Monecke Stefan, Jatzwauk Lutz, Müller Elke, Nitschke Hedda, Pfohl Katharina, Slickers Peter, Reissig Annett, Ruppelt-Lorz Antje, Ehricht Ralf
Institute for Medical Microbiology and Hygiene (IMMH), Technische Universität Dresden, Dresden, Germany.
Alere Technologies GmbH, Jena, Germany.
PLoS One. 2016 Sep 20;11(9):e0162654. doi: 10.1371/journal.pone.0162654. eCollection 2016.
SCCmec elements are very important mobile genetic elements in Staphylococci that carry beta-lactam resistance genes mecA/mecC, recombinase genes and a variety of accessory genes. Twelve main types and a couple of variants have yet been described. In addition, there are also other SCC elements harbouring other markers. In order to subtype strains of methicillin-resistant S. aureus (MRSA) based on variations within their SCCmec elements, 86 markers were selected from published SCC sequences for an assay based on multiplexed primer extension reactions followed by hybridisation to the specific probes. These included mecA/mecC, fusC, regulatory genes, recombinase genes, genes from ACME and heavy metal resistance loci as well as several genes of unknown function. Hybridisation patterns for published genome or SCC sequences were theoretically predicted. For validation of the microarray based assay and for stringent hybridisation protocol optimization, real hybridization experiments with fully sequenced reference strains were performed modifying protocols until yielded the results were in concordance to the theoretical predictions. Subsequently, 226 clinical isolates from two hospitals in the city of Dresden, Germany, were characterised in detail. Beside previously described types and subtypes, a wide variety of additional SCC types or subtypes and pseudoSCC elements were observed as well as numerous composite elements. Within the study collection, 61 different such elements have been identified. Since hybridisation cannot recognise the localisation of target genes, gene duplications or inversions, this is a rather conservative estimate. Interestingly, some widespread epidemic strains engulf distinct variants with different SCCmec subtypes. Notable examples are ST239-MRSA-III, CC5-, CC22-, CC30-, and CC45-MRSA-IV or CC398-MRSA-V. Conversely, identical SCC elements were observed in different strains with SCCmec IVa being spread among the highest number of Clonal Complexes. The proposed microarray can help to distinguish isolates that appear similar or identical by other typing methods and it can be used as high-throughput screening tool for the detection of putative new SCC types or variants that warrant further investigation and sequencing. The high degree of diversity of SCC elements even within so-called strains could be helpful for epidemiological typing. It also raises the question on scale and speed of the evolution of SCC elements.
葡萄球菌盒式染色体 mec 元件（SCCmec 元件）是葡萄球菌中非常重要的可移动遗传元件，携带β-内酰胺抗性基因 mecA/mecC、重组酶基因和多种辅助基因。目前已描述了 12 种主要类型和几种变体。此外，还有其他携带其他标记的 SCC 元件。为了基于耐甲氧西林金黄色葡萄球菌（MRSA）菌株的 SCCmec 元件内的变异对其进行亚型分类，从已发表的 SCC 序列中选择了 86 个标记，用于基于多重引物延伸反应并随后与特异性探针杂交的检测。这些标记包括 mecA/mecC、fusC、调控基因、重组酶基因、来自 ACME 和重金属抗性位点的基因以及几个功能未知的基因。理论上预测了已发表的基因组或 SCC 序列的杂交模式。为了验证基于微阵列的检测方法并优化严格的杂交方案，对完全测序的参考菌株进行了实际杂交实验，修改方案直至结果与理论预测一致。随后，对德国德累斯顿市两家医院的 226 株临床分离株进行了详细表征。除了先前描述的类型和亚型外，还观察到了各种各样的其他 SCC 类型或亚型以及假 SCC 元件以及大量复合元件。在研究收集的菌株中，已鉴定出 61 种不同的此类元件。由于杂交无法识别靶基因的定位、基因重复或倒位，这是一个相当保守的估计。有趣的是，一些广泛传播的流行菌株包含具有不同 SCCmec 亚型的确切变体。显著的例子是 ST239-MRSA-III、CC5-、CC22-、CC30-和 CC45-MRSA-IV 或 CC398-MRSA-V。相反，在不同菌株中观察到相同的 SCC 元件，其中 SCCmec IVa 在最多数量的克隆复合体中传播。所提出的微阵列有助于区分通过其他分型方法看起来相似或相同的分离株，并且它可以用作高通量筛选工具，用于检测需要进一步研究和测序的假定新 SCC 类型或变体。即使在所谓的菌株内，SCC 元件的高度多样性也可能有助于进行流行病学分型。这也提出了关于 SCC 元件进化的规模和速度的问题。
