinaria, 35 Universidad Complutense. Madrid. Spain; sServicio de Microbiología. Hospital Universitario 36 Donostia-IIS Biodonostia. San Sebastián. Spain; tServicio de Enfermedades Infecciosas. 1 37 Hospital Clínico. Barcelona. Spain; uFacultad de Ciencias y Tecnología, Área de Bioquímica y 38 Biología Molecular, Universidad de La Rioja. Logroño. Spain; wUnidad de Gestión Clínica de 39 Microbiología, Hospital Reina Sofía. Departamento de Microbiología, Universidad de Córdoba, 40 Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba. Spain. 41 42 Correspondencia. Dr. R. Cantón. Servicio de Microbiología. Hospital Universitario Ramón y 43 Cajal. 28034-Madrid. España. Correo electrónico: rafael.canton@salud.madrid.org 44 Phone number: +34913368832 45 46 Funding 47 This document did not have any specific funding. COESANT activities are partially 48 funded by the Agencia Española de Medicamentos y Productos Sanitarios (AEMPS) 49 and the Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica 50 (SEIMC). 51 Scientific and research activities of RC, AO, GB, JC, JC, MAD, FFC, LLC, FM, MIM, 52 JOI, AP, MPV, JV and LM-M are Supported by Plan Nacional de I+D+i 2013-2016 and 53 Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación 54 Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for 55 Research in Infectious Diseases (REIPI) (RD16/0016/0001; RD16/0016/0002; 56 RD16/0016/0003; RD16/0016/0004; RD16/0016/0005; RD16/0016/0006; 57 RD16/0016/0007; RD16/0016/0008; RD16/0016/0010; RD16/0016/0011; 58 RD16CIII/0004/002) ‐ cofinanced by European Development Regional Fund “A way 59 to achieve Europe”, Operative program Intelligent Growth 2014‐ 2020 and of CT by 60 Plan Nacional de I+D+i 2013‐ 2016, Ministerio de Economía, Industria y 61 Competitividad, Spain (SAF2016-76571-R). 62 63 Conflict of interest 64 RC has participated in educational activities sponsored by BD and bioMérieux and in 65 clinical validation studies of in vitro diagnostic medical devices from bioMérieux. AC 66 has participated in educational activities sponsored by BD. MAD has participated in 67 educational activities sponsored by Beckman Coulter. GB, NL and JV have participated 68 in educational activities sponsored by bioMérieux. LMM has participated in educational 69 activities sponsored by bioMérieux and an advisory board meeting with BD 70 Switzerland. Other authors declare no conflict of interest. 2 *Manuscrito (sin información de autores) 1 Recommendations of the Spanish Antibiogram Committee 1 2 2 (COESANT) for selecting antimicrobial agents and concentrations for 3 4 3 in vitro susceptibility studies using automated systems 5 6 4 7 8 5 Summary 9 10 6 Automated antimicrobial susceptibility testing devices are widely introduced in clinical 11 12 7 microbiology laboratories in Spain, mainly using EUCAST (European Committee on 13 14 8 Antimicrobial Susceptibility Testing) breakpoints. In 2007, a group of experts published 15 16 9 recommendations for including antimicrobial agents and selecting concentrations in 17 10 these systems. Under the patronage of the Spanish Antibiogram Committee (Comité 18 19 11 Español del Antibiograma, COESANT) and the Study Group on Mechanisms of Action 20 21 12 and Resistance to Antimicrobial Agents (GEMARA) from the Spanish Society of 22 23 13 Infectious Diseases and Clinical Microbiology (SEIMC), and aligned with the Spanish 24 25 14 Plan against Antimicrobial Resistance (PRAN), a group of experts have updated this 26 27 15 document. Main modifications from the previous version comprise the inclusion of new 28 16 antimicrobial agents, adaptation of the ranges of concentrations to cover the EUCAST 29 30 17 breakpoints and epidemiological cut-off values (ECOFFs), and the inference of new 31 32 18 resistance mechanisms. This proposal should be considered by different manufacturers 33 34 19 and users when designing new panels or cards. In addition, recommendations for 35 36 20 selective reporting are also included. With this approach, the implementation of 37 38 21 EUCAST breakpoints will be easier, increasing the quality of antimicrobial 39 22 susceptibility testing data and their microbiological interpretation. It will also benefit 40 41 23 surveillance as well as the clinical use of antimicrobials aligned with antimicrobial 42 43 24 stewardship programs. 44 45 25 46 47 26 Key words: Antibiogram; Automated susceptibility testing systems; Antimicrobial 48 27 concentrations; MICs. 49 50 28 51 52 29 53 54 55 56 57 58 59 60 61 62 1 63 64 65 30 Recomendaciones del Comité Español del Antibiograma (COESANT) para la 1 2 31 selección de antimicrobianos y sus concentraciones en el estudio in vitro de la 3 4 32 sensibilidad con métodos automáticos 5 33 6 7 34 Resumen 8 9 35 Los sistemas automáticos utilizados en el estudio de la sensibilidad a los 10 11 36 antimicrobianos están introducidos en la mayoría de los laboratorios de Microbiología 12 13 37 Clínica en España, utilizando principalmente los puntos de corte EUCAST (European 14 15 38 Committee on Antimicrobial Susceptibility Testing). En 2007, un grupo de expertos 16 39 publicó unas recomendaciones para incluir antimicrobianos y seleccionar 17 18 40 concentraciones en estos sistemas. Bajo el auspicio del Comité Español del 19 20 41 Antibiograma (COESANT) y del Grupo de Estudio de los Mecanismos de Acción y 21 22 42 Resistencia a los Antimicrobianos (GEMARA) de la Sociedad Española de 23 24 43 Enfermedades Infecciosas y Microbiología Clínica (SEIMC) y alineado con el Plan 25 44 Nacional frente a la Resistencia a los Antibióticos (PRAN), un grupo de expertos ha 26 27 45 actualizado dicho documento. Las principales modificaciones realizadas sobre la 28 29 46 versión anterior comprenden la inclusión de nuevos agentes antimicrobianos, la 30 31 47 adaptación de los rangos de concentraciones para cubrir los puntos de corte clínicos y 32 33 48 los puntos de corte epidemiológicos (ECOFF) definidos por el EUCAST, y para la 34 35 49 inferencia de nuevos mecanismos de resistencia. Esta propuesta debería ser considerada 36 50 por los diferentes fabricantes y los usuarios cuando se diseñen nuevos paneles o tarjetas. 37 38 51 Además, se incluyen recomendaciones para realizar informes selectivos. Con este 39 40 52 enfoque, la implementación de los puntos de corte del EUCAST será más fácil, 41 42 53 aumentando la calidad de los datos del antibiograma y su interpretación microbiológica. 43 44 54 También será de utilidad para los estudios de vigilancia epidemiológica, así como para 45 46 55 el uso clínico de los antimicrobianos, de acuerdo con los programas de optimización de 47 56 uso de antimicrobianos (PROA). 48 49 57 50 51 58 Palabras clave. Antibiograma; Sistemas automáticos de detección de sensibilidad; 52 53 59 concentraciones mínimas antimicrobianas; CMI. 54 55 60 56 61 57 58 62 59 60 61 62 2 63 64 65 63 Introduction 1 2 64 In 2007, the Study Group on Mechanisms of Action and Resistance to Antimicrobial 3 4 65 Agents (GEMARA) and the Spanish Committee on Antimicrobial Susceptibility testing 5 6 66 (named as MENSURA at that time) published, under the auspices of the Spanish 7 8 67 Society of Infectious Diseases and Clinical Microbiology (SEIMC), “Recommendations 9 68 for selecting antimicrobial agents for in vitro susceptibility studies using automatic and 10 11 69 semiautomatic systems”1. Since then, significant efforts in Europe for harmonization of 12 13 70 susceptibility testing methods and definition of breakpoint clinical criteria have been 14 15 71 done led by the European Committee of Antimicrobial Susceptibility Testing 16 17 72 (EUCAST)2 and Spain has created the COESANT (Comité Español del Antibiograma) 18 73 committee, which is the Spanish National Antimicrobial Committee (NAC) aligned 19 20 74 with EUCAST3. Ever since, several new antimicrobials have been marketed, new 21 22 75 resistance mechanisms have been described4,5, and health authorities have promoted 23 24 76 plans to address the problem of antimicrobial resistance6. In addition, professional 25 26 77 societies, such as the SEIMC, have designed antimicrobial stewardship programs, for 27 28 78 the better use of antimicrobial agents with the aim to curtail increasing prevalence of 29 79 resistance7. Within these programs, the importance of antimicrobial susceptibility 30 31 80 testing (AST), characterization of resistance mechanisms and analysis of clonal 32 33 81 relationship are highlighted. 34 35 82 Unlike Northern European countries, but in common with many other countries 36 37 83 worldwide, automated and semiautomated systems for AST are widely distributed in 38 39 84 Spanish clinical microbiology laboratories. In a recent survey performed by the SEIMC 40 85 in which 156 Spanish microbiology laboratories participated, 92.3% of them routinely 41 42 86 used these systems (unpublished data). These data are consistent with those reported in 43 44 87 recent multicentre quality control studies on antimicrobial susceptibility testing 45 46 88 performed in Spain8-10. This wide distribution may have several advantages such as 47 48 89 testing a high number of antimicrobial agents per isolate, and a better inference of 49 50 90 resistance phenotypes with the aid of the so-called “expert systems” incorporated in 51 91 these devices, the potential aggregation of data in MIC-based surveillance systems, and 52 53 92 the reporting of MIC values to adapt patients’ antimicrobial therapy applying 54 55 93 pharmacokinetics-pharmacodynamics (PK-PD) criteria. Nevertheless, different 56 57 94 manufacturers include diverse antimicrobials with different ranges of concentrations, 58 59 95 which hinder some of these advantages, particularly the data aggregation in surveillance 60 61 62 3 63 64 65 96 programs and in some cases, the inference of resistance mechanisms. In most cases, the 1 2 97 design of panels or cards used in these systems does not follow a consensus procedure 3 4 98 and only few documents address which antibiotics and concentrations should 5 99 specifically be included1,11,12. 6 7 100 In the current document we have updated the previous version of 8 9 101 “Recommendations for selecting antimicrobial agents for in vitro susceptibility studies 10 11 102 using automatic and semiautomatic systems”1. This new version has been led by 12 13 103 COESANT, SEIMC and its study group GEMARA in the context of the Spanish Plan of 14 15 104 Antimicrobial Resistance (PRAN, Plan Nacional de Resistencia a los Antimicrobianos) 16 105 coordinated by the Spanish Agency of Medicines and Sanitary Products (AEMPS, 17 18 106 Agencia Española de los Medicamentos y Productos Sanitarios)13. This manuscript was 19 20 107 prepared by a group of experts and was submitted for public consultation through the 21 22 108 COESANT and SEIMC websites. The manufacturers of automated AST devices 23 24 109 marketed in Spain were also included in this consultation. The final version was 25 110 constructed considering all these opinions. 26 27 111 28 29 112 Objectives and general recommendations for antimicrobial 30 31 113 susceptibility testing using automated and semiautomated systems 32 33 114 The main objective in the elaboration of this document was to update the general 34 35 115 recommendations for the selection of the antibiotics and their concentrations to be 36 37 116 included in the AST panels used by automated or semiautomated systems 38 39 117 commercialized in Spain that was published in 20071. Likewise, suggestions for 40 41 118 selective reporting of susceptibility testing results are also included (Table 1). The 42 43 119 participating experts have also agreed on these recommendations of selective reporting. 44 120 Recently, a European study has recognized this procedure as part of the stewardship 45 46 121 programs in which clinical microbiology laboratories should actively participate 47 48 122 through their informatics systems14. Obviously, this selective reporting can be 49 50 123 facilitated with appropriate recommendations for antimicrobial testing against different 51 52 124 microorganisms. In the European study, Spain was classified as a country with partial 53 54 125 implementation of this procedure and the present document can facilitate criteria to 55 126 enhance the number of laboratories with this practice. 56 57 127 However, although the document focuses on MIC-based automated systems, 58 59 128 most of the established criteria related to the selection of the antibiotics to be included 60 61 62 4 63 64 65 129 in the antibiogram and the reporting of the results can also be applied to the agar 1 2 130 diffusion-based methods, either with disc or with MIC gradient strips. Since the first 3 4 131 consensus document was published in 2007, a number of new antimicrobial agents have 5 132 been approved, several indications have been changed or expanded, and different 6 7 133 breakpoints have been significantly modified making it necessary to revise the previous 8 9 134 document and to include new antimicrobials (Supplementary tables S1-S9). Moreover, 10 11 135 the use of traditional susceptible clinical breakpoints does not necessarily recognize 12 13 136 isolates with low-level resistance mechanisms15,16 and recognition of wild-type 14 15 137 populations and the definition of the epidemiological cut-off values (ECOFF) have been 16 138 widely used. 17 18 139 More recently, EUCAST has modified definitions of interpretive clinical 19 20 140 categories [susceptible (S), intermediate (I) and resistant (R)]. These new definitions 21 22 141 mainly affect to the intermediate category, which is now interpreted as “susceptible, 23 24 142 increased exposure” which occurs when there is a high likelihood of therapeutic success 25 143 because exposure to the agent is increased by adjusting the dosing regimen or by its 26 27 144 concentration at the site of infection17. As a consequence, EUCAST has modified some 28 29 145 breakpoints and others only applied when high exposure of the microorganisms to the 30 31 146 agent is considered (i.e. most β-lactams and Pseudomonas aeruginosa)18. In addition, 32 33 147 EUCAST has introduced for some organism-agent combinations a new concept which 34 35 148 has been designed as an Area of Technical Uncertainty (ATU). It corresponds to an 36 37 149 MIC value and/or zone diameter interval where the categorisation is doubtful. Further 38 150 explanations and how to deal with results in the ATU are explained in the EUCAST 39 40 151 breakpoint tables18. 41 42 152 Automated and semiautomated systems should have a minimal set of 43 44 153 characteristics making them appropriate to fulfill the objectives for which they were 45 46 154 designed, allowing the application of the general criteria used in the antibiogram 47 48 155 interpretive reading19,20. These criteria are summarized in the following points: 49 156 a) Availability of the identification of the microorganism under study which is 50 51 157 necessary for the antibiogram interpretive reading and for the inference of the 52 53 158 resistance mechanisms18,19. This can be achieved through either biochemical 54 55 159 tests included in the same panel/card or an additional panel/card or through any 56 57 160 other method, including MALDI-TOF mass spectrometry. When the automated 58 59 161 AST systems are linked to MALDI-TOF mass spectrometry devices, it would be 60 162 desirable that this information could be also used for epidemiological purposes 61 62 5 63 64 65 163 in the identification of antimicrobial resistance mechanisms and bacterial 1 2 164 clones21. 3 4 165 b) Incorporate an informatics application with the capacity to interpret MIC values 5 166 (or inhibition zones) thus establishing the susceptible (S), intermediate (I) and 6 7 167 resistant (R) clinical categories. This software should apply criteria 8 9 168 recommended by EUCAST18, although it is recommended that it may allow the 10 11 169 access to the criteria established by other susceptibility testing committees, such 12 13 170 as CLSI12, or those specifically defined by COESANT (Supplementary tables 14 15 171 S1-S9). 16 172 c) Incorporate the so-called “expert systems” for antibiogram interpretive reading, 17 18 173 able to recognize phenotypes of resistance to multiple antibiotics from the same 19 20 174 or different families and inferring the underlying resistance mechanisms19,20. 21 22 175 d) Allow a bidirectional connection with the Laboratory Informatics System (LIS), 23 24 176 required not only for the transference of AST data but also to receive the 25 177 necessary information for the management of results, particularly with the aim 26 27 178 of conducting epidemiological analysis, infection control studies, and 28 29 179 antimicrobial stewardship programs7. Ideally, these systems should be 30 31 180 compatible for the connection to national and international surveillance 32 33 181 databases. The incorporation of these “expert” programs facilitates daily work 34 35 182 and decreases the workload. Moreover, these devices should also be able to 36 183 connect with programs using databases for infection control programs. 37 38 184 39 40 185 Antimicrobial selection criteria 41 42 186 The inclusion of antimicrobials in the panels of automated susceptibility testing systems 43 44 187 is mainly conditioned by their clinical interest. However, other points should also be 45 46 188 considered, such as the type of microorganism or the need of interpretation of resistance 47 48 189 mechanisms. In our document, the selection of the different compounds was performed 49 50 190 considering the following criteria: 51 191 Microbiological criteria 52 53 192 The antimicrobials to be included in the AST panels, regardless of the type of 54 55 193 automated system, are those required for the interpretive reading of the susceptibility 56 57 194 pattern and for the inference of underlying resistance mechanisms22-24.The selection of 58 59 195 antimicrobials is also intended to contribute to the inference of complex phenotypes 60 61 62 6 63 64 65 196 causing multidrug-resistant profiles, such as those derived from the simultaneous 1 2 197 presence of different resistance mechanisms affecting various members of a unique 3 4 198 family, e.g. β-lactam antibiotics19,25. Moreover, certain antimicrobials, such as 5 199 tetracycline or chloramphenicol, have been mainly selected for epidemiological 6 7 200 monitoring purposes. 8 9 201 In the case of antimicrobials belonging to families with several members, e.g. 10 11 202 cephalosporins and fluoroquinolones, selected compounds are considered as 12 13 203 representative of the antimicrobial activity of the group, additionally allowing the 14 15 204 deduction of the activity of those that are not included in the panels as well as the 16 205 assumption of the presence of resistance mechanisms23. The only purpose of including 17 18 206 certain antimicrobials, in some cases without clinical use such as nalidixic acid, have 19 20 207 the only purpose is to act as a marker of a primary resistance step which indicates the 21 22 208 presence of mutations that can preclude the use of fluoroquinolones in subsequent 23 24 209 rounds of topoisomerase mutations25. Similarly, kanamycin resistance alerts for the 25 210 presence of some aminoglycoside-modifying enzymes affecting amikacin while the 26 27 211 association of clavulanic acid with a third or fourth generation cephalosporin helps to 28 29 212 identify the presence of an extended-spectrum-β-lactamase26. Another example is 30 31 213 cefoxitin in panels for the study of Enterobacterales, which help to predict the presence 32 33 214 of AmpC β-lactamases (either chromosomally or plasmidic encoded) and/or a deficit in 34 35 215 outer membrane permeability24,27. In the case of staphylococci, cefoxitin has been 36 37 216 included as it performs better than oxacillin as a marker for detecting the presence of the 38 217 mec genes causing methicillin resistance28. 39 40 218 The emergence and sudden dispersion of a resistance mechanism may increase the 41 42 219 interest for the study of a particular compound. This is the case of the acquired 43 44 220 carbapenemases in gram-negative bacilli that has raised interest in aztreonam as an 45 46 221 indicator of the presence of metallo-β-lactamases, particularly when the study is 47 48 222 simultaneously performed with ceftazidime, the combination of ceftazidime-avibactam 49 223 and carbapenems29. Additionally, tigecycline, a glycylcycline derivative of minocycline, 50 51 224 has been included as it can be a therapeutic option against some multidrug-resistant 52 53 225 gram-negatives30. 54 55 226 In the case of staphylococci, the simultaneous presence of a concentration of 56 57 227 erythromycin together with one of clindamycin in the same well is intended to detect 58 59 228 inducible macrolide-clindamycin resistance31. Moreover, daptomycin and linezolid have 60 229 been included as they represent last-resort line therapeutic options against gram-positive 61 62 7 63 64 65 230 cocci32. More recently, certain panel/card manufacturers have also included ceftaroline, 1 2 231 a new cephalosporin with activity against methicillin-resistant Staphylococcus aureus33. 3 4 232 Pharmacokinetic/pharmacodynamic (PK/PD) criteria 5 233 EUCAST uses PK/PD Monte Carlo simulations as a key component of its breakpoints’ 6 7 234 setting process for old and new antimicrobials. The PK/PD breakpoint is the MIC value 8 9 235 considered necessary to achieve a probability of target attainment of >95% and applies 10 11 236 to specific dosage regimens34. The PD targets predicting maximum efficacy of the 12 13 237 antimicrobial, for example 50% for the percentage of the dosing interval during which 14 15 238 the serum concentration exceeds the MIC (%T>MIC) of a β-lactam, 100% for an area 16 239 under the concentration-time curve/MIC ratio (AUC24/MIC) of a fluoroquinolone, or 10 17 18 240 times for peak plasma concentration/MIC ratio (Cmax/MIC) of an aminoglycoside, 19 20 241 expressed as a function of the unbound drug concentration. 21 22 242 The magnitude of the PD target can vary among bacterial species35. A clinical 23 24 243 breakpoint setting process requires knowledge of the wild-type distribution of MICs, 25 244 assessment of the pharmacokinetic/pharmacodynamic (PK/PD) parameters, and study of 26 27 245 the clinical outcome of the infected patient when the antimicrobial agent is used34,36. 28 29 246 The use of PK parameters in the simulations considering different populations (healthy 30 31 247 volunteers or critically ill patients with different degrees of renal function), various dose 32 33 248 regimens and multiple infection sites (urinary concentrations of antimicrobial agents are 34 35 249 higher than serum concentrations over a dosing interval) will result in different 36 250 breakpoints. EUCAST has defined several breakpoints which are only valid for isolates 37 38 251 from uncomplicated urinary tract infections (e.g. amoxicillin-clavulanic acid MIC 39 40 252 breakpoint S ≤32 mg/L for Enterobacterales)21. 41 42 253 PK/PD data and MIC distributions comprise the primary data to support decisions 43 44 254 concerning revised breakpoints. For β-lactam antimicrobials and P. aeruginosa, 45 46 255 susceptible and intermediate (susceptible, increased exposure) breakpoints are 47 48 256 established to ensure optimal exposures with specific dosage regimens17. Additionally, 49 257 the MIC and associated breakpoints are a better means for guiding selection of therapy 50 51 258 for individual patients37-39. 52 53 259 It is important to consider that accuracy of the automated susceptibility tests 54 55 260 depends, among other factors, on the concentration of the antibiotics, as the lower the 56 57 261 concentration, the higher the error rates40. 58 59 262 Clinical criteria 60 61 62 8 63 64 65 263 Information about the bacterial susceptibility pattern is essential to guide the selection 1 2 264 of antibiotic treatment. Furthermore, it is well known that there are many important host 3 4 265 factors determining the clinical outcome. Several clinical data demonstrate that an in 5 266 vitro susceptible result often predicts therapeutic success. However, even in patients 6 7 267 with sepsis due to a microorganism with an in vitro resistant result, resistance in vivo 8 9 268 with concomitant clinical failure cannot be always predicted41-42. Therefore, and from a 10 11 269 clinical point of view, the most commonly used antibiotics or at least one representative 12 13 270 of the antibiotic family that predicts the activity of the other members, should be 14 15 271 included in the routine susceptibility report as occurs with first generation 16 272 cephalosporins. This subrogated use is also claimed in the case of new antimicrobials 17 18 273 when they are not yet included in testing devices. This is the case of tedizolid and 19 20 274 linezolid or dalbavancin and vancomycin 21 22 275 In addition, when the MIC is high but within the susceptibility range suggesting 23 24 276 the presence of a specific low-level resistance mechanism, or when clinical data indicate 25 277 worse outcome when the MIC is high, alternative antibiotics should be tested. For 26 27 278 instance, when MICs of carbapenems for Klebsiella spp. or E. coli are high, suggesting 28 29 279 the presence of a carbapenemase, alternative antibiotics including colistin, tigecycline or 30 31 280 fosfomycin should be tested43. A similar approach might occur when considering MICs 32 33 281 of vancomycin >1 and ≤2 mg/l for S. aureus causing bacteremia, which has been 34 35 282 associated in some studies to a worse outcome44, it is recommended to report data 36 283 concerning the susceptibility status of possible alternatives. 37 38 284 Nowadays, new antimicrobials, such as ceftazidime-avibactam or ceftolozane- 39 40 285 tazobactam for gram-negatives as well as dalbavancin, telavancin or oritavancin for 41 42 286 gram-positives, have been included in testing devices AST of these compounds are 43 44 287 recommended not only to obtain information of new therapeutic alternatives but also to 45 46 288 generate routine epidemiological data. 47 289 48 49 290 Criteria for the selection of antimicrobial concentrations 50 51 291 The selection of the concentrations proposed for each antimicrobial agent has been 52 53 292 made with the objective of covering the breakpoints used for defining clinical categories 54 55 293 (susceptible, intermediate and resistant) established by EUCAST17. For certain 56 57 294 antimicrobials, specific COESANT recommendations have been considered (specified 58 59 295 in the supplementary tables of this document). In addition, since the number of wells 60 61 62 9 63 64 65 296 available in the different panels or cards varies from one manufacturer to another, more 1 2 297 concentrations are also recommended. All these concentrations are classified in 3 4 298 different groups. The first one (indicated in bold in (Supplementary tables S1-S9).) 5 299 includes the concentrations that would be essential to respond to the previous objective 6 7 300 (covering EUCAST breakpoints) and therefore, should always be included in the 8 9 301 susceptibility testing panels. This range is mainly intended to include the concentration 10 11 302 defining the resistance breakpoint and one dilution below the susceptible breakpoint. In 12 13 303 addition, there are other concentrations (not indicated in bold) that could be added to 14 15 304 encompass the ECOFF value to detect wild-type populations or to facilitate 16 305 epidemiological surveillances, especially of microorganisms with low-level resistance 17 18 306 mechanisms. This approach also contributes to a better interpretive reading of the 19 20 307 antibiogram19,20. 21 22 308 23 24 309 Definition of categories and groups of antimicrobial agents tested in 25 26 310 the antibiogram 27 28 311 Five different categories of antimicrobials have been established (A to E) with the 29 30 312 recommendation of inclusion in the panels and selective reporting depending on the 31 32 313 clinical relevance of the antimicrobial tested, type of patient or type of infection. 33 314 Moreover, these recommendations also consider the interest of the antimicrobials for the 34 35 315 interpretive reading of the antibiogram and the inference of resistance mechanisms 36 37 316 (Table 1). A specific category (category D) has been defined for antimicrobials that are 38 39 317 recommended to be routinely studied and reported in urine isolates. These 40 41 318 antimicrobials normally have clinical breakpoints specifically adapted for non- 42 43 319 complicated urinary tract infections12,21, and some manufacturers offer specific panels 44 320 for microorganisms involved in these infections. 45 46 321 The last category (category E) is exclusively established for those 47 48 322 antimicrobials recommended to be studied but not reported. They are useful for the 49 50 323 detecion of antimicrobial resistance mechanisms, such as nalidixic acid and gyrA and 51 52 324 topoisomerase IV mutations in gram-negative organisms, application of an expert rule 53 54 325 or inference of a resistance mechanism, such as the combinations of third or fourth 55 326 generation cephalosporins with clavulanic acid, or as subrogated markers of the 56 57 327 susceptibility result of other antimicrobials19,20,25,26. Overall, they are not relevant for 58 59 328 clinical purposes. 60 61 62 10 63 64 65 329 1 2 330 Concluding remarks 3 331 4 332 Spain is a country where automated susceptibility testing systems are widely distributed 5 6 333 and every day, thousands of AST data are produced by clinical microbiology 7 8 334 laboratories. These data, as it is quoted in a European survey and in quality control 9 10 335 studies performed in Spain, are selectively reported by an important number of 11 336 laboratories using EUCAST breakpoints8-10,14. All these data are mainly used for 12 13 337 clinical purposes for patients’ treatments. Moreover, they should also be useful for 14 15 338 surveillance and for tracking the evolution of antimicrobial resistance at local or 16 17 339 national level if compiled in a common database, which is an objective of the Spanish 18 19 340 National Plan against Antimicrobial Resistance (PRAN)13. However, its development 20 21 341 might be complex due to the lack of homogeneity in the number of antibiotics tested for 22 342 each microorganism and also, importantly, in the concentrations tested for each 23 24 343 antimicrobial, which precludes not only fully implementation of the EUCAST 25 26 344 breakpoints but also data compilation. 27 28 345 Considering the criteria explained in the previous paragraphs, we propose in this 29 30 346 document those antimicrobial agents and concentrations to be used in the study of in 31 32 347 vitro susceptibility of the different microorganisms when automated systems are used 33 348 (Supplementary tables S1-S9). Different manufacturers and users should consider this 34 35 349 proposal when designing or using new panels. We believe that with this approach, the 36 37 350 implementation of EUCAST breakpoints will be easier, increasing the quality of data 38 39 351 and their microbiological interpretation44. Finally, it will benefit epidemiological 40 41 352 surveillances as well as the clinical use of antimicrobials aligned with the stewardship 42 43 353 programs. 44 354 45 46 355 47 48 356 Funding 49 50 357 This document did not have any specific funding. COESANT activities are partially 51 52 358 funded by the Agencia Española de Medicamentos y Productos Sanitarios (AEMPS) 53 54 359 and the Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica 55 360 (SEIMC). 56 57 361 58 59 60 61 62 11 63 64 65 362 Scientific and research activities of RC, AO, GB, JC, JC, MAD, FFC, LLC, FM, MIM, 1 2 363 JOI, AP, MPV, JV and LM-M are Supported by Plan Nacional de I+D+i 2013-2016 and 3 4 364 Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación 5 365 Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for 6 7 366 Research in Infectious Diseases (REIPI) (RD16/0016/0001; RD16/0016/0002; 8 9 367 RD16/0016/0003; RD16/0016/0004; RD16/0016/0005; RD16/0016/0006; 10 11 368 RD16/0016/0007; RD16/0016/0008; RD16/0016/0010; RD16/0016/0011; 12 13 369 RD16CIII/0004/002) ‐ cofinanced by European Development Regional Fund “A way 14 15 370 to achieve Europe”, Operative program Intelligent Growth 2014‐ 2020 and of CT by 16 371 Plan Nacional de I+D+i 2013‐ 2016, Ministerio de Economía, Industria y 17 18 372 Competitividad, Spain (SAF2016-76571-R). 19 20 373 21 22 374 Conflict of interest 23 24 375 RC has participated in educational activities sponsored by BD and bioMérieux and in 25 376 clinical validation studies of in vitro diagnostic medical devices from bioMérieux. AC 26 27 377 has participated in educational activities sponsored by BD. MAD has participated in 28 29 378 educational activities sponsored by Beckman Coulter. GB, NL and JV have participated 30 31 379 in educational activities sponsored by bioMérieux. LMM has participated in educational 32 33 380 activities sponsored by bioMérieux and an advisory board meeting with BD 34 35 381 Switzerland. Other authors declare no conflict of interest. 36 382 37 38 383 39 40 384 41 42 385 43 44 386 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 12 63 64 65 387 References 1 2 388 1. Cantón R, Alós JI, Baquero F, Calvo J, Campos J, Castillo J, et al. Recomendaciones 3 389 para la selección de antimicrobianos en el estudio de la sensibilidad in vitro con 4 5 390 sistemas automáticos y semiautomáticos. Enferm Infecc Microbiol Clin. 2007; 25:394- 6 7 391 400. 8 392 2. 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Larrosa MN, Benito N, Cantón R, Canut A, Cercenado E, Fernández-Cuenca F, et al. From 33 515 CLSI to EUCAST, a necessary step in Spanish laboratories. Enferm Infecc Microbiol Clin. 2018 34 35 516 Nov 5. pii: S0213-005X(18)30278-7. doi: 10.1016/j.eimc.2018.09.014. [Epub ahead of print] 36 517 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 16 63 64 65 518 Table 1. Categories used for the inclusion of the antimicrobial agents in 1 2 519 susceptibility testing panels for automated systems 3 4 520 5 Categories Definitions 6 A Antimicrobials that must be routinely studied and reported. They are relevant for both 7 8 clinical purpose and for the process of interpretive reading of the antibiogram. 9 10 B Antimicrobials that must be routinely studied but selectively reported. They are useful 11 for the process of interpretive reading of the antibiogram and should be selectively 12 13 reported according to the type of patient, type of infection or the inferred resistance 14 15 mechanism. 16 17 C Antimicrobials that should be selectively studied and reported acording to the type of 18 patient, type of infection or to the inferred resistance mechanism. 19 20 D Antimicrobials that are recommended to be routinely studied and reported in urine 21 22 isolates 23 E Antimicrobials that should be studied but not reported. They are useful for the detecion 24 25 of antimicrobial resistance mechanisms, aplication of an expert rule or as subrogate 26 27 markers of the susceptibility testing result of other antimicrobials. 28 29 521 30 522 31 523 32 33 524 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 17 63 64 65 Supplementary tables S1-S9 Recommendations of the Spanish Antibiogram Committee (COESANT) for selecting antimicrobial agents and concentrations for in vitro susceptibility studies using automated systems TABLE S1. Antibiotics and concentrations recommended for the susceptibility testing of Enterobacterales Antimicrobial agent Concentrations (mg/L) Category Comments ß-lactams Ampicillin 2-4-8-16-32 A Report as amoxicillin. Amoxicillin- 2/2-4/2-8/2-16/2-32/2 A For susceptibility testing purposes, the concentration of clavulanic acid is fixed at 2 mg/L. ECOFF has clavulanic acid not yet been defined. Breakpoints for uncomplicated urinary tract infections has been defined as S ≤32/2 mg/L and R >32/2. Ticarcillin 4-8-16-32-64 E It can be useful to infer the presence of resistance mechanisms, such as TEM-1, chromosomal AmpC hyperproduction or plasmid-mediated AmpC. Piperacillin- 4/4-8/4-16/4-32/4-64/4 A tazobactam Cefazolin 2-4-8-16-32 D It can be used as a surrogate test for uncomplicated urinary tract infection treated with oral cephalosporins. Breakpoints have not been defined by EUCAST; those shown are recommended by COESANT. ECOFF has not yet been defined. Cefuroxime 1-2-4-8-16-32 A Breakpoints for iv and oral (uncomplicated urinary tract infections) formulations are the same. iv defined for E. coli, K. pneumoniae and P. mirabilis only. Oral breakpoints defined for uncomplicated urinary tract infection only. Cefoxitin 4-8-16-32 E Breakpoints have not been defined by EUCAST. Cefoxitin MIC >8 mg/L may indicate high-level expression of AmpC β-lactamases (with the exception of ACC β-lactamases) or, in some organisms, porin deficiency. Ceftazidime 0.5-1-2-4-8-16-32 A Ceftazidime- 1/4-2/4-4/4-8/4 E Recommended for confirmation of ESBL production in Escherichia coli, Klebsiella spp., P. mirabilis, clavulanic acid Salmonella spp., and Shigella spp. Cefotaxime 0.25-0.5-1-2-4-8-16-32 A Cefotaxime- 1/4-2/4-4/4-8/4 E Recommended for confirmation of ESBL production in Escherichia coli, Klebsiella spp., Proteus clavulanic acid mirabilis, Salmonella spp., and Shigella spp. Cefixime 0.5-1-2-4-8-16 C Breakpoints defined for uncomplicated urinary tract infection only. ECOFF has not yet been defined. Cefepime 0.125-0.25-0.5-1-2-4-8-16-32 A Cefepime- 1/4-2/4-4/4-8/4 E Recommended for confirmation of ESBL production in Enterobacter spp., Citrobacter freundii complex, clavulanic acid Morganella morganii, Providencia stuartii, Serratia spp., and Hafnia alvei. It is also useful for E. coli hyperproducing chromosomal AmpC or producing plasmidic AmpC. Ceftolozane- 0.5/4-1/4-2/4-4/4-8/4 C ECOFF has not yet been defined. For susceptibility testing purposes, the concentration of tazobactam is tazobactam fixed at 4 mg/L. Ceftazidime- 0.5/4-1/4-2/4-4/4-8/4-16/4 C ECOFF has not yet been defined. It can be used to infer the presence of class A and class D avibactam carbapenemases in isolates that are resistant to carbapenems. For susceptibility testing purposes, the 1 Recommendations of the Spanish Antibiogram Committee (COESANT) for selecting antimicrobial agents and concentrations for in vitro susceptibility studies using automated systems concentration of avibactam is fixed at 4 mg/L. Aztreonam 0.25-0.5-1-2-4-8-16-32 A Imipenem 0.25-0.5-1-2-4-8-16 A >1 mg/L has been defined as screening cut-off for carbapenemase production. Breakpoints for M. morganii, Proteus spp. and Providencia spp. are S ≤ 0.125 mg/L and R >4 mg/L Meropenem 0.125-0.25-0.5-1-2-4-8-16 A >0.125 mg/L has been defined as screening cut-off for carbapenemase production. Meropenem- 0.125-0.25-0.5-1-2-4-8-16 C ECOFF has not yet been defined. It can be used to infer the presence of class A carbapenemases in vaborbactam isolates that are resistant to carbapenems. For susceptibility testing purposes, the concentration of vaborbactam is fixed at 8 mg/L. Ertapenem 0.06-0.125-0.25-0.5-1-2-4 A >0.125 mg/L has been defined as screening cut-off for carbapenemase production. ECOFF has not yet been defined. Aminoglycosides Gentamicin 2-4-8 A Breakpoints are based on once daily administration of high dose. Tobramycin 2-4-8 A Amikacin 2-4-8-16-32 A Quinolones Nalidixic acid 8-16-32 E Breakpoints have not been defined. It can be useful to infer the presence of mutations in topoisomerases and/or plasmid-mediated fluoroquinolone resistance genes. Ciprofloxacin 0.06-0.125-0.25-0.5-1-2 A Norfloxacin 0.25-0.5-1-2-4 D Breakpoints defined for uncomplicated urinary tract infection only. Tetracyclines Minocycline 0.5-1-2-4-8 C ECOFF has not yet been defined. Breakpoints have not been defined by EUCAST, those shown are recommended by COESANT. Tigecycline 0.25-0.5-1-2-4 B ECOFF has not yet been defined. Eravacycline 0.25-0.5-1-2-4 C ECOFF has not yet been defined. Others Azithromycin 16-32 C Only for Salmonella and Shigella spp. Breakpoints have not been defined by EUCAST, those shown are recommended by COESANT. Nitrofurantoin 32-64-128 D Breakpoints defined for E. coli in uncomplicated urinary tract infection only. Cotrimoxazole 1/19-2/38-4/76-8/152 A Fosfomycin 8-16-32-64-128 B Breakpoints for oral (uncomplicated urinary tract infections) and iv formulations are the same. Chloramphenicol 4-8-16-32 C It can be useful to infer the presence of certain efflux pumps or to study in multi-drug resistant isolates. Colistin 0.5-1-2-4-8 B Bold numbers indicate the minimum number of concentrations that are recommended to be included in the study of susceptibility testing to address the objectives explained in the text. Underlined numbers indicate the ECOFF values, when lacking, this is due to the absence of definition by EUCAST. When different ECOFF values exist for the different enterobacterial species, the E. coli ECOFF value is indicated in the table. Greyed numbers indicate clinical categories: light grey corresponds to concentrations within intermediate (I) category and dark gray corresponds to concentrations within resistant (R) category. 2 Recommendations of the Spanish Antibiogram Committee (COESANT) for selecting antimicrobial agents and concentrations for in vitro susceptibility studies using automated systems TABLE S2. Antibiotics and concentrations recommended for the susceptibility testing of Pseudomonas spp. Antimicrobial agent Concentrations (mg/L) Category Comments ß-lactams Ticarcillin 8-16-32-64 E Breakpoints are based on high dose therapy. Not currently used in the clinical setting but useful for the inference of resistance mechanisms such as acquired β-lactamases and/or efflux pump overexpression. ECOFF has not yet been defined. Piperacillin 4-8-16-32-64 C Breakpoints are based on high dose therapy. Piperacillin- 4/4-8/4-16/4-32/4-64/4 A Breakpoints are based on high dose therapy. For susceptibility testing purposes, the concentration of tazobactam tazobactam is fixed at 4 mg/L. Ceftazidime 1-2-4-8-16-32 A Breakpoints are based on high dose therapy. Cefepime 1-2-4-8-16-32 A Breakpoints are based on high dose therapy. Ceftolozane- 0.25/4-0.5/4-1/4-2/4-4/4-8/4-16/4 C Useful for the detection of resistance mechanisms, particularly acquired β-lactamases. For susceptibility tazobactam testing purposes, the concentration of tazobactam is fixed at 4 mg/L. Ceftazidime- 0.5/4-1/4-2/4-4/4-8/4-16/4-32/4 C ECOFF has not yet been defined. Useful for the detection of resistance mechanisms, particularly avibactam acquired β-lactamases. Aztreonam 1-2-4-8-16-32 A Breakpoints are based on high dose therapy. Useful for the detection of resistance mechanisms such as acquired MBLs. Imipenem 0.5-1-2-4-8-16 A Breakpoints are based on high dose therapy. Meropenem 0.25-0.5-1-2-4-8-16 A Meropenem- 0.125-0.25-0.5-1-2-4-8-16 C ECOFF has not yet been defined. For susceptibility testing purposes, the concentration of vaborbactam vaborbactam is fixed at 8 mg/L. Aminoglycosides Gentamicin 2-4-8 A Breakpoints are based on once daily administration of high dose therapy. Tobramycin 1-2-4-8 A Amikacin 2-4-8-16-32 A Fluoroquinolones Ciprofloxacin 0.125-0.25-0.5-1-2-4 A Breakpoints are based on high dose therapy. Levofloxacin 0.25-0.5-1-2-4-8 C Breakpoints are based on high dose therapy. Others Fosfomycin 16-32-64-128-256 C, D Breakpoints are not defined. Infections caused by wild type isolates (ECOFF 128 mg/L) have been treated with combinations of fosfomycin and other agents. Colistin 0.5-1-2-4-8 B Bold numbers indicate the minimum number of concentrations that are recommended to be included in the study of susceptibility testing to address the objectives explained in the text. Underlined numbers indicate the ECOFF values, when lacking is due to the absence of definition of this value by EUCAST. Greyed numbers indicate clinical categories: light grey corresponds to concentrations within intermediate (I) category and dark grey corresponds to concentrations within resistant (R) category. MBL: metallo-β-lactamases 3 Recommendations of the Spanish Antibiogram Committee (COESANT) for selecting antimicrobial agents and concentrations for in vitro susceptibility studies using automated systems TABLE S3. Antibiotics and concentrations recommended for the susceptibility testing of Acinetobacter spp. Antimicrobial agent Concentrations (mg/L) Category Comments ß-lactams Ampicillin- 4/2-8/4-16/8-32/16 B Breakpoints have not been defined by EUCAST, those shown are recommended by COESANT. sulbactam Piperacillin- 4/4-8/4-16/4-32/4-64/4 B Breakpoints have not been defined by EUCAST, those shown are recommended by COESANT. tazobactam Ceftazidime 2-4-8-16-32 B Breakpoints have not been defined by EUCAST, those shown are recommended by COESANT. Imipenem 0.5-1-2-4-8-16 A Breakpoints are based on high dose therapy. Meropenem 0.25-0.5-1-2-4-8-16 A Aminoglycosides Gentamicin 2-4-8 A Breakpoints are based on once daily administration of high dose therapy. Tobramycin 1-2-4-8 A Amikacin 2-4-8-16-32 A Fluoroquinolones Ciprofloxacin 0.06-0.125-0.25-0.5-1-2 A Levofloxacin 0.25-0.5-1-2-4-8 C Tetracyclines Doxycycline 2-4-8-16 A ECOFF has not yet been defined Breakpoints have not been defined by EUCAST, those shown are Minocycline 2-4-8-16 A recommended by COESANT. Tigecycline 0.25-0.5-1-2-4 A Others Cotrimoxazole 1/19-2/38-4/76-8/152 B Colistin 0.5-1-2-4-8 A Rifampicin 2-4-8 C Breakpoints have not been defined by EUCAST, those shown are recommended by COESANT. Bold numbers indicate the minimum number of concentrations that are recommended to be included in the study of susceptibility testing to address the objectives explained in the text. Underlined numbers indicate the ECOFF values, when lacking is due to the absence of definition of this value by EUCAST. Greyed numbers indicate clinical categories: light grey corresponds to concentrations within intermediate (I) category and dark gray corresponds to concentrations within resistant (R) category. 4 Recommendations of the Spanish Antibiogram Committee (COESANT) for selecting antimicrobial agents and concentrations for in vitro susceptibility studies using automated systems Table S4. Antibiotics and concentrations recommended for the susceptibility testing of Stenotrophomonas maltophilia. Antimicrobial agent Concentrations (mg/L) Category Comments ß-lactams Imipenem 0.5-1-2-4-8-16 E S. maltophilia is intrinsically resistant to all β-lactams. Imipenem MIC values >8 mg/L supports identification. Fluoroquinolones Levofloxacin 0.25-0.5-1-2-4-8 A ECOFF has not yet been defined. Breakpoints have not been defined by EUCAST, those shown are recommended by COESANT. Tetracyclines Minocycline 1-2-4-8-16 A Breakpoints have not been defined by EUCAST, those shown are recommended by COESANT. Others Cotrimoxazole 1/19-2/28-4/76-8/152 A Bold numbers indicate the minimum number of concentrations that are recommended to be included in the study of susceptibility testing to address the objectives explained in the text. Underlined numbers indicate the ECOFF values, when lacking is due to the absence of definition of this value by EUCAST. Greyed numbers indicate clinical categories: light grey corresponds to concentrations within intermediate (I) category and dark gray corresponds to concentrations within resistant (R) category. 5 Recommendations of the Spanish Antibiogram Committee (COESANT) for selecting antimicrobial agents and concentrations for in vitro susceptibility studies using automated systems Table S5. Antibiotics and concentrations recommended for the susceptibility testing of non-fermentative Gram-negative bacilli other than Pseudomonas spp., Acinetobacter spp. and Stenotrophomonas maltophilia. The ECOFF values are not indicated due to this table is for miscellaneous microorganisms for which in many cases ECOFFs have not been defined. Antimicrobial agent Concentrations (mg/L) Category Comments ß-lactams Ticarcillin 8-16-32-64 E Breakpoints have not been defined by EUCAST, those shown are recommended by COESANT. Piperacillin- 4/4-8/4-16/4-32/4-64/4 A For their definition, general criteria included in the EUCAST guidance document "Antimicrobial tazobactam susceptibility tests on groups of organisms or agents for which there are no EUCAST breakpoints Ceftazidime 1-2-4-8-16-32 A (http://www.eucast.org/clinical_breakpoints/when_there_are_no_breakpoints) have been followed Cefepime 1-2-4-8-16-32 B It is also recommended to consult the EUCAST intrinsic resistance tables for those species included in Aztreonam 0.5-1-2-4-8-16-32 B these tables (http://www.eucast.org/expert_rules_and_intrinsic_resistance/) Imipenem 0.5-1-2-4-8-16 A Meropenem 0.5-1-2-4-8-16 A Aminoglycosides Gentamicin 2-4-8 E Tobramycin 1-2-4-8 A Amikacin 2-4-8-16-32 A Fluoroquinolones Ciprofloxacin 0.125-0.25-0.5-1-2-4 A Levofloxacin 0.25-0.5-1-2-4-8 A Tetracyclines Minocycline 2-4-8-16 A Others Cotrimoxazole 1/19-2/38-4/76-8/152 A Chloranfenicol 4-8-16-32 C Colistin 0.5-1-2-4-8 C Bold numbers indicate the minimum number of concentrations that are recommended to be included in the study of susceptibility testing to address the objectives explained in the text. Greyed numbers indicate clinical categories: light grey corresponds to concentrations within intermediate (I) category and dark gray corresponds to concentrations within resistant (R) category. Breakpoints have not been defined by EUCAST for these microorganisms; PK/PD breakpoints were used when available and when not COESANT recommendations were followed. 6 Recommendations of the Spanish Antibiogram Committee (COESANT) for selecting antimicrobial agents and concentrations for in vitro susceptibility studies using automated systems TABLE S6. Antimicrobial agents and concentrations for testing and reporting the susceptibility for Staphylococcus spp. ECOFF values in this table are those from S. aureus. Antimicrobials Concentrations (mg/L) Category Comments ß-lactams Penicillin 0.06-0.125-0.25-0.5-1 A Oxacillin 0.25-0.5-1-2-4-8 A S. aureus, S. lugdunensis and S. saprophyticus with oxacillin MICs >2 mg/L are mostly methicillin (S. aureus, S. resistant due to the presence of the mecA or mecC genes. lugdunensis, S. saprophyticus) Oxacillin 0.25-0.5-1-2-4-8 A Coagulase-negative staphylococci other than S. saprophyticus and S. lugdunensis with oxacillin MICs (CNS other than >0.25 mg/L are mostly resistant due to the presence of the mecA gene. S. lugdunenis, S. saprophyticus) Cefoxitin 2-4-8 E S. aureus and S. lugdunensis with cefoxitin MIC values >4 mg/L and S. saprophyticus with cefoxitin MIC values >8 mg/L are methicillin resistant, mostly due to the presence of the mecA or mecC genes. For staphylococci other than S. aureus, S. lugdunensis and S. saprophyticus, the cefoxitin MIC is a poorer predictor of methicillin resistance than the disk diffusion test. Ceftaroline 0.25-0.5-1-2-4 B Methicillin-susceptible isolates can be reported susceptible to ceftaroline or ceftobiprole without further Ceftobiprole 0.5-1-2-4-8 B testing. Aminoglycosides Gentamicin 0.5-1-2-4-8 A Breakpoints are based on once daily administration of high dose therapy. Tobramycin 0.5-1-2-4-8 A Glycopeptides Vancomycin 0.5-1-2-4-8-16 A S. aureus with vancomycin MIC values of 2 mg/L are on the border of the wild type distribution and there may be an impaired clinical response. Teicoplanin (S. 1-2-4-8-16-32 A aureus) Teicoplanin 1-2-4-8-16-32 A ECOFFs have not yet been defined. (CNS) Lipoglycopeptides Telavancin 0.06-0.125-0.25 C Only approved for MRSA. MICs must be determined in the presence of polysorbate-80 (0.002% in the (MRSA) medium for broth dilution methods; agar dilution methods have not been validated). Dalbavancin 0.06-0.125-0.25 C MICs must be determined in the presence of polysorbate-80 (0.002% in the medium for broth dilution Oritavancin 0.06-0.125-0.25 C methods; agar dilution methods have not been validated). (S. aureus) 7 Recommendations of the Spanish Antibiogram Committee (COESANT) for selecting antimicrobial agents and concentrations for in vitro susceptibility studies using automated systems Lipopeptides Daptomycin 0.5-1-2-4 A MICs must be determined in the presence of Ca2+ (50 mg/L) in the medium for broth dilution methods; agar dilution methods have not been validated. Fluoroquinolones Ciprofloxacin 0.5-1-2-4 A Breakpoints are based on high dose therapy. Levofloxacin 0.5-1-2-4 A Moxifloxacin 0.125-0.25-0.5-1-2-4 C Macrolides and Erithromycin 0.5-1-2-4 A Erythromycin can be used to determine susceptibility to azithromycin, clarithromycin and lincosamides roxithromycin. Clindamycin 0.125-0.25-0.5-1-2 A Erithromycin- 4/0.5 E Inducible clindamycin resistance test. In a positive test, report as clindamycin resistant and consider Clindamycin adding this comment to the report: "Clindamycin may still be used for short-term therapy of less serious skin and soft tissue infections as constitutive resistance is unlikely to develop during such therapy". Tetracyclines Tetracycline 0.5-1-2-4-8 B Isolates susceptible to tetracycline are also susceptible to doxycycline and minocycline, although some resistant to tetracycline may still be susceptible to minocycline and/or doxycycline. Minocycline 0.125-0-25-0.5-1-2 C Tigecycline 0.25-0.5-1-2 C Eravacycline 0.125-0.25-0.5-1 C ECOFFs have not yet been defined. Oxazolidinones Linezolid 1-2-4-8 A Isolates susceptible to linezolid can be reported susceptible to tedizolid. Tedizolid 0.25-0.5-1-2 B Others Fosfomycin 8-16-32-64-128 B Use in combination in serious infections (i.e endocarditis). Breakpoints are not defined for oral use. Cotrimoxazole 0.25/4.75-0.5/9.5-1/19-2/38- A 4/76-8/152 Rifampicin 0.01-0.03-0.06-0.125-0.25-0.5- B 1-2 Mupirocin 0-5-1-2-4-256 B Breakpoint related to nasal decolonization of S. aureus. Intermediate isolates are associated to short term suppression (useful preoperatively) but unlike susceptible isolates, long-term eradication rates are low. Fusidic acid 0.25-0.5-1-2-4 B Nitrofurantoin 16-32-64-128 D Bold numbers indicate the minimum number of concentrations that are recommended to be included in the study of susceptibility testing to address the objectives explained in the text. Underlined numbers indicate the ECOFF values, when lacking is due to the absence of definition of this value by EUCAST. Greyed numbers indicate clinical categories: light grey corresponds to concentrations within intermediate (I) category and dark gray corresponds to concentrations within resistant (R) category. CNS: coagulase negative staphylococci. 8 Recommendations of the Spanish Antibiogram Committee (COESANT) for selecting antimicrobial agents and concentrations for in vitro susceptibility studies using automated systems Table S7. Antimicrobial agents and concentrations for testing and reporting the susceptibility for Streptococcus pneumoniae and other streptococci (including viridans streptococci and β-haemolytic groups A, B, C and G). Unless indicated in comments, breakpoints in this table are those recommended for S. pneumoniae. ECOFF values have not been indicated in this table as different values have been defined for different species/group. Category Comments Antimicrobial agents Concentrations (mg/L) S. pneumoniae β-haemolytic Viridans group streptococci streptococci ß-lactámicos Penicillin 0.06-0.12-0.25-0.5-1-2-4 A A A Breakpoints (S. pneumoniae) are those recommended for meningitis. For infections other than meningitis oral penicillin V breakpoints are S ≤0.06 mg/L / R>2 mg/L, and penicillin parenteral breakpoints are S ≤2 mg/L / R>4 mg/L. Ampicillin 0.25-0.5-1-2-4 A A A Breakpoints (S. pneumoniae) are those recommended for infections other than meningitis. Cefuroxime 0.125-0.25-0.5-1-2 C C C Breakpoints (S. pneumoniae) defined for oral administration are one dilution step lower than those for i.v administration. Cefotaxime 0.125-0.25-0.5-1-2-4 A A A Cefepime 0.25-0.5-1-2-4 C C C Ceftaroline 0.25-0.5-1 B C C Meropenem 0.125-0.25-0.5-1-2-4 B C C Meropenem is the only carbapenem recommended for meningitis. Ertapenem 0.25-0.5-1-2-4 C C B Imipenem 0.06-0.125-0.25-0.5-1-2 C C C Glycopeptides Vancomycin 0.5-1-2-4 C C A Lipopeptides Daptomycin 0.5-1-2 - C C Breakpoints have not been defined by EUCAST for S. pneumoniae, those shown are recommend by COESANT, which are also the same for β-haemolytic groups A, B, C and G. Lipoglycopeptides Dalbavancin 0.06-0.125-0.25-0.5 - C C Breakpoints have not been defined by EUCAST for S. pneumoniae, those shown are for S. anginosus group and β- heamolytic groups A, B, C and G. Quinolones Levofloxacin 0.5-1-2-4 A A A Breakpoints are based on high dose therapy. Breakpoints 9 Recommendations of the Spanish Antibiogram Committee (COESANT) for selecting antimicrobial agents and concentrations for in vitro susceptibility studies using automated systems have not been defined by EUCAST for viridans group streptococci, those shown are recommended for S. pneumoniae. Moxifloxacin 0.25-0.5-1-2 C C C Macrolides and Erithromycin 0.25-0.5-1-2 A A A Breakpoints have not been defined by EUCAST for viridans lincosamides group streptococci, those shown are recommended for S. pneumoniae. Erithromycin- 4/0.5 E E E Inducible clindamycin resistance test. Clindamycin Josamycin 0.5-1-2 C C C Breakpoints have not been defined by EUCAST, those shown are recommend by COESANT Clindamycin 0.5-1-2 A A A Tetracyclines Tetracycline 0.5-1-2-4 A C A Breakpoints have not been defined by EUCAST for viridans group streptococci, those shown are recommend for S. pneumoniae Others Linezolid 1-2-4-8 C C C Tedizolid 0.125-0.25-0.5 C C C Breakpoints have not been defined by EUCAST for S. pneumoniae, those shown are recommend for S. anginosus group Chloramphenicol 4-8-16 C C C Breakpoints have not been defined by EUCAST for viridans group streptococci, those shown are recommend for S. pneumoniae Cotrimoxazole 0.5/9.5-1/19-2/38-4/76 B B C Rifampicin 0.03-0.06-0.125-0.25-0.5-1-2 A B C Breakpoints have not been defined by EUCAST for viridans group streptococci, those shown are recommend for S. pneumoniae Bold numbers indicate the minimum number of concentrations that are recommended to be included in the study of susceptibility testing to address the objectives explained in the text. Greyed numbers indicate clinical categories: light grey corresponds to concentrations within intermediate (I) category and dark gray corresponds to concentrations within resistant (R) category. Unless indicated, breakpoints in this table are for S. pneumoniae. 10 Recommendations of the Spanish Antibiogram Committee (COESANT) for selecting antimicrobial agents and concentrations for in vitro susceptibility studies using automated systems TABLE S8. Antibiotics and concentrations recommended for the susceptibility testing of Enterococcus spp. Antimicrobial agent Concentrations (mg/L) Category Comments ß-lactams Ampicillin 1-2-4-8-16 A Susceptibility to ampicillin-sulbactam and to amoxicillin or piperacillin with and without β-lactamase inhibitors can be inferred from ampicillin. E. faecium resistant to penicillins can be considered resistant to all other ß-lactam agents including carbapenems. β-lactamase-producing isolates have been very unfrequently reported in some countries. These isolates may present ampicillin MIC values ≤4 mg/L and can be detected by the nitrocefin test. Aminoglycosides Gentamicin 128-500 A High-level resistance to gentamicin (MIC >128 mg/L) determines resistance to all aminoglycosides, except streptomycin. It also determines loss of synergism of all aminoglycosides (except streptomycin) with ß-lactams and glycopeptides. Streptomycin 512-1000 A High level-resistance to streptomycin (MIC >512 mg/L) determines the lost of synergy of this aminoglycoside with ß-lactams and glycopeptides. Kanamycin 1000 E This antibiotic can be used to predict high-level resistance to amikacin in non-high-level gentamicin resistant enterococci. Glycopeptides Vancomycin 1-2-4-8-16-32 A Teicoplanin 1-2-4-8-16-32 A Lipoglycopeptides Dalbavancin 0.125-0.25-0.5 C ECOFFs have not been defined. Breakpoints have not been defined by EUCAST, those shown are recommended by COESANT. Lipopeptides Daptomycin 1-2-4-8 B MICs must be determined in the presence of Ca2+ (50 mg/L in the medium for broth dilution methods; agar dilution methods have not been validated). Breakpoints have not been defined by EUCAST, those shown are recommended by COESANT. Quinolones Ciprofloxacin 1-2-4-8 D Defined only for uncomplicated urinary tract infections Levofloxacin 1-2-4-8 D Macrolides Erythromycin 0.5-1-2-4-8 E Breakpoints have not been defined by EUCAST. The ECOFF (4 mg/L) is used to infer resistant population for epidemiological purposes. Tetracyclines Tetracycline 2-4-8-16 E Breakpoints have not been defined by EUCAST. The ECOFF is used to infer resistant population for epidemiological purposes. Tigecycline 0.12-0.25-0.5-1-2-4 B Isolates with MIC values above the susceptible breakpoint are very rare. Eravacycline 0.06-0.125-0.25 C ECOFFs have not yet been defined. 11 Recommendations of the Spanish Antibiogram Committee (COESANT) for selecting antimicrobial agents and concentrations for in vitro susceptibility studies using automated systems Others Linezolid 0.5-1-2-4-8 A Fosfomycin 32-64-128-256 D ECOFFs have not yet been defined. Breakpoints have not been defined by EUCAST, those shown are recommended by COESANT. ECOFF has not yet been defined. Cotrimoxazole 0.5/9.5-1/19-2/38-4/76-8/152 E The activity of trimethoprim-sulfamethoxazole is uncertain against enterococci due to their ability to incorporate exogenously produced folates (which may be found in highly variable concentrations in the urine), so the wild type population is categorized as intermediate (susceptible, increased exposure). ECOFF value has been only defined for E. faecium. Nitrofurantoin 16-32-64-128 D Breakpoints apply to E. faecalis only. Bold numbers indicate the minimum number of concentrations that are recommended to be included in the study of susceptibility testing to address the objectives explained in the text. Underlined numbers indicate the ECOFF values (most of them from E. faecalis), when lacking is due to the absence of definition of this value by EUCAST. Greyed numbers indicate clinical categories: light grey corresponds to concentrations within intermediate (I) category and dark gray corresponds to concentrations within resistant (R) category. 12 Recommendations of the Spanish Antibiogram Committee (COESANT) for selecting antimicrobial agents and concentrations for in vitro susceptibility studies using automated systems TABLE S9. Antibiotics and concentrations recommended for the susceptibility testing of Haemophilus spp. These recommendations have been mainly performed for H. influenzae however they can be also applied for H. parainfluenzae Antimicrobial agent Concentrations (mg/L) Category Comments ß-lactams Ampicillin 0.25-0.5-1-2-4-8-16 A BLNAR* strains are usually referred to ampicillin. Breakpoints are based on intravenous administration. Amoxicillin 0.25-0.5-1-2-4-8-16 B Amoxicillin/ 0.25-0.5-1-2-4-8 A For susceptibility testing purposes, the concentration of clavulanic acid is fixed at 2 mg/L. clavulanic acid Cefuroxime 0.125-0.25-0.5-1-2-4-8-16 A This concentration range is useful both i.v and oral cefuroxime. Indicated breakpoints are those for oral administration. Breakpoints for i.v administration are S= 1 mg/L and R >2 mg/L. Cefotaxime 0.06-0.125-0.25-0.5-1-2-4 A Reported in invasive infections. Cefotaxime susceptibility can be used to infer that of ceftriaxone. Cefepime 0.06-0.125-0.25-0.5-1-2-4 B Meropenem 0.06-0.125-0.25-0.5-1-2-4 B This concentration range is useful for meningitis and other infections. Only reported in nervous central infections. Indicated breakpoints are those for meningitis. Breakpoints for infections other than meningitis are S ≤ 2 mg/L and R >2 mg/L. Quinolones Nalidixic acid 4 E Breakpoints have not been defined by EUCAST. Breakpoints for screening purposes have been defined Ciprofloxacin 0.03-0.06-0.125-0.25-0.5-1-2-4 A by COESANT. It can be useful to infer the presence of mutations in topoisomerases. Isolates categorized Levofloxacin 0.03-0.06-0.125-0.25-0.5-1-2-4 A as S to nalidixic acid (<4 mg/L) can be reported S to ciprofloxacin, levofloxacin and moxifloxacin. Isolates categorized as non-susceptible may have fluoroquinolone resistance and should be tested against the appropriate agent. Ciprofloxacin can better detect the presence of mutations in topoisomerases than levofloxacin. MLSB Azithromycin 0.125-0.25-0.5-1-2-4-8-16 A Correlation between macrolide MICs and clinical outcome is weak for H. influenzae. Therefore, breakpoints for macrolides and related antibiotics have been set to categorize wild type H. influenzae as intermediate (susceptible, increased exposure). Tetracyclines Tetracycline 0.25-0.5-1-2-4-8-16 B Isolates susceptible to tetracycline are also susceptible to doxycycline and minocycline, although some Minocycline 0.25-0.5-1-2-4-8-16 E resistant to tetracycline may still be susceptible to minocycline and/or doxycycline. Others Cotrimoxazole 0.25/4.75-0.5/9.5-1/19-2/38-4/76 A Rifampicin 0.5-1-2-4 C Only for prophylaxis. Chloramphenicol 0.5-1-2-4-8 C Bold numbers indicate the minimum number of concentrations that are recommended to be included in the study of susceptibility testing to address the objectives explained in the text. Underlined numbers indicate the ECOFF values, when lacking is due to the absence of definition of this value by EUCAST. Greyed numbers indicate clinical categories: light grey corresponds to concentrations within intermediate (I) category and dark gray corresponds to concentrations within resistant (R) category. BLNAR*: β-negative ampicillin-resistant. 13