Characterization of Multidrug Resistant <i>E</i>. <i>faecalis</i> Strains from Pigs of Local Origin by ADSRRS-Fingerprinting and MALDI -TOF MS; Evaluation of the Compatibility of Methods Employed for Multidrug Resistance Analysis

The aim of this study was to characterize multidrug resistant E. faecalis strains from pigs of local origin and to analyse the relationship between resistance and genotypic and proteomic profiles by amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI -TOF MS). From the total pool of Enterococcus spp. isolated from 90 pigs, we selected 36 multidrug resistant E. faecalis strains, which represented three different phenotypic resistance profiles. Phenotypic resistance to tetracycline, macrolides, phenicols, and lincomycin and high-level resistance to aminoglycosides were confirmed by the occurrence of at least one corresponding resistance gene in each strain. Based on the analysis of the genotypic and phenotypic resistance of the strains tested, five distinct resistance profiles were generated. As a complement of this analysis, profiles of virulence genes were determined and these profiles corresponded to the phenotypic resistance profiles. The demonstration of resistance to a wide panel of antimicrobials by the strains tested in this study indicates the need of typing to determine the spread of resistance also at the local level. It seems that in the case of E. faecalis, type and scope of resistance strongly determines the genotypic pattern obtained with the ADSRRS-fingerprinting method. The ADSRRS-fingerprinting analysis showed consistency of the genetic profiles with the resistance profiles, while analysis of data with the use of the MALDI- TOF MS method did not demonstrate direct reproduction of the clustering pattern obtained with this method. Our observations were confirmed by statistical analysis (Simpson’s index of diversity, Rand and Wallace coefficients). Even though the MALDI -TOF MS method showed slightly higher discrimination power than ADSRRS-fingerprinting, only the latter method allowed reproduction of the clustering pattern of isolates based on phenotypic resistance and analysis of resistance and virulence genes (Wallace coefficient 1.0). This feature seems to be the most useful for epidemiological purposes and short-term analysis.本研究旨在对本地来源生猪分离的多重耐药粪肠球菌（E. faecalis）菌株进行表型与基因型特征分析，并通过稀有酶切位点周围DNA片段扩增指纹技术（amplification of DNA fragments surrounding rare restriction sites, ADSRRS-fingerprinting）与基质辅助激光解吸电离飞行时间质谱（matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF MS），探究耐药性与基因型、蛋白质组型之间的关联。从90头生猪分离得到的肠球菌属（Enterococcus spp.）总菌株库中，本研究筛选出36株多重耐药粪肠球菌菌株，其涵盖3种不同的表型耐药谱。通过检测各菌株中至少一种对应耐药基因的存在，确认了受试菌株对四环素、大环内酯类、酰胺醇类及林可霉素的表型耐药性，以及高水平氨基糖苷类耐药性。基于受试菌株的基因型与表型耐药性分析，本研究共得到5种明确的耐药谱。作为本分析的补充，我们还测定了菌株的毒力基因谱，且该谱与表型耐药谱具有一一对应性。本研究中受试菌株对广谱抗菌药物表现出耐药性，这提示有必要开展分型研究，以在本地层面探明耐药性的传播态势。就粪肠球菌而言，耐药性的类型与范围似乎极大程度上决定了通过ADSRRS指纹技术得到的基因型谱。ADSRRS指纹分析显示，菌株的遗传谱与耐药谱具有高度一致性；而采用MALDI-TOF MS方法进行数据分析时，并未得到与该方法所得聚类模式直接吻合的结果。本研究的观察结果经统计学分析得到了验证，包括辛普森多样性指数（Simpson’s index of diversity）、兰德系数（Rand coefficient）与华莱士系数（Wallace coefficient）。尽管MALDI-TOF MS方法的区分能力略高于ADSRRS指纹技术，但仅后者能够基于表型耐药性重现分离株的聚类模式，且可同时用于耐药性与毒力基因的分析（华莱士系数为1.0）。该特性在流行病学研究与短期分析中似乎最为实用。