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Purpose The emergence of Extensively drug resistant (XDR) pathogens like Carbapenem Resistant Klebsiella pneumoniae (CR Kpn) and Carbapenem Resistant Escherichia coli (CR Eco) has limited therapeutic options for treating them. Fosfomycin a broad-spectrum antibiotic, has emerged as a potential treatment option in combination with other agents. It is therefore important that accurate drug susceptibility testing (DST) results of fosfomycin should be available to all clinical microbiology laboratories. Agar dilution which is the recommended method for fosfomycin DST is not convenient to adopt in a routine set-up. This study aimed to determine the susceptibility pattern of CR Kpn and CR Eco to fosfomycin and to evaluate the discrepancies of the available manual MIC based alternative methods. Methods Agar dilution (AD), broth microdilution (BMD), E-test and Ezy MIC test were performed on 235 CR-Kpn and Eco isolates respectively. Results Of 177 CR Kpn, 31.63% (n = 56/177) of the isolates were susceptible by AD. Categorical Agreement (CA) by BMD, E-test and Ezy MIC were lower than the acceptable limit while Very Major Errors (VMEs) and Major Errors (MEs) were beyond the acceptable limits. In the case of CR Eco, 96.55% (n = 56/58) were susceptible by AD. CA of 100% (n = 58/58) was shown by both BMD and Ezy MIC while 86.20% (n = 50/58) was shown by E-test, with no VME observed for CR Eco. ME was only observed for E-test method. Conclusion The alternative methods were in poor agreement with AD method for CR Kpn and for CR Eco, BMD and Ezy MIC have shown reliable results. Introduction Globally, the most important deterrent to antibiotic efficacy is the rapid emergence of drug resistant bacteria. Of major concern are the Carbapenem Resistant Enterobacteriaceae (CRE), as Carbapenems are the drugs of choice to treat infections by Multidrug Resistant (MDR) gram-negative organisms. Infections caused by CRE are associated with 50% mortality [1]. CR Kpn and CR Eco are the major causes of hospital and community acquired infections [1,2]. Carbapenem resistance has escalated from 35% in 2016 to 57% in 2021 among Kpn whereas Eco has seen an upsurge from 14% to 36% for the same year span [3]. The expeditious dissemination of Kpn producing New Delhi Metallo-beta-lactamase (NDM) have been reported in many countries from 2008 onwards [4]. In the Indian subcontinent, Kpn producing NDM was said to be endemic but is now being accompanied by the escalating incidences of OXA-48 [5,6]. Fosfomycin is a bactericidal agent that has a broad-spectrum activity [7]. It is known for its immunomodulating activity and activity against bacterial biofilms [8]. Oral formulation of fosfomycin is commonly used to treat uncomplicated urinary tract infections. There is renewed interest in the intravenous formulation of fosfomycin as it can be a part of an effective combination therapy for managing critical infections due to XDR Enterobacteriaceae [9]. Breakpoints for fosfomycin differs from Clinical and Laboratory Standard Institute (CLSI) to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines depending on the formulation. CLSI mentions breakpoints for the oral formulation of fosfomycin, for its use against urinary tract infections caused by Eco [10]. While EUCAST mentions guidelines for intravenous fosfomycin and for its use against Enterobacteriaceae infections [11]. Both the guidelines have recommended agar dilution (AD) as the gold standard method for fosfomycin susceptibility. However, AD is not suitable for its use in routine clinical laboratories. Also, for fosfomycin susceptibility, glucose-6-phosphate (G6P) is required to enhance the hexose-6-phosphate uptake system [8]. Moreover, automated methods have not shown acceptable results [9]. Therefore, we have considered to evaluate the manual based MIC methods; Broth microdilution (BMD), E-test (bioMerieux) and Ezy MIC (HiMedia). In this study we aim to determine the in vitro activity of fosfomycin against CR Kpn and CR Eco and to evaluate the discrepancies of the available fosfomycin susceptibility testing manual based MIC methods. Section snippets Bacterial isolates A total of 235 pathogenic non-duplicating clinical isolates of CR Kpn (n = 177) and CR Eco (n = 58) isolated from urine (n = 50), blood (n = 89), respiratory tract (n = 10), sterile body fluids (n = 38), wound (n = 10), pus (n = 22) and tissue (n = 16) were tested in this study. Antimicrobial agent Fosfomycin sodium (Sigma Aldrich, Switzerland), E-test (bioMerieux, France) and Ezy MIC impregnated with G6P (HiMedia Pvt Ltd, Pune, India) were procured. Susceptibility testing The in vitro susceptibility of the test isolates Results Isolates were categorized as susceptible or resistant in accordance with EUCAST breakpoints. Of the 177 CR Kpn, 31.63% of the isolates were susceptible to fosfomycin by AD method and 68.36% were resistant. For CR Eco, 96.55% of the isolates were susceptible and 3.44% were resistant. On calculating MIC50 and MIC90, E-test showed the lowest values as compared to the other methods (Table 1). According to the EUCAST guidelines, AD is the reference method for the in vitro susceptibility testing of Discussion In this era with the emergence of XDR pathogens, therapeutic options for treating serious infections due to CRE are limited. This therefore necessitated the need for revaluating older drugs. Fosfomycin due to its bactericidal effect against both gram-negative and gram-positive organisms has shown to be a potential option in treating infections caused by these pathogens [8]. In recent studies, fosfomycin has shown to be susceptible to many CRE isolates and the fact that it exhibits synergistic Conclusion On evaluating the performances of the alternative manual based MIC methods, for CR Kpn the methods were in poor agreement with the reference method. Clinical laboratories should therefore gear up to perform agar dilution if fosfomycin has to be evaluated for XDR pathogens, especially for CR Kpn. CRediT authorship contribution statement Joanna Valanie Pereira: Conceptualization, Validation, Formal analysis, Investigation, Data Curation, Writing - Original Draft, Writing - Review & Editing, Visualization. Anurag Kumar Bari: Methodology, Validation, Investigation, Resources, Data Curation, Writing - Review & Editing, Supervision, Project administration. Rashmi Kokare: Validation, Investigation, Data Curation, Writing - Review & Editing. Aruna Poojary: Writing - Review & Editing, Supervision, Project administration. Funding None. Ethical approval This study was approved by the Breach Candy Medical Research Centre (BCMRC) Ethics Committee (No: P18-2016). Declaration of competing interest None declared. Acknowledgement We thank BCMRC for its financial assistance. References (21) K.Z. Vardakas et al. Susceptibility of contemporary isolates to fosfomycin: a systematic review of the literature Int J Antimicrob Agents (2016) P.V. Saiprasad et al. Exploring the hidden potential of fosfomycin for the fight against severe Gram-negative infections Indian J Med Microbiol (2016) G. Camarlinghi et al. Discrepancies in fosfomycin susceptibility testing of KPC-producing Klebsiella pneumoniae with various commercial methods Diagn Microbiol Infect Dis (2019) B. Grabein et al. Intravenous fosfomycin-back to the future. Systematic review and meta-analysis of the clinical literature Clin Microbiol Infect (2017) A.M. Kelly et al. Carbapenem-resistant Enterobacteriaceae in the community: a scoping review Int J Antimicrob Agents (2017) T. Xiao et al. A retrospective, comparative analysis of risk factors and outcomes in carbapenem-susceptible and carbapenem-nonsusceptible Klebsiella pneumoniae bloodstream infections: tigecycline significantly increases the mortality Infect Drug Resist (2018) Annual report 2021 (2021) M. Berrazeg et al. New Delhi Metallo-beta-lactamase around the world : an eReview using Google Maps Eurosurviellance (2014) P. Nordmann et al. Global spread of carbapenemase-producing Enterobacteriaceae Emerg Infect Dis (2011) B. Veeraraghavan et al. Carbapenem resistant Klebsiella pneumoniae isolated from bloodstream infection : Indian experience Pathog Glob Health (2017) There are more references available in the full text version of this article. Cited by (0) Recommended articles (0) View full text © 2023 Indian Association of Medical Microbiologists. Published by Elsevier B.V. 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