Gutiérrez Diana, Vandenheuvel Dieter, Martínez Beatriz, Rodríguez Ana, Lavigne Rob, García Pilar
Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Departamento de Tecnología y Biotecnología de Productos Lácteos Villaviciosa, Asturias, Spain.
Laboratory of Gene Technology, KU Leuven, Leuven, Belgium.
Appl Environ Microbiol. 2015 May 15;81(10):3336-48. doi: 10.1128/AEM.03560-14. Epub 2015 Mar 6.
Phage therapy is a promising option for fighting against staphylococcal infections. Two lytic phages, vB_SauM_phiIPLA-RODI (phiIPLA-RODI) and vB_SepM_phiIPLA-C1C (phiIPLA-C1C), belonging to the Myoviridae family and exhibiting wide host ranges, were characterized in this study. The complete genome sequences comprised 142,348 bp and 140,961 bp and contained 213 and 203 open reading frames, respectively. The gene organization was typical of Spounavirinae members, with long direct terminal repeats (LTRs), genes grouped into modules not clearly separated from each other, and several group I introns. In addition, four genes encoding tRNAs were identified in phiIPLA-RODI. Comparative DNA sequence analysis showed high similarities with two phages, GH15 and 676Z, belonging to the Twort-like virus genus (nucleotide identities of >84%); for phiIPLA-C1C, a high similarity with phage phiIBB-SEP1 was observed (identity of 80%). Challenge assays of phages phiIPLA-RODI and phiIPLA-C1C against planktonic staphylococcal cells confirmed their lytic ability, as they were able to remove 5 log units in 8 h. Exposure of biofilms to phages phiIPLA-RODI and phiIPLA-C1C reduced the amount of adhered bacteria to about 2 log units in both monospecies and dual-species biofilms, but phiIPLA-RODI turned out to be as effective as the mixture of both phages. Moreover, the frequencies of bacteriophage-insensitive mutants (BIMs) of Staphylococcus aureus and S. epidermidis with resistance to phiIPLA-RODI and phiIPLA-C1C were low, at 4.05 × 10(-7) ± 2.34 × 10(-9) and 1.1 × 10(-7) ± 2.08 × 10(-9), respectively. Overall, a generally reduced fitness in the absence of phages was observed for BIMs, which showed a restored phage-sensitive phenotype in a few generations. These results confirm that lytic bacteriophages can be efficient biofilm-disrupting agents, supporting their potential as antimicrobials against staphylococcal infections.
噬菌体疗法是对抗葡萄球菌感染的一种有前景的选择。在本研究中对两种裂解性噬菌体进行了特性分析，即属于肌尾噬菌体科且具有广泛宿主范围的vB_SauM_phiIPLA - RODI（phiIPLA - RODI）和vB_SepM_phiIPLA - C1C（phiIPLA - C1C）。其完整基因组序列分别为142,348 bp和140,961 bp，分别包含213个和203个开放阅读框。基因组织是葡萄球菌病毒亚科成员的典型特征，具有长的直接末端重复序列（LTRs），基因被分组为彼此没有明显分隔的模块，并且有几个I组内含子。此外，在phiIPLA - RODI中鉴定出四个编码tRNA的基因。比较DNA序列分析表明，它与属于Twort样病毒属的两种噬菌体GH15和676Z具有高度相似性（核苷酸同一性>84%）；对于phiIPLA - C1C，观察到与噬菌体phiIBB - SEP1具有高度相似性（同一性为80%）。噬菌体phiIPLA - RODI和phiIPLA - C1C对浮游葡萄球菌细胞的挑战试验证实了它们的裂解能力，因为它们能够在8小时内清除5个对数单位的细菌。将生物膜暴露于噬菌体phiIPLA - RODI和phiIPLA - C1C下，在单物种和双物种生物膜中，使附着细菌的数量减少到约2个对数单位，但phiIPLA - RODI被证明与两种噬菌体的混合物一样有效。此外，金黄色葡萄球菌和表皮葡萄球菌对phiIPLA - RODI和phiIPLA - C1C具有抗性的噬菌体不敏感突变体（BIMs）的频率较低，分别为4.05×10(-7)±2.34×10(-9)和1.1×10(-7)±2.08×10(-9)。总体而言，观察到BIMs在没有噬菌体的情况下适应性普遍降低，并且在几代后显示出恢复的噬菌体敏感表型。这些结果证实裂解性噬菌体可以是有效的生物膜破坏剂，支持了它们作为抗葡萄球菌感染抗菌剂的潜力。
