Low levels of β-lactam antibiotics induce extracellular DNA release and biofilm formation in Staphylococcus aureus.
Low levels of β-lactam antibiotics induce extracellular DNA release and biofilm formation in Staphylococcus aureus.
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作者：
Kaplan JB;Izano EA;Gopal P;Karwacki MT;Kim S;Bose JL;Bayles KW;Horswill AR
抗生素的亚最低抑制浓度已被证明可诱导细菌生物膜的形成。很少有研究调查抗生素诱导的金黄色葡萄球菌（一种重要的人类病原体）生物膜的形成。我们的目标是测量低水平 β-内酰胺抗生素存在下金黄色葡萄球菌生物膜的形成。采用了 15 种系统发育多样化的耐甲氧西林金黄色葡萄球菌 (MRSA) 和甲氧西林敏感金黄色葡萄球菌 (MSSA) 菌株。将甲氧西林、氨苄青霉素、阿莫西林和氯唑西林添加到培养物中，浓度范围为 0× 至 1× MIC。使用结晶紫结合测定在 96 孔微量滴定板中测量生物膜的形成。使用视觉试管沉降测定来测量自聚集。使用琼脂糖凝胶电泳对细胞外 DNA 进行定量。所有四种抗生素都诱导某些菌株形成生物膜。生物膜诱导量高达10倍，并且与不存在抗生素时菌株产生的生物膜量成反比。 USA300、USA400 和 USA500 谱系的 MRSA 菌株表现出最高水平的甲氧西林诱导的生物膜诱导。低浓度甲氧西林诱导的生物膜形成被 DNase 抑制。低浓度的甲氧西林还会诱导 DNase 敏感的自动聚集和细胞外 DNA 释放。在自溶素（atl）缺陷的菌株中不存在生物膜诱导表型。我们的研究结果表明，在某些金黄色葡萄球菌菌株中，亚最小抑制浓度的 β-内酰胺抗生素可显着诱导自溶素依赖性细胞外 DNA 释放和生物膜形成。在农业中广泛使用抗生素作为生长促进剂可能会使细菌接触低浓度的药物。本研究的目的是调查低水平抗生素对细菌自动聚集和生物膜形成的影响，这两个过程已被证明可以促进遗传交换和抗生素耐药性。我们发现，低水平的β-内酰胺抗生素（一类在临床和农业环境中常用的抗生素）会导致重要的人类病原体金黄色葡萄球菌发生显着的自聚集和生物膜形成。这两个过程都依赖于细胞裂解和 DNA 释放到环境中。这种效应在被称为耐甲氧西林金黄色葡萄球菌 (MRSA) 的多重耐药菌株中最为明显。这些结果可能有助于揭示临床环境中某些细菌感染对抗生素治疗的顽抗以及农业环境中抗生素耐药细菌的进化。
Subminimal inhibitory concentrations of antibiotics have been shown to induce bacterial biofilm formation. Few studies have investigated antibiotic-induced biofilm formation in Staphylococcus aureus, an important human pathogen. Our goal was to measure S. aureus biofilm formation in the presence of low levels of β-lactam antibiotics. Fifteen phylogenetically diverse methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains were employed. Methicillin, ampicillin, amoxicillin, and cloxacillin were added to cultures at concentrations ranging from 0× to 1× MIC. Biofilm formation was measured in 96-well microtiter plates using a crystal violet binding assay. Autoaggregation was measured using a visual test tube settling assay. Extracellular DNA was quantitated using agarose gel electrophoresis. All four antibiotics induced biofilm formation in some strains. The amount of biofilm induction was as high as 10-fold and was inversely proportional to the amount of biofilm produced by the strain in the absence of antibiotics. MRSA strains of lineages USA300, USA400, and USA500 exhibited the highest levels of methicillin-induced biofilm induction. Biofilm formation induced by low-level methicillin was inhibited by DNase. Low-level methicillin also induced DNase-sensitive autoaggregation and extracellular DNA release. The biofilm induction phenotype was absent in a strain deficient in autolysin (atl). Our findings demonstrate that subminimal inhibitory concentrations of β-lactam antibiotics significantly induce autolysin-dependent extracellular DNA release and biofilm formation in some strains of S. aureus. The widespread use of antibiotics as growth promoters in agriculture may expose bacteria to low levels of the drugs. The aim of this study was to investigate the effects of low levels of antibiotics on bacterial autoaggregation and biofilm formation, two processes that have been shown to foster genetic exchange and antibiotic resistance. We found that low levels of β-lactam antibiotics, a class commonly used in both clinical and agricultural settings, caused significant autoaggregation and biofilm formation by the important human pathogen Staphylococcus aureus. Both processes were dependent on cell lysis and release of DNA into the environment. The effect was most pronounced among multidrug-resistant strains known as methicillin-resistant S. aureus (MRSA). These results may shed light on the recalcitrance of some bacterial infections to antibiotic treatment in clinical settings and the evolution of antibiotic-resistant bacteria in agricultural settings.