Fatty acid kinase A is an important determinant of biofilm formation in Staphylococcus aureus USA300
Fatty acid kinase A is an important determinant of biofilm formation in Staphylococcus aureus USA300
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DOI：
10.1186/s12864-015-1956-8
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发表时间：
2015-10-26
期刊：
影响因子：
4.4
通讯作者：
Malhotra-Kumar, S.
中科院分区：
文献类型：
--
作者：
Sabirova, J. S.;Hernalsteens, J-P;Malhotra-Kumar, S.
背景资料：耐甲氧西林金黄色葡萄球菌（MRSA）-USA 300因其引起社区和医疗保健获得性感染的能力而臭名昭著，当与生物膜表型相关时，这些感染甚至更难治疗。我们的目的是描述在USA 300皮肤脓肿分离株（UAS 391）中生物膜形成的遗传决定因素，该分离株形成了多产的生物膜。基于全基因组作图（ArgusTM光学作图系统，OpgenInc，盖瑟斯堡，美国），发现金黄色葡萄球菌菌株TCH 1516和FPR 3757与UAS 391密切相关（96.3- 99.1%相似性，P=0.0151），然而在动态测定（BioFlux 200，Fluxion Biosciences，美国）上，生物膜形成显著不同（P=0.0001）。通过比较这些菌株的全基因组序列，共发现了18个基因的差异。这些靶基因中相应的Tn（携带法氏囊）敲除突变体从相同克隆谱系（USA 300-JE 2）的公开可用的突变体文库获得，并表征生物膜形成的表型。在生物膜形成方面显示出显著差异的Tn突变体用于转导至UAS 391的质粒固化红霉素敏感性衍生物中并用于互补实验。在动态测定法上测试所有菌株，并将17小时生物膜染色（SYT 09，Life Technologies），并通过显微镜（Zeiss，ImageJ）定量荧光强度。通过定量逆转录酶PCR（StepOnePlusTM，Applied Biosystems（R））研究Tn和转导突变体中的基因表达水平。结果：比较TCH 1516、FPR 3757和UAS 391的测序基因组产生了有限数量的变异基因（n=18），假设这些变异基因解释了观察到的生物膜形成能力的差异。筛选在这些靶基因中被破坏的Tn突变体，鉴定出一种在SAUSA300_1119（fakA）中携带转座子插入的突变体（NE 229），其表现出与UAS 391相似的生物膜形成增加（P=0.9320）。转导实验证实，fakA：：Tn对应于生物膜形成的1.9- 4.6倍增加，这取决于USA 300菌株背景（P
Background: Methicillin-resistant Staphylococcus aureus (MRSA)-USA300 is notorious for its ability to cause community- and healthcare-acquired infections, which are even more difficult to treat when associated with a biofilm phenotype. We aimed to characterize the genetic determinants of biofilm formation in a USA300 skin abscess isolate (UAS391) that formed prolific biofilms.Methods: USA300 S. aureus strains, TCH1516 and FPR3757, were found to be closely related based on whole genome mapping (Argus (TM) Optical Mapping System, Opgen Inc, Gaithersburg, USA) to UAS391 (96.3-99.1 % similarity, P=0.0151), however differed markedly in biofilm formation (P=0.0001) on a dynamic assay (BioFlux 200, Fluxion Biosciences, USA). Comparison of whole genome sequences of these strains identified differences in a total of 18 genes. Corresponding Tn (bursa aurealis-bearing) knockout mutants in these target genes were obtained from a publicly available mutant library of the same clonal lineage (USA300-JE2) and were characterized phenotypically for biofilm formation. Tn mutants showing significant differences in biofilm formation were utilized for transduction into a plasmid-cured erythromycin-sensitive derivative of UAS391 and for complementation experiments. All strains were tested on the dynamic assay, and 17h-biofilms were stained (SYTO9, Life Technologies) and fluorescence intensity quantified by microscopy (Zeiss, ImageJ). Gene expression levels in Tn and transduced mutants were studied by quantitative reverse transcriptase PCR (StepOnePlusTM, Applied Biosystems (R)).Results: Comparison of the sequenced genomes of TCH1516, FPR3757 and UAS391 yielded a limited number of variant genes (n=18) that were hypothesized to account for the observed difference in biofilm-forming capacity. Screening of Tn mutants disrupted in these target genes identified one mutant (NE229) bearing a transposon insertion in SAUSA300_1119 (fakA), which exhibited increased biofilm formation similar to UAS391 (P=0.9320). Transduction experiments confirmed that fakA:: Tn corresponded to 1.9- to 4.6-fold increase in biofilm formation depending on the USA300 strain background (P