Sewell Max, Farley Caitlin, Portal Edward A R, Lindsay Diane, Ricci Maria Luisa, Jarraud Sophie, Scaturro Maria, Descours Ghislaine, Krøvel Anne Vatland, Barton Rachael, Boostom Ian, Ure Roisin, Kese Darja, Gaia Valeria, Golob Matej, Paukner Susanne, Ginevra Christophe, Afshar Baharak, Nadarajah Sendurann, Wybo Ingrid, Michel Charlotte, Echahdi Fedoua, González-Rubio Juana María, González-Camacho Fernando, Mentasti Massimo, Flountzi Anastasia S, Petzold Markus, Moran-Gilad Jacob, Uldum Søren, Winchell Jonas, Wooton Mandy, Bernard Kathryn, Jones Lucy C, Chalker Victoria J, Spiller Owen B
Medical Microbiology, Division of Infection and Immunity, Cardiff University, Cardiff, UK.
Medical Microbiology, Division of Infection and Immunity, Cardiff University, Cardiff, UK; Ineos Oxford Institute for Antimicrobial Research (IOI), Department of Biology, University of Oxford, UK.
J Microbiol Methods. 2025 Jan;228:107071. doi: 10.1016/j.mimet.2024.107071. Epub 2024 Dec 18.
Currently there is no detailed, internationally agreed protocol defined to evaluate antimicrobial susceptibility testing (AST) for Legionella pneumophila (required to establish epidemiological cut-off value or "ECOFF" boundaries); therefore, antimicrobial resistance in these isolates cannot be defined. AST methods utilising media containing activated charcoal as an ingredient, to enable Legionella growth, are unreliable as noted in an internationally authored opinion paper and a new gold standard is required. Here we define a detailed protocol for broth microdilution (BMD) using defined cell culture collection-deposited control reference strains (Philadelphia-1 and Knoxville-1) as well as two accessible reference strains with moderately (lpeAB-carrying) and markedly (23S rRNA mutation-carrying) elevated azithromycin minimum inhibitory concentration (MIC). The defined protocol enables up to eight L. pneumophila strains to be set up on a single 96-well plate per antimicrobial tested. Initial ranges to routinely capture an MIC for these reference strains using clinically relevant antimicrobials azithromycin (0.01-0.25 mg/L), levofloxacin (0.008-0.03 mg/L), lefamulin (0.01-2 mg/L), rifampicin (0.0002-0.0008 mg/L) and doxycycline (0.25-16 mg/L) following incubation for 48 h at 37 °C in a shaking incubator have been empirically determined. Establishment of this internationally agreed protocol sets the scene for the next step: validation and comparison of antimicrobial ranges between international Legionella reference laboratories to establish putative resistance cut-off thresholds for these clinically relevant antimicrobials.
目前，尚无详细的、国际公认的方案来评估嗜肺军团菌的抗菌药敏试验（AST）（确定流行病学临界值或“ECOFF”界限所必需）；因此，无法确定这些分离株中的抗菌耐药性。正如一篇国际作者撰写的意见书所指出的，使用含有活性炭作为成分的培养基以促进军团菌生长的AST方法不可靠，需要一种新的金标准。在此，我们定义了一种肉汤微量稀释（BMD）的详细方案，使用已定义的细胞培养保藏对照参考菌株（费城-1和诺克斯维尔-1）以及两种可获取的参考菌株，其阿奇霉素最低抑菌浓度（MIC）中度（携带lpeAB）和显著（携带23S rRNA突变）升高。所定义的方案允许在每个测试抗菌药物的单个96孔板上最多设置8株嗜肺军团菌菌株。使用临床相关抗菌药物阿奇霉素（0.01 - 0.25mg/L）、左氧氟沙星（0.008 - 0.03mg/L）、左法莫林（0.01 - 2mg/L）、利福平（0.0002 - 0.0008mg/L）和强力霉素（0.25 - 16mg/L），在37°C的振荡培养箱中孵育48小时后，凭经验确定了常规捕获这些参考菌株MIC的初始范围。这一国际公认方案的建立为下一步奠定了基础：国际军团菌参考实验室之间对抗菌药物范围进行验证和比较，以确定这些临床相关抗菌药物的假定耐药临界阈值。
