Antimicrobial Susceptibility Testing: A Comprehensive Review PDF

antibiotics Review Antimicrobial Susceptibility Testing: A Comprehensive Review of Currently Used Methods Ina Gajic 1, * , Jovana Kabic 1 , Dusan Kekic 1 , Milos Jovicevic 1 , Marina Milenkovic 2 , Dragana Mitic Culafic 3 , Anika Trudic 4,5 , Lazar Ranin 1 and Natasa Opavski 1...

antibiotics Review Antimicrobial Susceptibility Testing: A Comprehensive Review of Currently Used Methods Ina Gajic 1, * , Jovana Kabic 1 , Dusan Kekic 1 , Milos Jovicevic 1 , Marina Milenkovic 2 , Dragana Mitic Culafic 3 , Anika Trudic 4,5 , Lazar Ranin 1 and Natasa Opavski 1 1 Institute of Microbiology and Immunology, Faculty of Medicine, University of Belgrade, 11000 Belgrade, Serbia; [email protected] (J.K.); [email protected] (D.K.); [email protected] (M.J.); [email protected] (L.R.); [email protected] (N.O.) 2 Department of Microbiology and Immunology, Faculty of Pharmacy, University of Belgrade, 11000 Belgrade, Serbia; [email protected] 3 Faculty of Biology, University of Belgrade, 11000 Belgrade, Serbia; [email protected] 4 Faculty of Medicine, University of Novi Sad, 21000 Novi Sad, Serbia; [email protected] 5 Institute for Pulmonary Diseases of Vojvodina, Sremska Kamenica, 21204 Novi Sad, Serbia * Correspondence: [email protected]; Tel.: +381-629-726-331 Abstract: Antimicrobial resistance (AMR) has emerged as a major threat to public health globally. Accurate and rapid detection of resistance to antimicrobial drugs, and subsequent appropriate antimicrobial treatment, combined with antimicrobial stewardship, are essential for controlling the emergence and spread of AMR. This article reviews common antimicrobial susceptibility testing (AST) methods and relevant issues concerning the advantages and disadvantages of each method. Although accurate, classic technologies used in clinical microbiology to profile antimicrobial susceptibility


are time-consuming and relatively expensive. As a result, physicians often prescribe empirical
 antimicrobial therapies and broad-spectrum antibiotics. Although recently developed AST systems Citation: Gajic, I.; Kabic, J.; Kekic, D.; have shown advantages over traditional methods in terms of testing speed and the potential for Jovicevic, M.; Milenkovic, M.; Mitic providing a deeper insight into resistance mechanisms, extensive validation is required to translate Culafic, D.; Trudic, A.; Ranin, L.; Opavski, N. Antimicrobial these methodologies to clinical practice. With a continuous increase in antimicrobial resistance, Susceptibility Testing: A additional efforts are needed to develop innovative, rapid, accurate, and portable diagnostic tools Comprehensive Review of Currently for AST. The wide implementation of novel devices would enable the identification of the optimal Used Methods. Antibiotics 2022, 11, treatment approaches and the surveillance of antibiotic resistance in health, agriculture, and the 427. https://doi.org/10.3390/ environment, allowing monitoring and better tackling the emergence of AMR. antibiotics11040427 Academic Editor: Eleonora Nicolai Keywords: antimicrobial susceptibility testing; antimicrobial resistance; methods Received: 28 February 2022 Accepted: 18 March 2022 Published: 23 March 2022 1. The Emergence of Antimicrobial Resistance and Overlooked Pandemic Publisher’s Note: MDPI stays neutral Antimicrobial resistance (AMR) remains the world’s most urgent public health con- with regard to jurisdictional claims in cern. According to the World Health Organization (WHO), Geneva, Switzerland, antibiotic published maps and institutional affil- resistance is rising to dangerously high levels in all parts of the world, leading to increased iations. morbidity and mortality. Hence, the six leading mortality-causing pathogens—Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter bauman- nii, and Pseudomonas aeruginosa—were responsible for 929,000 deaths attributable to AMR and 3.57 million deaths associated with AMR in 2019. This number could rise to 10 million by Copyright: © 2022 by the authors. 2050 according to estimates by the WHO. Furthermore, the SARS-CoV-2 pandemic has Licensee MDPI, Basel, Switzerland. exacerbated the existing global crisis of AMR, mostly due to the mis- and over-use of antibi- This article is an open access article distributed under the terms and otics, treatments that induce immunosuppression, and prolonged hospitalisation. Besides, conditions of the Creative Commons during the COVID-19 pandemic, limited ability to work with AMR partnerships, decreases in Attribution (CC BY) license (https:// funding, and reduced availability of nursing, medical, and public health staff affected AMR creativecommons.org/licenses/by/ surveillance, prevention, and control. In addition, increased use of disinfectants, including 4.0/). hand sanitisers and surface cleaners, is anticipated to cause increased rates of antimicrobial Antibiotics 2022, 11, 427. https://doi.org/10.3390/antibiotics11040427 https://www.mdpi.com/journal/antibiotics Antibiotics 2022, 11, 427 2 of 26 resistance in pathogenic microbes in the coming years. Replacement of first-line antibiotics by more expensive medications, a longer duration of illness, and treatment-related to AMR increases healthcare costs as well as the economic burden on patients and societies. The World Bank estimates that drug-resistant infections could cause a global economic crisis, leading to 28 million people who could be pushed into extreme poverty every year by 2050, with an overall cost to the global economy of USD 1 trillion per year. Throughout their evolution, bacteria have developed versatile resistance mechanisms to antibiotics. The four main mechanisms of AMR are enzymatic inactivation of antimicro- bial compounds, alteration of a drug target, reduced permeability of the outer membrane, and active drug efflux. Hydrolases (e.g., beta-lactamases encoding by bla genes, such as extended-spectrum beta-lactamases, ESBL; cephalosporinases; and carbapenemases), passivation, and modified enzymes are three of the most important drug-inactivating enzymes. An altered target site is a major cause of Gram-positive bacteria’s drug resistance (e.g., PBP2a in methicillin-resistant S. aureus, MRSA by the acquisition of the mecA gene and other homologues), as well as polymyxin-resistant bacteria. The membrane permeability is a key in the level of susceptibility to antibiotics in some bacteria, such as Enterobacterales. Modification of the bacterial envelope by decreasing the porin production or increasing the expression of efflux pump systems (e.g., M phenotype in Streptococcus spp. encoding by mefA gene) has been reported. The causes of antimicrobial resistance are complex and multifaceted. In countries where antibiotics are sold without a prescription or used as growth-promoting substances or prophylactic additives in livestock farming, antibiotic-resistant bacteria develop especially fast. Administration of antibiotics to patients with suspected moderate to severe bacterial infections has been deemed inappropriate in at least half of the cases. Antimicrobial stewardship (AMS) is one of the key strategies for combatting resistance. Implementation of such programs is therefore recommended across the globe. The present review provides an updated overview of the various antimicrobial sus- ceptibility testing (AST) methods that are currently used or potentially applicable in the foreseeable future, as well as their advantages and disadvantages. 2. The Rationale for Performing Susceptibility Testing The choice of the best therapeutic option for the treatment of bacterial infections relies on the results of AST, a part of the routine work of all clinical microbiological laboratories. These reports provide insight into local patterns of antimicrobial susceptibility, helping physicians to choose the most effective antibiotic therapy. For instance, if the AMR rate of a pathogen is above 20%, that drug should not be administered as a single empiric ther- apy for infection treatment. Evaluation of the effectiveness of prevention and infection control measures relies as well on the results of AST, e.g., monitoring of resistant pathogens such as MRSA (methicillin-resistant Staphylococcus aureus), VRE (vancomycin-resistant enterococci), extended-spectrum beta-lactamase (ESBL)- and carbapenemase-producing En- terobacterales, carbapenem-resistant Acinetobacter baumannii (CRAB), carbapenem-resistant Pseudomonas aeruginosa (CRPA), colistin-resistant bacteria, etc.. Finally, surveillance of antimicrobial resistance is based on routine clinical antimicrobial susceptibility data from microbiological laboratories. Numerous AMR surveillance systems exist, of which the WHOs Global Antimicrobial Resistance and Use Surveillance System (GLASS), European Antimicrobial Resistance Surveillance Network (EARS-Net), and Antibiotic Resistance Laboratory Network (AR Lab Network) of the Centers for Disease Control and Prevention are the most recognizable networks of national surveillance systems providing information on the actual burden of resistance at the international level. Policymakers and health administrators revise the recommendations for empirical treatment for community or hospital-acquired infections according to the local, national, and international AMR data. In addition, prevention and infection control measures are implemented based on the same data as a part of AMS programs [16,17]. Likewise, continuous monitoring provides early warnings of emerging threats and identifies long-term resistance trends. Antibiotics 2022, 11, 427 3 of 26 Although resistance surveillance at the national and international levels is of great benefit to public health, knowledge of the local resistance rates is of even greater practical importance to physicians. An antibiogram represents a convenient and widely available measurement of an institution’s pathogens and susceptibilities. Therefore, it is in- creasingly suggested that there is the necessity to create local (hospital or institutional) antibiograms specific for each hospital and even ward, annually. This principle applies especially to certain hospital departments where resistance rates are high, such as intensive care units. Additionally, this is particularly relevant for secondary and tertiary hospitals that treat chronically ill patients who have already received multiple antibiotic courses and thus increase antimicrobial selective pressure. Klinker et al. provide the rationale for why hospital AMS programs should implement alternative antibiograms, including combination and syndromic antibiograms, in addition to traditional antibiograms. A combination antibiogram is used to determine in vitro rates of susceptibility to potential antibacterial combination regimens consisting of a first-choice antibiotic plus alternatives. A syndromic antibiogram displays the likelihood of adequate coverage for a specific infection syndrome, considering the weighted incidence of pathogens causing that syndrome. It was developed by Hebert et al. as a weighted-incidence syndromic combination antibiogram. While combination antibiograms are useful in determining combined empiric antibiotic regimens for multidrug-resistant pathogens , syndromic antibiograms provide effective antibiotic therapy for a specific infectious syndrome, such as hospital- and ventilator-associated pneumonia. The Clinical and Laboratory Standards Institute (CLSI) has developed guidelines (M39-A4) to provide a standardised template for the preparation of institu- tional antibiograms. In a retrospective study by Puzniak et al. , the utility of combination antibiograms in identifying optimal anti-P. aeruginosa drug regimens in US hospitals was evaluated. They found that adding an aminoglycoside to backbone antibiotic, such as extended-spectrum cephalosporin, carbapenem, or piperacillin-tazobactam, resulted in higher susceptibility rates than adding a fluoroquinolone. They concluded that local insti- tutional use of combination antibiograms ensures optimisation and timely administration of appropriate empiric therapy of infections caused by difficult-to-treat pathogens. Clinical laboratories currently employ several AST methods depending on the equip- ment and laboratory test menu that they provide. Conventional AST based on phenotypic testing examines the bacterial response in the presence of an antimicrobial agent. Classical culture-dependent methods (e.g., a disk diffusion test, gradient diffusion method) are firmly established in the diagnostic routine, and their main limitation is that the results are obtained for most clinically important bacteria within at least 18–24 h or 48 h, including prior bacterial isolation and identification. The turnaround time is prolonged for anaerobes or some slow-growing fastidious bacteria such as the HACEK group (Haemophilus species, Aggregatibacter species, Cardiobacterium hominis, Eikenella corrodens, and Kingella species), Brucella spp. etc.. For many years, clinical laboratories have been equipped with automated systems based on microdilution trays to provide faster results (6–24 h after initial isolation). However, the time required to obtain the results is similar in comparison with the broth microdilution (BMD) method. Molecular AST is based on the detection of resistance determinants in bacterial isolates or directly in clinical specimens by molecular methods with a turnaround time of approximately 1–6 h. Besides high costs, major drawbacks of molecular methods are detection of the resistance genes targeted only by the known probes and overestimating resistance because the resistance gene is not neces- sarily associated with the expression of a resistance phenotype. Because of a significant rise in multi- and pan-drug-resistant infections, there is an urgent need for a more rapid and reliable test to improve infection diagnosis and support evidence-based antimicrobial prescribing. The currently used methods for AST are summarised in Figure 1. Antibiotics 2022, 11, x FOR PEER REVIEW 4 of 27 Antibiotics 2022, 11, 427 antimicrobial prescribing. The currently used methods for AST are summarised in 4 of 26 Figure 1. Figure 1. Current methods for antimicrobial susceptibility testing and turnaround time (created with FigureBioRender.com, 1. Current methods foron accessed antimicrobial 27 Februarysusceptibility testingofand 2022. Reproduction thisturnaround time figure requires (created from permission with BioRender.com., accessed on 27 February 2022. Reproduction of this figure requires permission BioRender.com). PCR—polymerase chain reaction. qPCR—quantitative polymerase chain reaction. from BioRender.com). PCR—polymerase chain reaction. qPCR—quantitative polymerase chain re- NGS—next-generation sequencing. MALDI-TOF MS—matrix-assisted laser desorption/ionization action. NGS—next-generation sequencing. MALDI-TOF MS—matrix-assisted laser desorption/ion- izationtime-of-flight time-of-flightmass massspectrometry. spectrometry. 3. Commonly Used Techniques 3. Commonly Used 3.1. Classical Techniques Methods 3.1. Classical MethodsMethods: Broth Dilution and Agar Dilution 3.1.1. Dilution 3.1.1. Dilution Methods: Although newBroth Dilution have technologies and Agar beenDilution introduced to obtain data on bacterial sus- ceptibilitynew Although to antimicrobial technologies have agents, conventional been introducedtechnologies to obtain dataare onstill in widespread bacterial suscep- use. tibility to antimicrobial agents, conventional technologies are still in widespread use. Be-broth Besides disc diffusion susceptibility tests, the most widely used methods include sides macro- and microdilution disc diffusion susceptibility andtests, agar dilution, the most representing widely usedthe reference methods methods include. By broth macro- using and broth and agar and microdilution dilution agarmethods, the minimumthe dilution, representing inhibitory referenceconcentrations methods.(MICs)By of usingantimicrobial broth and agar agents (i.e.,methods, dilution the lowest theconcentration at whichconcentrations minimum inhibitory the agent inhibits the of (MICs) growth of microorganisms) antimicrobial canlowest agents (i.e., the be determined [29,30]. concentration The MIC at which valueinhibits the agent servesthe as growth the basis for of microorganisms) can be determined [29,30]. The MIC value serves as the basis for as- that assessing the susceptibility category of the pathogen to a given antibiotic, of organisms sessinggive theambiguous results, susceptibility and especially category when no of the pathogen to aclinical breakpoints given antibiotic, of for disk diffusion organisms that are give ambiguous results, and especially when no clinical breakpoints for disk diffusiondegree available. Contrary to a qualitative method, the MIC value allows assessing the are of susceptibility or resistance to the antibiotic. Besides the determination available. Contrary to a qualitative method, the MIC value allows assessing the degree of of MICs, the ad- vantage of susceptibility or broth dilution resistance methods to the is the. antibiotic possibility Besidesofthe obtaining the minimum determination of MICs, bactericidal the advantage of broth dilution methods is the possibility of obtaining the minimum bacteri- that concentration (MBC), which is the lowest concentration of an antimicrobial substance cidal kills 99.9% of bacteria concentration (MBC),. which is the lowest concentration of an antimicrobial sub- The macrodilution stance that kills 99.9% of bacteria method,.also known as the in-tube dilution test, uses serial two-fold dilution of antimicrobial substances in corresponding media. A known concentration of suspended bacteria is added to the tubes prepared, as described in. After 24 h of incubation at 37 ◦ C, bacterial growth is measured by turbidity of media, allowing visual Antibiotics 2022, 11, 427 5 of 26 determination of MIC values. Another macrodilution method is the time-kill methodol- ogy. This test allows monitoring of the effect of different concentrations of antimicrobial substances by examining the rate at which antimicrobials lead to bacterial death—i.e., the bactericidal activity of antimicrobial agents is determined depending on the concentration and time. Bacterial viability is determined by counting colonies on agar plates at regular time points for 24 h. The rate of bacterial growth is monitored via changes in log CFU/mL during the first 24 h time-kill test. Based on the results, experimental curves which represent the absence of growth or the killing effect can be constructed and give us insight into the interaction between the bacteria and the antimicrobial agent. The data can be further analysed using different mathematical models [30,34,35]. The BMD method is standardised, accurate, and inexpensive. Since it is performed in 96-well microtiter plates, it allows the testing of several antimicrobial substances in a row and eight series of two-fold dilutions of antimicrobial agents in one plate. After the dilutions are made, each well is inoculated with standardised bacterial inoculum and incubated for at least 16–24 h. Although this procedure is used as a reference method, it has been improved by the addition of a resazurin colour redox indicator. Resazurin is a blue colour that turns into pink, fluorescent resorufin in the presence of metabolically active bacterial cells. The reduction of resazurin to fluorescent resorufin can be measured fluorimetrically [27,32,36–38]. Nowadays, there are several commercially available easy to perform BMD systems such as MBD Sensititre System (Thermo Fisher Scientific, Waltham, MA, USA) and ComASP Colistin (Liofilhem, Roseto degli Abruzzi, Italy), formerly Sen- siTest Colistin. The MBD Sensititre System can be performed manually or automatically. ComASP Colistin is a compact panel containing the antibiotic colistin in seven two-fold serial dilutions and allows for four samples to be tested simultaneously with the BMD method. The agar dilution method involves adding different concentrations of antimicrobial substances to the non-selective medium before solidification. Afterwards, the stan- dardised bacterial inoculum is spotted on the agar surface. Following overnight incubation, plates are evaluated visually, determining whether growth has occurred at the inoculated sites. The lowest concentration of antibiotics that prevent bacterial growth is considered to be the MIC. This method allows simultaneous testing of different bacterial strains. 3.1.2. Antimicrobial Gradient Method The gradient strip test is a combination of disk-diffusion and dilution method of AST, having advantageous properties of both methods. It allows the MIC to be determined while keeping it simple and easy to use. The method is based on the diffusion of an antibiotic through agar with a continuous gradient. A concordance of the susceptibility categories and MIC values obtained by gradient test and BMD method, a “gold standard” recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and CLSI, were observed [28,42]. For example, the new ceftazidime–avibactam and ceftolozane– tazobactam gradient tests (Etests, bioMérieux, Marcy-l’Étoile, France) have shown a high categorical agreement between gradient test and BMD, of 96% and 94%, respectively [43,44]. On the other hand, for some antibiotics, such as colistin and tigecycline , controversial results have been obtained. Some agar-related factors, i.e., the content of divalent cations, can affect the diffusion of colistin, resulting in false susceptibility. Consequently, BMD remains the only appropriate method for MIC determination for certain antibiotics. Currently, a few commercial gradient strip tests, such as Etest (bioMérieux, France), MIC Test Strip (Liofilchem, Roseto degli Abruzzi, Italy), M.I.C.Evaluator (Thermo Scientific, Waltham, MA, USA), and Ezy MIC Strip (HiMedia Laboratories, Mumbai, India), are available. They can be used for susceptibility testing of microorganisms to antibiotics and antifungals [48–50]. A gradient strip test is performed according to the manufacturer’s instructions: a short plastic or paper strip impregnated with antibiotic is placed on inoculated agar (Figure 2). On the standardised 100 mm Petri dish, two strips may be placed, while on the larger  Antibiotics 2022, 11, x FOR PEER REVIEW 6 of 27 available. They can be used for susceptibility testing of microorganisms to antibiotics and antifungals [48–50]. Antibiotics 2022, 11, 427 6 of 26 A gradient strip test is performed according to the manufacturer’s instructions: a short plastic or paper strip impregnated with antibiotic is placed on inoculated agar (Fig- ure 2). On the standardised 100 mm Petri dish, two strips may be placed, while on the 150 mm150 larger Petri mm dish, updish, Petri to sixupantibiotics may be tested to six antibiotics may be simultaneously. The MICThe tested simultaneously. of a MIC tested of agent is determined by the intersection of a zone of inhibition with the strip and a tested agent is determined by the intersection of a zone of inhibition with the strip and measured using labelled measured concentrations using on the strip.on labelled concentrations If the the intersection strip. If the is between two intersection values on is between twoa scale, a higher value is reported as MIC. In addition, if beta-haemolysis is values on a scale, a higher value is reported as MIC. In addition, if beta-haemolysis is present on the plate, careful present examination on the of the plate, careful strip is required examination sinceisthe of the strip reporting required ofthe since thereporting intersection of of the haemolysis leads to false higher MICs values. Automated systems for reading the results intersection of haemolysis leads to false higher MICs values. Automated systems for read- of gradient tests are also available (ADAGIO Automated System, Bio-Rad Laboratories, ing the results of gradient tests are also available (ADAGIO Automated System, Bio-Rad Hercules, CA, USA). Laboratories, Hercules, CA, USA). Figure 2. Disk diffusion and gradient test of various bacterial isolates. (A)—Antimicrobial suscepti- Figure 2. Disk diffusion and gradient test of various bacterial isolates. (A)—Antimicrobial bility of Streptococcus pyogenes showing iMLS phenotype, using disk diffusion method. (B)—Anti- susceptibility of Streptococcus microbial susceptibility pyogenes showingbeta-lactamase-producing of extended-spectrum iMLS phenotype, using disk diffusionaeruginosa, Pseudomonas method. (B)—Antimicrobial using disk diffusion susceptibility of extended-spectrum method. (C)—Gradient beta-lactamase-producing test of Enterococcus Pseudomonas aerugi- spp. iMLS phenotype—inducible nosa, using disk macrolide, diffusion method. lincosamide, (C)—Gradient and streptogramin test of Enterococcus spp. iMLS phenotype—inducible phenotype. macrolide, lincosamide, and streptogramin phenotype. A variation of gradient tests exists for the detection of various AMR phenotypes. A variation Currently, Etestsofforgradient testsdetection phenotypic exists forofthe ESBLdetection of various production AMR phenotypes. in enterobacteria are avail- Currently, able, suchEtests forwith as strips phenotypic detection of ESBL cefotaxime+clavulanic production acid, ceftazidimein with enterobacteria clavulanicare avail- acid, and able, such as strips with cefotaxime+clavulanic acid, ceftazidime with clavulanic acid, and cefepime with clavulanic acid. The gradient tests for ESBL detection are two-sided cefepime with clavulanic acid. The gradient tests for ESBL detection are two-sided strips that contain antibiotic on one end, while on the other is the same antibiotic with strips that contain antibiotic on one end, while on the other is the same antibiotic with clavulanic acid. Reduction in MIC equal to or greater than eight times by the combination clavulanic acid. Reduction in MIC equal to or greater than eight times by the combination of antibiotic and clavulanate refers to ESBL production. Similar to the double-disk of antibiotic and clavulanate refers to ESBL production. Similar to the double-disk synergy test, the phantom zone below the clavulanic end also indicates a positive result. synergy test, the phantom zone below the clavulanic end also indicates a positive result. Identification of metallo-beta-lactamase (MBL)-producing bacteria can be carried out us- Identification of metallo-beta-lactamase (MBL)-producing bacteria can be carried out using ing a gradient test. These tests contain carbapenem antibiotic on one side of the strip and a gradient test. These tests contain carbapenem antibiotic on one side of the strip and the the same carbapenem with EDTA on the other side. Imipenem with EDTA for detection same carbapenem with EDTA on the other side. Imipenem with EDTA for detection of MBL of MBL in Acinetobacter spp. and Pseudomonas spp. is available, although sensitivity and in Acinetobacter spp. and Pseudomonas spp. is available, although sensitivity and specificity specificity may vary [53,54]. For detection of AmpC beta-lactamase-producing enterobac- may vary [53,54]. For detection of AmpC beta-lactamase-producing enterobacteria, Etest teria, Etest impregnated impregnated with cefotetanwithoncefotetan one end andon one end and cefotetan–cloxacillin cefotetan–cloxacillin on the otheronendthe other can be end can be used. Gradient tests with a predefined gradient of vancomycin used. Gradient tests with a predefined gradient of vancomycin and teicoplanin on each and teicoplanin side on can of the strip eachbeside usedoffor thethe strip can beofused detection for the detection glycopeptide resistanceof in glycopeptide re- Staphylococcus sistance aureus in Since. Staphylococcus these testsaureus.toSince are easy thesethey perform, testscould are easy to perform, be used they could as “screening” be tests used as “screening” tests for the detection of emerging resistance patterns for the detection of emerging resistance patterns among clinically relevant bacteria. among clini- callyPlenty relevant bacteria. of gradient tests are known: simple performance, flexibility in of advantages the testing of any combination of antibiotics, and the fact that they do not require expertise and special technologies. Moreover, their use is suited when only a couple of antibiotics are needed to be tested. The price of each strip is relatively high, compared with the price of disks; therefore, gradient tests are usually used to test only a few antibiotics per strain. The incubation length of 16–24 h for gradient tests may represent a disadvantage, as more rapid automated systems are available with the reliable determination of MIC. Antibiotics 2022, 11, 427 7 of 26 3.1.3. Disk Diffusion Test Since its development in 1940, the disk diffusion (DD) test has remained the most widely used routine AST in clinical microbiological laboratories. It has been stan- dardised to test the susceptibility of the most common and clinically relevant bacteria that cause human diseases [42,58]. The standardisation is a continuous process, and DD for many microorganisms/antimicrobials is an ongoing process. The method is based on placing different antibiotic-impregnated disks on previously inoculated agar with bacterial suspension. The antibiotic diffuses radially outward through the agar medium, producing an antibiotic concentration gradient. After the inhibition zones are established within 24 h of incubation at 35 ± 1 ◦ C, the zone diameters of each tested antibiotic are measured by the naked eye or using an automated system. Obtained results should be interpreted and categorised according to the recommended clinical breakpoint of the standard in use [42,61]. Disk diffusion is the most widely used AST method in microbiology laboratories because of its low cost and ease of performance and applicability of numerous bacterial species and antibiotics. The choice of antibiotic disks is flexible and enables the clinical laboratory to make different combinations according to the bacterial species and the type of sample the isolate was obtained from. Simple interpretation allows the detection of atypical phenotypes and visibility of contamination. However, the main disadvantages are the inability to determine the MIC and delays in getting the results. Reduction in turnaround time and timely treatment are of great importance for critically ill patients. In addition, the biological properties of lag and log phase of bacterial growth and their expression on antibiotic influence should be considered. Nevertheless, methods to reduce incuba- tion time for DD were suggested decades ago [64–66]. A revival of that idea led to the development of automated systems (WASPLab, Copan, Murrieta, CA, USA and BD Kiestra, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) for an acceleration of AST by DD [67,68]. The automatisation of the AST by DD leads to a shortening of the required time to obtain results and produce the final report. EUCAST has defined a methodol- ogy of disk diffusion rapid AST (RAST), which is performed directly from positive blood culture bottles, with breakpoints for short incubations of 4, 6, and 8 h [69–71]. RAST can be implemented in routine laboratories without major investments. The method has been validated for a limited number of bacterial species and antibiotics so far. Furthermore, the combined use of a MALDI-TOF MS for the identification of bacteria and RAST directly from positive blood bottles enables reporting AST results within less than 24 h, which significantly reduced the turnaround time compared with the 24–48 h needed for culturing and classical AST methods, such as DD. The predictive value of direct DD testing from positive blood cultures has been reported to have an important influence on AMS. Quality control testing of media and antibiotics is of great importance for ensuring that disk diffusion is providing accurate and reliable results [59,74]. In some cases, the DD method is more reliable than MIC determination. For instance, in the case of detecting penicillinase-producing S. aureus strains, the inhibition zone diameter combined with the EUCAST-based zone edge test is the most sensitive and specific phenotypic method [42,75]. The DD method can be used for screening of susceptibility to a larger number of antibiotics or a whole class of antibiotics; detection of certain important resistance phenotypes, such as ESBL, carbapenemases, inducible resistance to macrolides (Figure 2); or the presence of a heteroresistant population of bacterial species in a sample that cannot be detected by other phenotypic AST methods. The above-mentioned suggests that the DD method will remain a widely used AST method in the future. 3.1.4. Chromogenic Agar Media for Detection of Antimicrobial-Resistant Bacteria Since the introduction of the first chromogenic media for the detection of antibiotic- resistant bacteria 20 years ago, a variety of different media for the detection of clinically important resistant pathogens—such as MRSA, VRE, and ESBL- and carbapenemases- producing or colistin-resistant Gram-negative bacteria—have been developed [76–80]. Antibiotics 2022, 11, 427 8 of 26 The main purpose of the development of chromogenic media was to enable more rapid detection and identification of resistant microorganisms. The target organisms are characterised by specific enzyme systems that metabolise the substrates to release the chromogen. The chromogen can then be visually detected by direct observation of a distinct colour change in the medium. Thus, these selective and differential media enable target pathogens to grow as coloured colonies. Compared with the use of conventional culture media, the use of chromogenic agar often reduces the costs and labour time. Their primary use is for screening of patients colonised with various pathogens, and therefore they are valuable in infection prevention and control of hospital-acquired infections. The sensitivity and specificity of chromogenic media depend on the manufacturer and the type of microorganism detected; thus, additional identification confirmation of the resistant bacteria is sometimes needed. Because of their wide applicability, new chromogenic media are being developed [83,84]. 3.1.5. Colourimetric Tests for Detection of Antimicrobial-Resistant Bacteria Colourimetric tests represent phenotypic methods developed for the detection of AMR. They are based upon the bacterial enzymes hydrolysing test component, which is detected by the changes in pH values and the colour of chromogenic substances. Briefly, bacterial suspension or bacterial lysate suspension is added to a detection solution containing antibiotic and pH indicator dyes, such as phenol red, and incubated for a short period of time, no longer than a couple of hours. The pH of the detection solution changes due to the growth of antibiotic-resistant bacteria, or bacterial enzymes activity, and subsequently, the colour of the solution changes, which can then be visually observed. These tests were shown to be fast, easy to perform and interpret, and highly sensitive and specific. A good example of such colourimetric tests is Carba NP (bioMérieux, Marcy-l’Étoile, France), which detects carbapenemase-producing bacteria. The test gives reliable results in 30 min to 2 h, making it the quick and easy way to control carbapenemase producers. 4. Current Technologies for Rapid AST 4.1. Automated and Semi-Automated Devices Based on Microdilution Susceptibility Testing Clinical microbiology laboratories are under increasing pressure to provide fast and reliable microbial identification (ID) and AST. Automated and semi-automated devices for bacterial ID and AST are worthy of the task and have significantly improved laboratory efficiency. Nowadays, automation has been successfully implemented in most clinical microbiological laboratories to reduce turnaround times, increase efficiency, and improve cost-effectiveness [86,87]. Various test systems—such as the VITEK 2 (bioMérieux, France), MicroScan Walkaway (Dade-Behring MicroScan, Deerfield, IL, USA), and Phoenix system (BD Diagnostic Systems, Baltimore, MD, USA)—have been widely used over the last decades. These instruments, using optical systems for measuring subtle changes, determine bacterial growth and antimicrobial susceptibility and can produce results in a shorter time (6–12 h) than conventional manual assessment. VITEK 2 Systems—The first generation of VITEK system with a turnaround time of 13 h was developed for enumeration and identification of bacteria and yeasts in 1973. The VITEK 2 System, the next-generation of an instrument, is a BMD-based AST system that uses 64-well plastic cards containing 17–20 antimicrobial agents. If the bacterial isolate is not previously identified, one card is used for bacterial identification (ID card) and the other for antimicrobial susceptibility testing (AST card). Two Vitek 2 instruments are available with test card (ID and AST) capacities of 60 cards (Vitek 2) and 120 cards (Vitek 2 XL). Results are reported in 4–18 h, containing MIC and category of susceptibility, whereas the detection of AMR is facilitated by the Advanced Expert System (AES). The currently available Vitek 2 Compact instruments can use 15, 30, and 60 cards. The main advantage of the Vitek 2 system with computer software is the determination of susceptibility of clinically important resistant pathogens, such as Staphylococcus aureus and Enterococcus faecalis, to an additional four to ten antibiotics [86,89,90]. Antibiotics 2022, 11, 427 9 of 26 Phoenix System—The Phoenix System is widely accepted and used in clinical mi- crobiology laboratories for identification testing (ID) and antimicrobial susceptibility testing (AST). The principle of determining the susceptibility is based on the use of an oxidation-reduction indicator (resazurin dye or Alamar blue) and the detection of bacterial growth in the presence of various concentrations of the antimicrobial agent. In the Phoenix instrument, a maximum of 100 tests can be performed by using Phoenix ID/AST combination panels (51 for ID and 85 for AST). The instrument performs automatic reading at 20 min intervals during incubation for up to 18 h and provides accurate and rapid susceptibility results with easy workflow for the laboratory worker. In 2014, the new panel for susceptibility of Gram-negative bacteria was introduced for the Phoenix system to be used in combination with the BD Bruker MALDI-TOF. MicroScan WalkAway plus System—The MicroScan WalkAway plus System provides accurate and rapid identification and susceptibility results for a wide range of Gram- positive and Gram-negative aerobic bacteria. The instrument utilises three types of panel configurations: combo panels, breakpoint combo panels, and MIC panels. There are two types of system: 40- and 96-panel capacity models. The panels are manually inoculated, rehydrated by the RENOK inoculator, and read automatically. The results are obtained after 4.5–18 h by reading of rapid panels. MicroScan AutoScan 4—The AutoScan 4 is a semiautomated instrument mostly used in smaller laboratories or for the testing of supplemental antimicrobial agents. The instrument provides simplified ID/AST testing in a highly reliable and affordable package. The system uses the off-line incubation of the conventional MicroScan AST panels. The panels are manually inoculated or with the MicroScan Renok instrument and read automatically. MicroScan WalkAway System—The first generation of the MicroScan WalkAway System available on the market is the AutoSCAN-3. The new versions of instruments Auto-ACAN-4 and AutoSCAN-WalkAway are improved and use dry panels that do not need refrigeration. The AutoSCAN-WalkAway system detects bacterial enzymatic activity and can process 96 panels at the same. Each of the above-mentioned systems has inherent advantages and limitations, and the results vary widely by antimicrobial drugs, software versions, and cards used. Hence, some of the systems are not reliable for correct categorisation of susceptibility profiles for certain drugs, leading to wrong classifications of susceptibility categories. It seems that low inoculum size has a major influence on the outcome of these systems, with false susceptibilities being reported. Additionally, software updates and synchronisation of breakpoints according to the current standards are mandatory. Thus, it is incumbent upon the instrument manufacturer to keep pace with the breakpoint updates and make relevant improvements, such as extending the detection limit and verifying the performance of the AST system with the revised breakpoints internally, to avoid the problem of uncategorised results. Panels usually contain only several concentrations of each antimicrobial agent, and the resulting MIC is not always given as an exact value. In contrast, classical BMD con- tains a wide range of doubling dilution antimicrobial concentrations for the determination of the MIC, thus obtaining the more precise value. In addition, according to the previously published reports, many of the resistance phenotypes are not easily detected using the automated susceptibility testing methods so prevalent in today’s clinical laboratories. Nonetheless, interestingly, the ability of automated systems to detect inducible resistance to clindamycin in 524 isolates of Staphylococcus spp. revealed sensitivity and specificity of 100% and 99.6%, respectively, for Phoenix, and 91.1% and 99.8%, respectively, for Vitek 2. The multicentre evaluation showed that categorical agreement between the Phoenix system and a BMD reference method for 2013 streptococcal isolates including Streptococcus pneumoniae, viridans group streptococci, and beta-haemolytic Streptococcus groups A, B, C, and G ranged from 92% to 100%, with one exception for viridans streptococci and penicillin, which was 87%. However, according to the results of the evaluation of ASTs obtained  ity and specificity of 100% and 99.6%, respectively, for Phoenix, and 91.1% and 99.8%, respectively, for Vitek 2. The multicentre evaluation showed that categorical agree- ment between the Phoenix system and a BMD reference method for 2013 streptococcal isolates including Streptococcus pneumoniae, viridans group streptococci, and beta-haemo- lytic Streptococcus groups A, B, C, and G ranged from 92% to 100%, with one exception for Antibiotics 2022, 11, 427 10 of 26 viridans streptococci and penicillin, which was 87%. However, according to the re- sults of the evaluation of ASTs obtained using Vitek 2, Phoenix, and MicroScan, caution should be taken for AST of Stenotrophomonas maltophilia, as a high rate of errors may be using 2, Phoenix, and MicroScan, caution should be taken for AST of Stenotrophomonas Vitek. observed maltophilia, as a high rate of errors may be observed. 4.2. Molecular-Based Techniques for Resistance Detection 4.2. Molecular-Based Techniques for Resistance Detection Molecular AST directly detects specific resistance genes, as well as mutations in and Molecular AST directly detects specific resistance genes, as well as mutations in and expression of these genes. These molecular methods have been developed and tested as expression of these genes. These molecular methods have been developed and tested as an an alternative for or complementary to conventional AST and are generally faster than alternative for or complementary to conventional AST and are generally faster than classic classic culture-based culture-based assays, assays, with the with the test test results results available available within onewithin onehours to a few to a few hours (Figure 3). (Figure 3). Most of the molecular AST methods fall into one of the three categories: Most of the molecular AST methods fall into one of the three categories: amplification-based, ampli- fication-based, hybridization-based, hybridization-based, or sequence-based. or sequence-based. In amplification-based In amplification-based methods, methods, the target gene the target gene sequence is amplified to allow detection; in hybridization-based sequence is amplified to allow detection; in hybridization-based techniques, hybridized tech- niques, hybridized nucleic acid probes target gene sequences allowing nucleic acid probes target gene sequences allowing detection; and in sequence-based detection; and in sequence-based approaches, approaches, genome sequences genome sequences are analysed are analysed to detect to detect resistance-confer- resistance-conferring mutations or ring mutations resistance genes. or resistance genes. Figure3. Figure 3. The Thebasic basicworkflow workflowofofmolecular-based molecular-based techniques techniques forfor antimicrobial antimicrobial susceptibility susceptibility testing. testing. The The routes from a clinical specimen to a final result are indicated by arrows (created routes from a clinical specimen to a final result are indicated by arrows (created with BioRender.com, with accessed on 27 February 2022. Reproduction of this figure requires permission from BioRender.com). 4.2.1. Polymerase Chain Reaction The most widely used nucleic acid amplification-based method for the detection of specific resistance genes is polymerase chain reaction (PCR). Both real-time and conven- tional PCR rely on the amplification of nucleic acid sequences that encode resistance to an antibiotic. New PCR-based methods are being developed for the detection of genetic determinants of resistance to a variety of antibiotics for various bacterial species, as our knowledge about the genetic basis of antibiotic resistance increases. Multiplex assays for simultaneous testing of multiple genetic determinants in various bacterial species have also been developed, i.e., multiplex assays for identifying numerous cephalosporinase- and carbapenemase-encoding genes, such as blaKPC , blaNDM , blaIMP , blaVIM , blaAmpC , blaTEM , blaSHV , and blaOXA , or mecA gene-encoding methicillin resistance in MRSA [100,101]. Op- Antibiotics 2022, 11, 427 11 of 26 Gen, Inc. (Rockville, MD, USA) has recently released the multiplex-based Acuitas® AMR Gene Panel that detects 28 genetic AMR markers, covering select drugs in nine classes of antibiotics, from 26 different pathogens. The advantage of this test in comparison with other commercially available molecular tests is that it also detects non-beta-lactam resistance genes and those for what would be considered “last-resort antibiotics”, such as colistin. Real-time PCR (quantitative PCR, qPCR) is one of the most ubiquitous methods found throughout clinical microbiology. Although costlier, qPCR offers several advantages over conventional PCR, including the measurement of data in real-time, greater sensitivity, reduced risk of carryover contamination, and greater amenity to multiplexing. Further advantages are that many systems are partially or even completely automated, such as GeneXpert® Instrument Systems (Cepheid Corp., Sunnyvale, CA, USA) and BD MAX System platform (Becton Dickinson, Franklin Lakes, NJ, USA), which are easily operated and can be used for the detection of carbapenemases, ESBLs, MRSA, VRE, etc. [103–105]. The downside is that they are limited to using test assays only from specific manufacturers, with GeneXpert® Instrument Systems requiring GeneXpert assays (Cepheid Corp., Sun- nyvale, CA, USA) and BD MAX System using Check-Points® qPCR assays (Wageningen, The Netherlands). Tests based on qPCR can also be used for phenotypic differentiation of resistant and susceptible strains due to its ability to measure genome copy numbers during bacterial growth in the presence of antibiotics. The major disadvantage is that the system cannot provide information about the mechanism of resistance and that it requires the previous culture, meaning that the primary clinical samples cannot be used. 4.2.2. DNA-Microarrays DNA-microarrays are used to identify the presence of specific nucleic acid sequences using complementary short oligonucleotides immobilised on a solid surface. Since these oligonucleotides can be assembled onto solid surfaces in close proximity, this method could detect numerous sequences in a single assay, which would allow simultaneous, in parallel detection of different pathogens and detection of vast numbers of different resistance genes, as well as detecting numerous distinct mechanisms of resistance or variants of a single mechanism present in bacterial isolates, as opposed to PCR-based approaches. The Verigene system (Luminex Corporation, Austin, TX, USA) has developed Blood Culture Multiplex Microarray-Based Molecular assays for rapid diagnostics of 12 Gram-positive and 9 Gram-negative bacteria, along with their associated resistance genes (i.e., mecA, vanA, vanB, blaKPC , blaNDM , blaIMP , and blaVIM ). In addition to qPCR-based assays, Check- Points® has also developed the CHECK-MDR CT103 DNA microarray for the detection of the clinically most prevalent ESBLs and carbapenemases, as well as mobile colistin resistance (mcr) genes in Gram-negative bacteria. Both of these microarray tests have shown high sensitivity (94–100%) and specificity (94–98%), and CHECK-MDR CT103 DNA microarray also showed the ability to discriminate between carbapenemase and ESBL variants of GES-type beta-lactamase [108,109]. The advantages of currently available molecular-based methods are that they are direct, rapid, highly sensitive, and specific, thus potentially allowing the earlier administration of targeted therapy. Furthermore, for some methods, direct clinical samples can be used. However, it should be noted that the presence of a resistance marker does not always have to correlate with phenotypic resistance. Additionally, the extent and intensity of gene expression are important parameters, as some genes need different expression levels to produce resistance. A potential solution to this issue would be the use of reverse transcription qPCR, which relies on the measurement of gene transcripts (RNA levels) instead of the presence of a gene [110,111]. Another drawback is that these methods can only detect resistances that are searched for, and not novel or uncharacterised mechanisms of resistance, which could lead to false-negative results and inappropriate classification of resistant isolates as susceptible. A final consideration is that these methods are not capable of defining MIC values. As such, these methods have to be validated against phenotypic Antibiotics 2022, 11, 427 12 of 26 data to be useful, and extensive resistance marker databases and innovative bioinformatics methodologies are mandatory requirements. Nevertheless, molecular-based AST methods are a safe, efficient, and reliable screening tool in clinical settings. As experience with these tests grows, and as data are gathered on their efficacy and clinical impact, they will likely be more widely adopted. 4.2.3. Whole-Genome Sequencing in Antimicrobial Susceptibility Testing As DNA sequencing technology and bioinformatics pipelines for genome assembly and analysis advance, the possibility of using these techniques for the detection of an- tibiotic resistance opens. Applying whole-genome sequencing (WGS) would essentially enable the detection of all genes involved in AMR, which would help make comprehen- sive databases of all species-specific resistance factors (i.e., CARD-Comprehensive Antibi- otic Resistance Database—https://card.mcmaster.ca, ResFinder—https://cge.cbs.dtu.dk/ services/ResFinder (accessed on 27 February 2022)) and make in silico AMR detection possible. Recent studies showed high concordance between the resistance profiles obtained using WGS and those obtained using phenotypic susceptibility testing, demonstrating that data obtained from genome sequences can correlate well with phenotypic resistance in some cases [112,113]. In addition to genome-based resistome analyses, RNA-mediated tran- scriptomic approaches have also been described [114,115]. Despite all of the advantages, WGS is not routinely performed in clinical practice. Considering the turnaround times of WGS, the existence of unknown resistance mechanisms, and the elevated cost compared with traditional and emerging techniques, the use of WGS for AST is not yet part of routine practice in clinical microbiology [116,117]. 4.3. Mass Spectrometry Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI- TOF MS) was discovered in the 1980s and introduced into the microbiological routine as an effective tool for bacterial and yeast identification about 15 years ago. It has been applied to classify the specific bacterial protein contents and their matching protein biomarkers because of its rapid turnaround time, low sample volume requirements, and per-sample costs. Several MALDI-TOF MS-based methods have been proposed for rapid detec- tion of antimicrobial resistance, including monitoring antibiotic modification by bacterial culture (e.g., beta-lactam hydrolysis [65,119]), acetylation of fluoroquinolones , direct detection of proteins involved in specific resistance mechanisms [121,122], and detection of stable isotope labelling that requires expensive, isotopically labelled media [123,124]. The hydrolysis of the target beta-lactam antibiotic, as shown by peak disappearance, is used to detect beta-lactamase-producing bacteria using MALDI-TOF MS. As a result, the assay for detecting carbapenemase production automatically determines sensitivity or resistance depending on the degree of antibiotic hydrolysis. The method had 98% sensi- tivity and 100% specificity after 30 min of incubation of bacteria with the antibiotic, with both reaching 100% after 60 min of incubation [126,127]. However, beta-lactam resistance is only recognized when it is mediated by beta-lactamases; alternative resistance mechanisms have not been elucidated; therefore, other tests should confirm negative results. Another assay for the detection of carbapenemases is a rapid and novel method using detonation nanodiamonds (DNDs) as a platform for the concentration and extraction of A. baumannii carbapenemase-associated proteins before MALDI-TOF MS analysis. The sensitivity and the specificity of the proposed platform could reach 96% and 73%, as compared with traditional imipenem susceptibility testing, and 100% compared with PCR results. This method may detect the carbapenemases produced by A. baumannii in 90 min and does not require the addition of a carbapenemase substrate, as other mass spectrometric methods do. It is efficient for detecting other carbapenemase-producing bacteria. MALDI Biotyper-Antibiotic Susceptibility Test Rapid Assay (MBT-ASTRA) is an al- ternative MS-based method for AST which utilises semi-quantitative MALDI-TOF MS to measure the relative growth rates of bacterial isolates exposed to antibiotics compared Antibiotics 2022, 11, 427 13 of 26 with untreated controls during a short incubation step. A software tool calculates and compares the area under the curves (AUCs) of spectra of bacteria either exposed or not to an antibiotic. In this method, if the microbial strain is susceptible, the AUC of the bacterial suspension with the antibiotic will be reduced compared with that without antibiotics, whereas with a resistant strain the AUCs with or without antibiotics will be comparable. The main advantage of the MBT-ASTRA is that the assay does not depend on the resistance mechanism and is utilisable with any antibiotic. Moreover, it does not require specialised media or instrumentation, beyond the MALDI-TOF mass spec- trometer. However, a drawback of the MBT-ASTRA assay is that the concentration of antibiotics used and the incubation time must be optimised for each species and antibiotic combination. MBT-Resist assay, based on the detection of peak shift after stable isotope labelling, is an approach that uses the following principle: bacteria are grown in parallel in two distinct culture mediums, one containing 12C as a carbon component and the other containing 13C. The system compares the mass spectrum of bacteria grown on an isotope-labelled medium with antibiotics to the mass spectrum of the same strain grown on an unlabelled medium without antibiotics. Resistant strains can thrive in the presence of antibiotics, incorporating 13C into the polypeptide, causing a shift in the peak to a higher m/z in the mass spectrum. Antibiotic resistance by direct-on-target microdroplet growth assay (DOT-MGA) is a novel approach for detecting antimicrobial susceptibility in bacteria treated with break- point concentrations of antibiotic on the target plate of MALDI-TOF MS. The best performance was obtained by recovering bacteria from positive blood cultures and after a 4 h incubation of microdroplets with or without meropenem at the breakpoint concentra- tion. Under these conditions, 96.3% validity, 91.7% sensitivity, and 100% specificity were achieved. Recently, a screening panel for the detection of ESBL and AmpC beta-lactamase activity was developed. Compared with the PCR results, positive percentage agree- ment values for ESBL, AmpC, and ESBL + AmpC resistance were 94.4%, 94.4%, and 100%, and negative percentage agreement values were 100%, 93.7%, and 100%, respectively. The accuracy of the DOT-MGA achieved results incomparable with those of the BMD assay, with a time saving of about 14 h, and higher than combination disk tests. According to Yoon et al., due to the great speed and simple application, MALDI- TOF MS would be the most suitable for endemic AMR clinical strains in specific set- tings, i.e., MRSA, VRE, CRAB, CRPA, and ESBL-, AmpC-, and carbapenemase-producing Enterobacterales. The advantages and disadvantages of the commonly used methods of antimicrobial susceptibility testing were summarised in Table 1. Table 1. Advantages and disadvantages of the common methods of antimicrobial susceptibility testing. Method Advantage Disadvantage Comments Broth dilution Well-standardised Time-consuming Quantitative ** Harmonised Individual mistakes Commercially available tests are easy to perform Agar Dilution Well-standardised Time-consuming Quantitative Limited concentration of Possible automation Suitable for testing a large number of isolates antimicrobial agents in part Disk diffusion Simple to perform Time-consuming Qualitative * Low cost No MIC value The inability for some Simple and fast interpretation antibiotics to be tested The high number of test antibiotics per test High flexibility in antibiotic selection Detection of resistance patterns Mass use and the possibility of automatisation A number of a different use (AST, identification, screening, etc.) Detection of heteroresistant population or contamination Antibiotics 2022, 11, 427 14 of 26 Table 1. Cont. Method Advantage Disadvantage Comments Gradient test Convenient and flexible Relatively expensive Quantitative Relatively Simple to perform long incubation Does not require expertise Detection of resistance patterns Automated Simple to perform Relatively expensive Semi-quantitative *** systems Qualitative with no Chromogenic Not completely Mass use and the possibility of automatisation interpretation media susceptible and specific criteria (S, I, R) Simple to perform Time-consuming Limited spectra or Simple and fast interpretation single antibiotic Relatively expensive Screening only or required confirmatory identification No MIC value High cost of the MALDI-TOF MS Rapid turnaround time MALDI-TOF MS Need further optimisation for each Simple to perform species and antibiotic combination Low sample volume requirements No MIC value Low per-sample costs Genetic methods Rapid Limited spectra Qualitative Highly accurate Limited throughput Semi-quantitative Sensitive High cost Reproducible Increased ability to detect slow-growing or non-cultivable organisms Genomic methods Highly accurate High cost Qualitative Sensitive Time-consuming Increased ability to detect slow-growing or Challenging non-cultivable organisms interpretation of results * Qualitative; results are expressed as susceptible (S), susceptible, increased exposure (I), or resistant (R) based on established criteria from EUCAST. ** Quantitative; results are expressed as minimal inhibitory concentration (MIC) for each drug. Susceptibility reports should include interpretation of MIC, such as S, I, or R. *** Semi- quantitative; results are expressed as MIC using three to four antimicrobial dilutions for each drug. Precise MIC values cannot be established if the MIC falls below or above the three to four dilutions used in the test panel. Susceptibility reports include interpretation of breakpoint MIC as S, I, or R. MALDI-TOF MS—matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. 5. Selection of Antimicrobial Drugs for Susceptibility Testing, Interpretation,