Flórez Ana B, Vázquez Lucía, Mayo Baltasar
Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias - Consejo Superior de Investigaciones Científicas, IPLA - CSICAsturias, Spain.
Front Microbiol. 2017 May 24;8:907. doi: 10.3389/fmicb.2017.00907. eCollection 2017.
Metagenomic techniques have been successfully used to monitor antibiotic resistance genes in environmental, animal and human ecosystems. However, despite the claim that the food chain plays a key role in the spread of antibiotic resistance, metagenomic analysis has scarcely been used to investigate food systems. The present work reports a functional metagenomic analysis of the prevalence and evolution of tetracycline resistance determinants in a raw-milk, blue-veined cheese during manufacturing and ripening. For this, the same cheese batch was sampled and analyzed on days 3 and 60 of manufacture. Samples were diluted and grown in the presence of tetracycline on plate count milk agar (PCMA) (non-selective) and de Man Rogosa and Sharpe (MRS) agar (selective for lactic acid bacteria, LAB). DNA from the cultured bacteria was then isolated and used to construct four fosmid libraries, named after the medium and sampling time: PCMA-3D, PCMA-60D, MRS-3D, and MRS-60D. Clones in the libraries were subjected to restriction enzyme analysis, PCR amplification, and sequencing. Among the 300 fosmid clones analyzed, 268 different EcoRI restriction profiles were encountered. Sequence homology of their extremes clustered the clones into 47 groups. Representative clones of all groups were then screened for the presence of tetracycline resistance genes by PCR, targeting well-recognized genes coding for ribosomal protection proteins and efflux pumps. A single tetracycline resistance gene was detected in each of the clones, with four such resistance genes identified in total: (A), (L), (M), and (S). (A) was the only gene identified in the PCMA-3D library, and (L) the only one identified in the PCMA-60D and MRS-60D libraries. (M) and (S) were both detected in the MRS-3D library and in similar numbers. Six representative clones of the libraries were sequenced and analyzed. Long segments of all clones but one showed extensive homology to plasmids from Gram-positive and Gram-negative bacteria. (A) was found within a sequence showing strong similarity to plasmids pMAK2 and pO26-Vir from and , respectively. All other genes were embedded in, or near to, sequences homologous to those of LAB species. These findings strongly suggest an evolution of tetracycline resistance gene types during cheese ripening, which might reflect the succession of the microbial populations. The location of the tetracycline resistance genes in plasmids, surrounded or directly flanked by open reading frames encoding transposases, invertases or mobilization proteins, suggests they might have a strong capacity for transference. Raw-milk cheeses should therefore be considered reservoirs of tetracycline resistance genes that might be horizontally transferred.
宏基因组学技术已成功用于监测环境、动物和人类生态系统中的抗生素抗性基因。然而，尽管有人声称食物链在抗生素抗性传播中起关键作用，但宏基因组分析几乎未被用于研究食品系统。本研究报告了对一种生乳蓝纹奶酪在制造和成熟过程中四环素抗性决定因素的流行情况和演变进行的功能宏基因组分析。为此，在制造的第3天和第60天对同一批奶酪进行采样和分析。将样品稀释后，在四环素存在的情况下，在平板计数乳琼脂（PCMA）（非选择性）和德氏乳杆菌培养基（MRS）琼脂（对乳酸菌有选择性）上培养。然后分离培养细菌的DNA，并用于构建四个fosmid文库，根据培养基和采样时间命名为：PCMA - 3D、PCMA - 60D、MRS - 3D和MRS - 60D。对文库中的克隆进行限制性内切酶分析、PCR扩增和测序。在分析的300个fosmid克隆中，遇到了268种不同的EcoRI限制性图谱。其末端的序列同源性将克隆聚为47组。然后通过PCR筛选所有组的代表性克隆中是否存在四环素抗性基因，靶向编码核糖体保护蛋白和外排泵的公认基因。在每个克隆中检测到一个四环素抗性基因，总共鉴定出四个这样的抗性基因：（A）、（L）、（M）和（S）。（A）是在PCMA - 3D文库中鉴定出的唯一基因，（L）是在PCMA - 60D和MRS - 60D文库中鉴定出的唯一基因。（M）和（S）在MRS - 3D文库中均被检测到，且数量相似。对文库中的六个代表性克隆进行了测序和分析。除一个克隆外，所有克隆的长片段与革兰氏阳性和革兰氏阴性细菌的质粒显示出广泛的同源性。（A）分别在与来自[具体文献1]和[具体文献2]的质粒pMAK2和pO26 - Vir具有高度相似性的序列中被发现。所有其他基因都嵌入在与乳酸菌物种同源的序列中或其附近。这些发现强烈表明在奶酪成熟过程中四环素抗性基因类型发生了演变，这可能反映了微生物种群的演替。四环素抗性基因位于质粒中，周围或直接侧翼是编码转座酶、转化酶或移动蛋白的开放阅读框，这表明它们可能具有很强的转移能力。因此，生乳奶酪应被视为可能水平转移的四环素抗性基因的储存库。
