Franssens, B. T.

Harryvan, T. J.

Melles, D. C.

Rentenaar, R. J.

Schaftenaar, E.

Utrecht University Repository

J Antimicrob Chemother 2024; 79: 2718–2719 https://doi.org/10.1093/jac/dkae245Advance Access publication 16 July 2024EUCAST ciprofloxacin area of technical uncertainty in Escherichia coli and Klebsiella pneumoniae using BD Phoenix™ and VITEK® 2 automated susceptibility systemsT. J. Harryvan 1*, E. Schaftenaar2, B. T. Franssens3, D. C. Melles4 and R. J. Rentenaar11Department of Medical Microbiology, University Medical Centre Utrecht, Utrecht, The Netherlands; 2Department of Medical Microbiology and Immunology, St Antonius Hospital, Utrecht/ Nieuwegein, The Netherlands; 3Department of Medical Microbiology, St Jansdal Hospital, Harderwijk, The Netherlands; 4Department of Medical Microbiology and Medical Immunology, Meander Medical Centre, Amersfoort, The Netherlands*Corresponding author. E-mail: t.j.harrijvan@umcutrecht.nlCiprofloxacin is frequently used in the treatment of Enterobacterales infections and has high efficacy and excellent oral bioavailability. Oral antibiotic step-down therapy with ciprofloxacin was shown to be effective in patients with Enterobacterales-associated infec-tions1,2 and reduced the length of hospital admission.2EUCAST has determined MIC and disc diffusion (DD) growth inhibition zone (GIZ) breakpoints for interpreting ciprofloxacin susceptibility in Enterobacterales. In Enterobacterales, a cipro-floxacin MIC of 0.5 mg/L or GIZ of 22–24 mm constitutes the area of technical uncertainty (ATU) and exactly overlaps with the ‘susceptible, increased exposure (I)’ category. In this case, EUCAST suggests, amongst other things, to report the results as unreliable, downgrade the susceptibility category or perform an alternative test to confirm the initial findings.3Automated susceptibility systems, like BD Phoenix™ and VITEK® 2 do not qualify as EUCAST methodologies. Nevertheless, these methods are frequently used and results are often interpreted using EUCAST breakpoints. Therefore, in four laboratories, we inves-tigated a total of 861 unique Enterobacterales isolates (Figure S1a and b, available as Supplementary data at JAC Online) with either BD Phoenix™ or VITEK® 2 ciprofloxacin antimicrobial susceptibility testing (AST) MICs of 0.5 mg/L (ATU/I) by using ciprofloxacin gradi-ent strip tests (GSTs) and, in a subset of isolates (n = 91, laboratory 1), ciprofloxacin DD was included to compare with a true EUCAST methodology (Supplementary methods).Primary and secondary susceptibility testing and reporting strategies per laboratory are described in Figure 1(a). The majority of Enterobacterales isolates (63/91; 69%) determined to be in the ATU/I category by BD Phoenix AST had a GIZ within the susceptible (S) category (Figure S2a, yellow bars). Only, 21/91 (23%) and 7/91 (8%) of tested Enterobacterales had a GIZ corresponding to the ATU/I and resistant (R) categories, respectively (Figure S2a, yellow bars). EUCAST historic data of Enterobacterales with broth micro-dilution (BMD) ciprofloxacin MICs of 0.5 mg/L displayed the follow-ing ciprofloxacin GIZ distribution: S (5/24; 21%); ATU/I (16/24; 67%); and R (3/24; 13%) (Figure S2a, green bars). Restricting this analysis to E. coli, BD Phoenix ATU category E. coli isolates had ci-profloxacin GIZ corresponding to S in 33/37 (89%), ATU/I in 3/37 (8%) and R in 1/37 (3%) (Figure S2b, yellow bars). E. coli isolates from the historic EUCAST cohort, with BMD ciprofloxacin MICs of 0.5 mg/L, had GIZ corresponding to S in 4/17 (24%), ATU/I in 11/17 (65%) and R in 2/17 (12%) (Figure S2b, green bars). Altogether, Enterobacterales with a BD Phoenix ciprofloxacin AST MIC of 0.5 mg/L (ATU/I) appeared susceptible using DD in the majority of cases, especially in Escherichia coli.In the same Enterobacterales isolates (laboratory 1), ciprofloxa-cin GST resulted in 58%, 36% and 6% S, ATU/I or R categorizations, respectively (Figure 1b, left panel), compared with 69%, 23% and 8% for DD (Figure S2a). In laboratories 2, 3 and 4, Enterobacterales isolates with VITEK® 2 ciprofloxacin AST MICs of 0.5 mg/L were predominantly S (range 37%–59%; Figure 1c–e, left panels) or ATU/I upon retesting using GST. The percentage of ci-profloxacin resistance, measured by GST among these isolates in la-boratories 2, 3 and 4 was equally low (ranges: 1%, 8–4%, 9%), and a ciprofloxacin MIC of 1 mg/L occurred in the majority (Figure 1b–e, left panels). Subgroup analyses of E. coli isolates with ciprofloxacin AST MIC results of 0.5 mg/L showed low resistance rates of cipro-floxacin by GST (range 0%–2.8%, Figure 1b–e, top-right panels) but in Klebsiella pneumoniae isolates, ciprofloxacin-resistant iso-lates were found in 2.3%–9.1% (Figure 1b–e, bottom-right panels).Altogether, our data indicate that Enterobacterales with auto-mated ciprofloxacin AST measurements in the ATU (0.5 mg/L) tend to underestimate susceptibility in comparison with ciprofloxa-cin DD and GST. Therefore, the main finding of this study is that, re-gardless of laboratory or primary semi-automated AST system, retesting Enterobacterales with a ciprofloxacin MIC of 0.5 mg/L using a different method frequently results in classification to the S or I category and rarely in classification to the R category.Clinical microbiology laboratory practices currently differ in handling the ciprofloxacin ATU in Enterobacterales, illustrated by the participating laboratories in this study (Figure 1a). © The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.2718Downloaded from https://academic.oup.com/jac/article/79/10/2718/7714438 by guest on 15 October 2024Retesting ATU category isolates identified by automated AST, using ciprofloxacin DD or GST, seems a viable approach since a significant portion of these isolates are susceptible. Therefore, we feel that direct R categorization of these isolates will lead to disqualification of an adequate treatment option in most of these isolates. Simply categorizing the isolates as I, ignoring the ATU, might also be an option in the case of E. coli, since resistance measured by an alternative method is infrequent. However, our findings may not apply to regions with different epidemiology, or in the case of changes in coverage of antibiotics by manufac-turers of the AST panels. Importantly, infection type and alterna-tive antibiotic options should be taken into consideration. Finally, our data demonstrate that automated AST and EUCAST reference methods are not always comparable, as has been previously demonstrated for gentamicin.4 This warrants careful evaluation of other ‘bug–drug’ combinations before extrapolating semi- automated AST results to EUCAST methodologies.AcknowledgementsWe would like to thank Dr P. J. Haas and Dr O. Karatuna for their help with data acquisition. Figure 1a has been created using BioRender.com.FundingThis study was supported by internal funding and was conducted as part of our routine work.Transparency declarationsThe authors declare no conflicts of interest and no source of external funding.Author contributionsT.J.H. and R.J.R. designed the study. T.J.H., E.S., B.T.F., D.C.M. and R.J.R. col-lected and analysed the data. T.J.H. and R.J.R. co-wrote the manuscript. All authors amended and approved the final version of the manuscript.Supplementary dataFigures S1 and S2 are available as Supplementary data at JAC Online.References1 GBD 2019 Antimicrobial Resistance Collaborators. Global mortality as-sociated with 33 bacterial pathogens in 2019: a systematic analysis for the global burden of disease study 2019. Lancet 2022; 400: 2221–48. https://doi.org/10.1016/S0140-6736(22)02185-72 Tamma PD, Conley AT, Cosgrove SE et al. Association of 30-day mor-tality with oral step-down vs continued intravenous therapy in pa-tients hospitalized with Enterobacteriaceae bacteremia. JAMA Intern Med 2019; 179: 316–23. https://doi.org/10.1001/jamainternmed. 2018.62263 Kahlmeter G; EUCAST Steering Committee. EUCAST proposes to change the definition and usefulness of the susceptibility category ‘intermediate’. Clin Microbiol Infect 2017; 23: 894–5. https://doi.org/10.1016/j.cmi.2017. 08.0154 Klein Klouwenberg PMC, Altorf-van der Kuil W, van Griethuysen AJ et al. False aminoglycoside resistance in Enterobacterales and non- fermenters by an automated testing system: a descriptive study. Microbiol Spectr 2023; 11: e0309323. https://doi.org/10.1128/ spectrum.03093-23Figure 1. (a) Ciprofloxacin ATU handling strategies of Enterobacterales isolates, stratified for the different participating laboratories. *Ciprofloxacin ATU Enterobacterales isolates from laboratory 1 were reported as ‘R’ for clinical purposes, and stored in the freezer for subsequent further analysis by DD and GSTs. (b–e) GST ciprofloxacin MICs and SIR categorizations of all Enterobacterales isolates (left panels), or E. coli (top-right panels, frequencies of SIR categorizations only) and K. pneumoniae (bottom-right panels, frequencies of SIR categorizations only) specifically, stratified according to the dif-ferent participating laboratories (laboratories 1, 2, 3 and 4). Laboratory 1, UMC Utrecht; laboratory 2, Meander Medical Centre; laboratory 3, St. Jansdal hospital; laboratory 4, St. Antonius hospital.Research letters                                                                                                                                                2719Downloaded from https://academic.oup.com/jac/article/79/10/2718/7714438 by guest on 15 October 2024

EUCAST ciprofloxacin area of technical uncertainty in Escherichia coli and Klebsiella pneumoniae using BD Phoenix™ and VITEK® 2 automated susceptibility systems

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EUCAST ciprofloxacin area of technical uncertainty in Escherichia coli and Klebsiella pneumoniae using BD Phoenix™ and VITEK® 2 automated susceptibility systems

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