Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Cátedra de Microbiología, Laboratorio de Resistencia Bacteriana, Ciudad de Buenos Aires, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina; Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Hospital de Clínicas 'José de San Martín', Departamento de Bioquímica Clínica, Laboratorio de Bacteriología Clínica, Ciudad de Buenos Aires, Argentina.
J Glob Antimicrob Resist. 2018 Sep;14:233-237. doi: 10.1016/j.jgar.2018.03.015. Epub 2018 Apr 9.
The aim of this study was to characterise OXA-258 variants and other features that may contribute to carbapenem resistance in Achromobacter ruhlandii.
Kinetic parameters for purified OXA-258a and OXA-258b were determined measuring the rate of hydrolysis of a representative group of antimicrobial agents. Whole-genome shotgun sequencing was performed on A. ruhlandii 38 (producing OXA-258a) and A. ruhlandii 319 (producing OXA-258b), and in silico analysis of antimicrobial resistance determinants was conducted. Substrates of the AxyABM efflux pump were investigated by inhibition assays using phenylalanine-arginine β-naphthylamide (PAβN). Outer membrane protein profiles were resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Kinetic measurements of purified OXA-258 variants displayed an overall weak catalytic efficiency toward β-lactams. A detectable hydrolysis of imipenem was observed. In silico genomic analysis confirmed the presence of 32 and 35 putative efflux pump-encoding genes in A. ruhlandii strains 38 and 319, respectively. Complete sequences for AxyABM and AxyXY efflux pumps, previously described in Achromobacter xylosoxidans, were detected. Decreases in the MICs for chloramphenicol, nalidixic acid and trimethoprim/sulfamethoxazole were observed in the presence of the inhibitor PAβN, suggesting that these antibiotics are substrates of AxyABM. AxyXY-encoding genes of A. ruhlandii 38 and A. ruhlandii 319 displayed 99% identity. No differences were observed in the outer membrane protein profiles.
The contribution of OXA-258 enzymes to the final β-lactam resistance profile may be secondary. Further studies on other putative resistance markers identified in the whole-genome analysis should be conducted to understand the carbapenem resistance observed in A. ruhlandii.
本研究旨在描述在鲁氏不动杆菌中产 OXA-258 变体和其他可能导致碳青霉烯类耐药的特征。
通过测量一组代表性抗菌药物的水解速率来确定纯化的 OXA-258a 和 OXA-258b 的动力学参数。对产生 OXA-258a 的鲁氏不动杆菌 38 株和产生 OXA-258b 的鲁氏不动杆菌 319 株进行全基因组鸟枪法测序，并进行抗菌药物耐药决定因素的计算机分析。通过使用苯丙氨酸-精氨酸 β-萘基酰胺（PAβN）进行抑制试验来研究 AxyABM 外排泵的底物。通过 12%十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）解析外膜蛋白图谱。
纯化的 OXA-258 变体的动力学测量显示对β-内酰胺类药物的总体催化效率较弱。观察到亚胺培南的可检测水解。计算机基因组分析证实，鲁氏不动杆菌株 38 和 319 分别存在 32 和 35 个推定的外排泵编码基因。先前在木糖氧化不动杆菌中描述的 AxyABM 和 AxyXY 外排泵的完整序列被检测到。在抑制剂 PAβN 的存在下，氯霉素、萘啶酸和甲氧苄啶/磺胺甲恶唑的 MIC 降低，表明这些抗生素是 AxyABM 的底物。鲁氏不动杆菌 38 和鲁氏不动杆菌 319 的 AxyXY 编码基因具有 99%的同一性。在外膜蛋白图谱中未观察到差异。
OXA-258 酶对最终β-内酰胺类耐药谱的贡献可能是次要的。应对全基因组分析中鉴定的其他假定耐药标记进行进一步研究，以了解在鲁氏不动杆菌中观察到的碳青霉烯类耐药现象。
