Towards better antimicrobial susceptibility testing: impact of the Journal of Antimicrobial Chemotherapy

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1 J Antimicrob Chemother : doi: /jac/dkw494 Towards better antimicrobial susceptibility testing: impact of the Journal of Antimicrobial Chemotherapy Mandy Wootton 1 *, Alasdair P. MacGowan 2 and Robin A. Howe 1 1 Welsh Antimicrobial Resistance Programme, Public Health Wales, University Hospital of Wales, Cardiff CF14 4XW, UK; 2 Bristol Centre for Antimicrobial Research & Evaluation (BCARE), Department of Infection Sciences, Pathology Sciences Building, Southmead Hospital, Westbury-on-Trym, Bristol BS10 5NB, UK Introduction *Corresponding author. Tel: þ ; Fax: þ ; mandy.wootton@wales.nhs.uk Susceptibility testing of bacteria is one of the most important tests performed in a clinical microbiology laboratory. Improvements in laboratory techniques, especially the move towards standardized susceptibility testing, has provided better consistency and accuracy of testing. When used in conjunction with the most recently developed interpretative criteria, the result is better prediction of the outcome of antimicrobial therapy for infected patients. Throughout the last four decades this Journal has published numerous articles evidencing improvements and new techniques, a valuable source of information for microbiology laboratories. From the moment antimicrobial agents began to be used to treat patients suspected of having infectious diseases it was obvious that there was a need to predict efficacy. Clearly, in vitro susceptibility tests were required and initial testing was based upon determining the MIC, which was first developed by Alexander Fleming and is well described in his 1929 paper. 1 Since then there has been considerable work to develop practical, reliable and reproducible laboratory methods for susceptibility testing, methods to correlate in vitro laboratory tests to clinical outcome, and methods to identify new resistance mechanisms of clinical importance. In 1960, an expert committee of the WHO met in Geneva and recommended the definition of standard routine methods for antibiotic susceptibility testing. 2 This led to an international collaborative study sponsored by the WHO that examined almost all aspects of susceptibility testing over the next 10 years and finally published recommendations in Although the original WHO committee of 1960 had intended the development of a single, universally adopted method, the 1971 report suggested that there should be national reference laboratories with the duty of preparing complete descriptions of current recommended methods and interpretative schemes. It is perhaps no coincidence that the BSAC was founded to facilitate the acquisition and dissemination of knowledge in the field of antimicrobial chemotherapy, and one way in which it has aimed to achieve this has been by setting standards for antimicrobial susceptibility testing, both within the UK and overseas. The Journal of Antimicrobial Chemotherapy (JAC) has been an important vehicle through which the Society has encouraged the dissemination of knowledge, encouraged debate and facilitated the international moves towards standardized testing methods and interpretive criteria. Methods of susceptibility testing From the first days of susceptibility testing in the 1920s 1940s, the serial dilution method (in broth and agar) was in use in laboratories and became widespread during the 1950s and 1960s. The 1970s Serial dilution methods were labour intensive and time consuming and a method replacing serial dilutions with one or more critical concentrations that separated organisms into resistant and susceptible categories was devised: the breakpoint technique, a term first used by Ericsson and Sherris. 3 The first disc diffusion method was developed in the 1950s to replace the MIC. 4 The zone diameters of disc diffusion tests were correlated to MICs extensively during this decade to consolidate their usefulness in the laboratory. 5,6 The report by Ericsson and Sherris (1971) described an international collaborative study that established the basis for standardized susceptibility testing methods, in particular disc diffusion tests, and also explained the relationship between MIC and zone diameter. 3 By 1972 the National Committee for Clinical Laboratory Standards (NCCLS) [now the Clinical Laboratory Standards Institute (CLSI)] had published a tentative standard that recommended the Kirby Bauer disc diffusion method. 7 As with any new technique, variations in how the disc diffusion was performed abounded and the effect of these variations on testing phenomena required VC The Author Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please journals.permissions@oup.com. 323

2 explanation. Scientists generally turned to published papers to ask and answer these burning questions; in 1975, the JAC was set up to disseminate knowledge in the area of antimicrobial chemotherapy, and in its first year published its first susceptibility testing burning question paper. Marshall and Field 8 tested the variations within the disc diffusion method to show that growth build-up at the zone edge was not predictive of resistance of an organism. They tested a range of antimicrobial agents and incorporated a variety of testing factors to conclude that growth build-up at the edge of the zone of inhibition was not consistently affected by anything. This importantly discounted this phenomenon as a predictor of resistance and gave clinicians clear guidance on how to read their disc diffusion tests. During the 1970s the disc diffusion method became more established throughout the world as the laboratory method to test antimicrobial agent susceptibility of organisms. However, there was a move to standardize the test and hence improve reproducibility and surveillance. Two method types were dominant; in the first, the zone diameter was interpreted using a regression line between zone diameter and MIC. Examples were the Ericsson method, based on recommendations of the WHO-sponsored International Collaborative Study of antibiotic-susceptibility testing and commonly used in Sweden; and a modified Kirby Bauer method which was widely used in the USA and recommended by the FDA and the predecessor of the CLSI, the NCCLS. 3,7 This second method was developed by Joan Stokes and Pamela Waterworth and was designed to allow both the performance of the test to be monitored and the results to be interpreted by placing a control isolate on the same plate. Typical of the period, Stokes and Waterworth never published the method in a peer-reviewed article but rather promulgated it in a broadsheet published by the Association of Clinical Pathologists. 9,10 At this time, only a few laboratories had expertise in susceptibility testing, despite the disc diffusion test being performed widely throughout the world to predict antimicrobial therapy outcome. In 1978 two papers published in the JAC not only looked at how well four disc methods compared (comparative, Stokes, Ericsson and Kirby Bauer), but also how well they were performed in diagnostic laboratories that were not experts in disc diffusion testing. 11,12 These two papers comprehensively analysed the current disc diffusion methods and provided a further push towards a standardized disc diffusion method. The 1980s The 1980s saw the disc diffusion test (Stokes and Kirby Bauer methods) in common use in laboratories and papers in the JAC were instrumental in developing the methods further and resolving a number of testing irregularities. Brown and Ribeiro 13 showed how media affected disc diffusion tests for amoxicillin/ clavulanate (Augmentin VR ) and Crosse et al. 14 determined the most appropriate loading of ceftazidime discs to use. Another report in the JAC suggested that inoculum could be standardized using a spectrophotometer. 15 However, other methods were frequently used in laboratories and in 1991 the BSAC Working Party published a comprehensive guide to the various susceptibility testing techniques used in UK laboratories with clear instructions on how to perform them plus details of factors that could affect the results. 16 This report provided an in-depth understanding of susceptibility testing to laboratories, including how to prevent inaccurate resultssuchasthosecausedbyincorrect disc storage. The main susceptibility tests were MIC testing using agar dilution, and broth macrodilution and microdilution, the comparative and Stokes disc diffusion test, and breakpoint methods. The broad coverage of methods ensured that all laboratories were able to perform their favoured method and, in the UK, the report probably led to more consistent testing within laboratories, irrespective of the technique employed. Although the majority of laboratories in the UK at this time still used the Stokes disc diffusion method, the breakpoint method was also used in a number of laboratories. First developed in the USA, it is a truncated MIC method but has the advantage of enabling the testing of large numbers of organisms and lent itself to semiautomation. It can be performed in either broth or agar bases and correlated well with disc diffusion methods. 17 At the same time, the Epsilometer VR gradient strip test (Etest VR ) was developed and provided an attractive alternative to performing agar or broth dilution to determine an MIC. This test involves applying a strip containing a predefined gradient of antimicrobial agent to a seeded agar plate. The MIC of the agent is read from where the growth intersects the strip. The paper by Brown and Brown 18 was one of the first published to evaluate this new technique, but it was followed by publications evaluating its performance for many organisms whether or not the test performed satisfactorily These papers provided laboratories with a very valuable source of information that allowed them to change the tests they performed on a daily basis 21 making the gradient strip the most common method used worldwide for determining MICs. The 1990s From the 1990s onwards, there were repeated calls, published in JAC, to move from comparative methods to a standardized method. 22,23 In Brown s Leading article in the JAC 22 he highlighted the limitations of the Stokes method and suggested it might be time to move to a more standardized method but noted the change might cause controversy. In 1995 there was another call to standardize the disc diffusion test in the UK, especially as it was felt that in Northern America and parts of Europe disc diffusion standardization was more advanced. 24 In that paper, Wise proposed that the BSAC Working Party on susceptibility testing provide laboratories with a manual for a standardized method of disc diffusion susceptibility testing. To start this process a survey of the susceptibility testing methods employed by laboratories around the UK was undertaken. 25 At that time, 71.4% used disc diffusion as a sole method of susceptibility testing with 97% using the Stokes method. Overall, 26.1% of laboratories used a combination of disc diffusion and breakpoint methods, with none using just the breakpoint method alone. 19.2% of laboratories additionally used the Etest VR to determine MICs. The survey indicated that 90.6% of laboratories would consider a standardized method if the methodology was supplied. Standardization of the disc diffusion method was now becoming increasingly important to achieve as the survey highlighted some of the inconsistencies with the current methods. A comparison of MIC results and BSAC clinical breakpoints with the Stokes method 324

3 JAC showed the latter produced an unacceptable number of major errors though minor errors were in the acceptable range to the present In 2001 the BSAC published its first standardized method after months of trialling different aspects of disc diffusion methodology. 27 The resulting standardized method for disc diffusion testing reduced variability and yielded reproducible results. The method set out the antimicrobial agent disc content, inoculum preparation, media type, incubation temperatures and times, breakpoints or disc diffusion criteria for interpretation and control strain ranges for quality control. The method was part of a Supplement published in the JAC devoted to susceptibility testing; also contained within was the quality assurance guidance for the disc diffusion method and guidance for fastidious organisms. 28 This now meant that laboratories were furnished with a thoroughly tried and tested standardized technique which was not too far removed from contemporaneous methods. The standardized method was continually extended to include Bacillus testing and to compare media for Pseudomonas testing. 29,30 Laboratories quickly took up the challenge of implementing this standardized method and in the same year an evaluation was published that suggested that it performed better than the Stokes method. 31 A further report in the JAC stated that the BSAC disc diffusion method was good for confirming susceptibility to guide therapy and to monitor resistance trends. The authors highlighted a concern about the overestimation of resistance, and frequent zone MIC disagreements for isolates with borderline susceptibility. 32 In the following years the BSAC standardized disc diffusion method became the most widely used method in the UK and larger studies and surveillance research employed the method to compare resistance rates. A study in 2002/2003 showed that resistance within community-acquired respiratory tract isolates was low in the UK. 33 This study also found that a comparison of the BSAC disc diffusion method and MIC correlated well, but it also highlighted a methodological problem with macrolides particularly against Haemophilus influenzae. As the BSAC disc diffusion method for susceptibility testing gained ground, it became clear that further work was required to understand the effects of variability in media and disc content. Publications in JAC indicated that media purporting to be equivalent to IsoSensitest VR (Oxoid Ltd, UK) did not all yield the same results. 27 In addition, the impact of variable drug concentrations in high- and low-content discs was reported. 34 From 2001 onwards, the JAC and the BSAC Working Party on susceptibility testing published annual versions of the disc diffusion method and breakpoint tables, each time updating and improving the method and interpretation. One such example was replacing the oxacillin disc with a cefoxitin disc to determine methicillin resistance in Staphylococcus aureus, one of the major pathogens in UK hospitals. An article in the JAC initially using the Swedish standardized method but extending to the BSAC method suggested that cefoxitin was a better indicator of MRSA than oxacillin; it had the added advantage of being incubated at the normal Cinstead of 30 C for an oxacillin disc. 35,36 This work was subsequently confirmed by the BSAC Standardised Method Development Centre. 37 Skov et al. 38 alsoreportedin2009thatincubationtemperatures should be restricted to 36 C for accurate testing, improving the method further for MRSA detection. At the same time, the BSAC set up its respiratory and blood stream pathogen/antimicrobial resistance surveillance programmes (respiratory , bloodstream ). These programmes helped embed BSAC susceptibility testing methods as they mandated their use by the central testing laboratories in the programmes. Subsequently, there has been an ongoing flow of data on resistance in key pathogens in the UK reported in a number of JAC publications The BSAC disc diffusion method became the most widely used disc method in the UK during the 2000s, and even some continental European countries adopted it. At the same time the UK and other European countries began to adopt EUCAST MIC breakpoints, even though disc diffusion methods were still used routinely. This was perhaps no great surprise since Ian Phillips, former Editor-in- Chief of JAC ( ), and also President of the BSAC ( ), had become the first chair of EUCAST in 1997 and was joined by Derek Brown as the secretary. Furthermore, EUCAST was a standing committee jointly organized by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) and various European national breakpoint committees, including the BSAC. Although it was not initially intended that EUCAST develop a disc diffusion method, it made sense in the context of harmonizing breakpoints. A unified method of disc diffusion testing would provide standardization across Europe. In 2009 a EUCAST disc diffusion method was first published, with the latest version (6.0) published in The EUCAST disc diffusion method has now been developed for more antimicrobial agent/organism combinations than any other method, and is the main method used in European laboratories according to both the NEQAS and EARS-Net external quality assurance schemes. It was widely introduced into laboratories throughout Europe (and beyond, e.g. Australia) and currently the method has been adopted by >50% of laboratories in just over 40% of countries, and between 10% and 50% of laboratories in just under 40% of countries in Europe. In 2014, the departing chair of EUCAST, Gunnar Kahlmeter, summarized the status of susceptibility testing in the BSAC Garrod Lecture: EUCAST are we heading towards international agreement? 42 After the BSAC decision in 2016 to adopt the EUCAST disc diffusion method in the UK, laboratories have been rapidly following suit. 43 Indeed, the move to EUCAST disc diffusion now includes laboratories in the USA. Other hot topics published in the JAC include the consequences of testing fixed concentration clavulanate instead of a ratio. 44,45 As we head for 2020, it may be that most laboratories in the world will be using the same disc diffusion method, with experts from around the globe helping to improve the method. Better comparison of susceptibility testing data will also improve surveillance and speed up detection of resistance at a time when we need it the most. Development of interpretive criteria The main function of a susceptibility test is to produce a result of susceptible, intermediate (or indeterminate) or resistant for an antimicrobial agent/organism combination. In order to do this, criteria are required to interpret the test to devise clinical breakpoints. In the 1950s these breakpoints were commonly used throughout the world, but very often were different in different countries and even in different laboratories in the same country. The term susceptibility breakpoint was first coined by Ericsson and Sherris 3 in 1971 and then adopted in 1972 by the NCCLS, which published MIC 325

4 breakpoints and disc diffusion criteria. 46 These breakpoints were based solely on frequency distributions which would separate susceptible and resistant phenotypes. By the mid-1980s the outcome of treatment had been incorporated into breakpoints but the final piece of the jigsaw came with the inclusion of the pharmacological attributes of the antimicrobial agent itself. A significant contribution to the understanding of the role of pharmacology in informing susceptibility testing and the development of more accurate breakpoints was published in a JAC Supplement. 16 This approach, though widely criticized at the time, was truly innovative as it was an open and transparent process and based clinical breakpoints on pharmacokinetic rather than purely microbiological factors. Protein binding was also taken into consideration, which was itself a novel departure. Sections in the JAC Supplement described in detail a definition of breakpoints along with how breakpoints are calculated and presented a table of proposed breakpoints. Throughout the 1990s the pharmacodynamics and pharmacokinetics of many antimicrobial agents were established, with some commonly used ones published in the JAC. 47 In 2001, MacGowan and Wise reviewed the philosophy behind the setting of the BSAC breakpoints, including newly determined data, and updated those breakpoints that had been published earlier. 48 In 2005, the terminology used for pharmacodynamic/pharmacokinetics was being standardized 49 with publications in the JAC leading the way. The plethora of pharmacokinetic and pharmacodynamic data from both old and new antimicrobial agents was now being used to review old breakpoints and generate new ones, but the diversity of breakpoints in different countries remained. In 2003, an article in the JAC called for the harmonization of breakpoints in Europe. 50 This paper described the start of the harmonization process in Europe through EUCAST. Along the way there have been many open discussions on certain controversial topics such as the view that the cephalosporin MIC is a better predictor of the therapeutic outcome than categorization according to the resistance mechanism. For instance, the EUCAST recommendation is to report the third-generation cephalosporins as determined, even if ESBLs are present. Many experts criticized this view and published their articles in the JAC to foster open discussion. 51,52 Although still ongoing, the harmonization of breakpoints in Europe has essentially been achieved and EUCAST breakpoints are now used in the majority of European laboratories. Consequently, thoughts are now moving towards harmonizing breakpoints throughout the world. For instance, USCAST was formed in 2015 to represent the USA. Scrutiny and comparison of CLSI and EUCAST breakpoints and disc zone diameters to facilitate further harmonization have been published in the JAC In one such study, a comparison of EUCAST and CLSI breakpoints for third-generation cephalosporins found that there were significant differences in susceptibility rates, leading to further calls for more harmonization. 57 EUCAST has also extended its approach to include antifungal agents as well as antimicrobial agents used in veterinary practice, each forming a subcommittee of EUCAST, namely the subcommittee on Antifungal Susceptibility Testing (AFST) and the EUCAST Veterinary Subcommittee (VetCAST). 58,59 Emerging resistance Susceptibility testing has improved our ability to predict the outcome of therapy but has been paralleled by increasing resistance to antimicrobial agents. Research into the mechanisms of antimicrobial resistance coincided with better means of detection. Although the disc diffusion test and MIC testing could detect resistance, some hospitals needed to detect specific mechanisms of resistance for infection control purposes and these proved harder for the diagnostic laboratory to determine. In the early days of the JAC,major resistance mechanisms were explained to eager scientists, for instance, in 1975, trimethoprim resistance in Klebsiella pneumoniae was described even before the drug was introduced to the market. 60 In the following year, Sykes and Matthew 61 showed the effect of b-lactamases produced by the Enterobactericeae on b- lactam antibiotics. This would prove to be one of the most important resistance mechanisms. Indeed, the trend of resistance mechanism elucidation continued with the description of the activity of TEM-1, the original b-lactamase in Escherichia coli andthenmutation of TEM to become a cephalosporinase. 62,63 Throughout the 1990s and into the 2000s the emergence of ESBLs such as CTX-M was reported in the JAC. Papers were published detailing the mechanism of resistance and how to detect ESBLs in the laboratory, with some publications detailing the very beginning of the emergence of ESBL-producing strains in the UK as a clinical problem from 2000 onwards Subsequently, the spread of ESBLs throughout the UK and Europe was described. 67 As more and more complex resistance mechanisms were being reported, laboratory scientists needed a way of detecting the affected isolates. A paper published in 2001 set out a philosophy called interpretive reading of antibiograms that provided a guide on how to interpret them and infer resistance due to ESBLs and AmpC b-lactamases. 68 More recently carbapenemase-producing organisms, especially Enterobacteriaceae, have become a major concern as they markedly limit the treatment options for patients. The JAC was instrumental in reporting some of the first metallo b-lactamases (MBLs) such as a new variety of the VIM (Verona imipenemase) gene and published many papers on other MBLs, including evaluations of new rapid detection techniques. 69,70 The emergence of non-mbl carbapenemases such K. pneumoniae carbapenemase (KPC) and OXA-48 carbapenemases created further alarm. KPC initially emerged in the USA but is now found across the globe. Similarly, OXA-48 is now prevalent worldwide. JAC published papers with advice on using the antibiograms of suspect isolates and detection methods Resistance mechanism detection was problematic for diagnostic laboratories and reports of testing anomalies were instructive for laboratories; a report by Ratkai et al. 75 in 2009 advised on false positives produced when detecting MBLs in Pseudomonas species. Reviews on classes of carbapenemases such as OXA-48-like enzymes continue to be of significant value as they emerge throughout the UK. 76 In Gram-positive bacteria, the JAC published the first report of glycopeptide intermediate resistance in S. aureus, named later as heterogeneous and homogeneous vancomycin intermediately resistant S. aureus (hvisa and VISA, respectively). 77 The clinical relevance of these strains was debated for many years, with some authors linking the reduced susceptibility to treatment failure. Even more problematic was detection of these isolates in the diagnostic laboratory, since disc diffusion diameters and MICs were normally in the susceptible range. In 2001 a modified population analysis technique was developed to detect these hvisa and VISA isolates. 78 This method was not suitable for diagnostic laboratories. Moreover, testing for hvisa/visa by disc diffusion was proven 326

5 JAC to be inefficient, causing both BSAC and EUCAST to remove the disc diffusion criteria. Hence, detection can only be achieved reliably by reference laboratories. Susceptibility testing of new antimicrobial agents is essential as agents progress from the bench to the clinic and is now being done by a network of laboratories throughout the world, which supply them to EUCAST. The JAC provides a suitable vehicle for conveying this information to clinicians such as the publication of susceptibility breakpoints for ceftaroline and temocillin. 79,80 Future of susceptibility testing Susceptibility testing is one of the most important tasks undertaken by a diagnostic microbiology laboratory. It not only informs patient therapy but is also necessary to monitor resistance trends and measure the impact of infection control. These tests are unusual in always providing an answer, namely susceptible or resistant, whether or not they are performed correctly. Like any other laboratory test, susceptibility tests have an inherent error rate but using a standardized method helps keep this to a minimum, thereby providing, in most cases, accurate and useful results. As the identification of bacteria has become easier than ever with the advent of MALDI-TOF MS, perhaps it is now a good time to place more emphasis on susceptibility testing. Given its long history in publishing papers on susceptibility testing in the UK and beyond, the JAC should continue to attract publications that improve techniques, evaluate new technologies, inform laboratories of pitfalls as well as report on antimicrobial surveillance and patient outcomes. Transparency declarations A. P. M.: Speakers bureau for Astellas, Grant investigator for Cubist/ Merck, The Medicine Company, Bayer Healthcare, Achaogen and Tetraphase. The other authors have none to declare. References 1 Fleming A. 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