Marti Hanna, Kim Hoyon, Joseph Sandeep J, Dojiri Stacey, Read Timothy D, Dean Deborah
Center for Immunobiology and Vaccine Development, University of California at San Francisco/Benioff Children's Hospital Oakland Research Institute, Oakland CA, USA.
Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, AtlantaGA, USA; Department of Human Genetics, Emory University School of Medicine, AtlantaGA, USA.
Front Microbiol. 2017 Feb 7;8:156. doi: 10.3389/fmicb.2017.00156. eCollection 2017.
is a swine pathogen that has also recently been found to cause zoonotic infections of the human eye, pharynx, and gastrointestinal tract. Many strains contain a tetracycline class C gene [(C)] cassette that confers tetracycline resistance. The cassette was likely originally acquired by horizontal gene transfer from a Gram-negative donor after the introduction of tetracycline into animal feed in the 1950s. Various research groups have described the capacity for different species to exchange DNA by homologous recombination. Since over 90% of strains are tetracycline resistant, they represent a potential source for antibiotic-resistance spread within and between species. Here, we examined the genetics of (C)-transfer among strains. Tetracycline-sensitive strain S45 was simultaneously or sequentially co-infected with tetracycline-resistant strains in McCoy cells. Potential recombinants were clonally purified by a harvest assay derived from the classic plaque assay. strain Rogers132, lacking transposases IS and IS, was the most efficient donor, producing two unique recombinants detected in three of the 56 (5.4%) clones screened. Recombinants were found to have a minimal inhibitory concentration (MIC) of 8-16 μg/mL for tetracycline. Resistance remained stable over 10 passages as long as recombinants were initially grown in tetracycline at twice the MIC of S45 (0.032 μg/mL). Genomic analysis revealed that (C) had integrated into the S45 genome by homologous recombination at two unique sites depending on the recombinant: a 55 kb exchange between F and G, and a 175 kb exchange between A and Q. Neither site was associated with inverted repeats or motifs associated with recombination hotspots. Our findings show that cassette transfer into S45 has low frequency, does not require IS/IS transposases, is stable if initially grown in tetracycline, and results in multiple genomic configurations. We provide a model for stable cassette transfer to better understand the capability for cassette acquisition by species that infect humans, a matter of public health importance.
是一种猪病原体，最近还被发现可引起人类眼睛、咽部和胃肠道的人畜共患感染。许多菌株含有赋予四环素抗性的四环素C类基因[(C)]盒。该盒式结构可能最初是在20世纪50年代四环素引入动物饲料后通过水平基因转移从革兰氏阴性供体获得的。不同的研究小组描述了不同物种通过同源重组交换DNA的能力。由于超过90%的菌株对四环素耐药，它们是抗生素耐药性在物种内部和物种之间传播的潜在来源。在这里，我们研究了菌株间(C)转移的遗传学。四环素敏感菌株S45在 McCoy 细胞中同时或依次与四环素耐药菌株共同感染。潜在的重组体通过源自经典噬菌斑测定的收获测定进行克隆纯化。缺乏转座酶IS和IS的菌株Rogers132是最有效的供体，在筛选的56个克隆中的3个(5.4%)中产生了两个独特的重组体。发现重组体对四环素的最低抑菌浓度(MIC)为8 - 16μg/mL。只要重组体最初在四环素中以S45的MIC(0.032μg/mL)的两倍生长，抗性在10代中保持稳定。基因组分析表明，根据重组体的不同，(C)通过同源重组在两个独特位点整合到S45基因组中：F和G之间的55 kb交换，以及A和Q之间的175 kb交换。这两个位点均与反向重复序列或与重组热点相关的基序无关。我们的研究结果表明，盒式结构转移到S45中的频率较低，不需要IS/IS转座酶，如果最初在四环素中生长则是稳定的，并导致多种基因组构型。我们提供了一个稳定盒式结构转移的模型，以更好地理解感染人类的物种获取盒式结构的能力，这是一个具有公共卫生重要性的问题。
