INIBIC-Complejo Hospitalario Universitario A Coruña, Unidad de Genética, As Xubias 84, 15006- A Coruña, Spain.
BMC Microbiol. 2011 Aug 25;11:191. doi: 10.1186/1471-2180-11-191.
Antibiotics which inhibit bacterial peptidoglycan biosynthesis are the most widely used in current clinical practice. Nevertheless, resistant strains increase dramatically, with serious economic impact and effects on public health, and are responsible for thousands of deaths each year. Critical clinical situations should benefit from a rapid procedure to evaluate the sensitivity or resistance to antibiotics that act at the cell wall. We have adapted a kit for rapid determination of bacterial DNA fragmentation, to assess cell wall integrity.
Cells incubated with the antibiotic were embedded in an agarose microgel on a slide, incubated in an adapted lysis buffer, stained with a DNA fluorochrome, SYBR Gold and observed under fluorescence microscopy. The lysis affects the cells differentially, depending on the integrity of the wall. If the bacterium is susceptible to the antibiotic, the weakened cell wall is affected by the lysing solution so the nucleoid of DNA contained inside the bacterium is released and spread. Alternatively, if the bacterium is resistant to the antibiotic, it is practically unaffected by the lysis solution and does not liberate the nucleoid, retaining its normal morphological appearance. In an initial approach, the procedure accurately discriminates susceptible, intermediate and resistant strains of Escherichia coli to amoxicillin/clavulanic acid. When the bacteria came from an exponentially growing liquid culture, the effect on the cell wall of the β-lactam was evident much earlier that when they came from an agar plate. A dose-response experiment with an E. coli strain susceptible to ampicillin demonstrated a weak effect before the MIC dose. The cell wall damage was not homogenous among the different cells, but the level of damage increased as dose increased with a predominant degree of effect for each dose. A microgranular-fibrilar extracellular background was evident in gram-negative susceptible strains after β-lactam treatment. This material was digested by DNase I, hybridised with a specific whole genome probe, and so recognized as DNA fragments released by the bacteria. Finally, 46 clinical strains from eight gram-negative and four gram-positive species were evaluated blind for susceptibility or resistance to one of four different β-lactams and vancomycin, confirming the applicability of the methodology.
The technique to assess cell wall integrity appears to be a rapid and simple procedure to identify resistant and susceptible strains to antibiotics that interfere with peptidoglycan biosynthesis.
目前临床上应用最广泛的抗生素是抑制细菌肽聚糖生物合成的抗生素。然而，耐药菌株的数量急剧增加，对经济和公共健康造成了严重影响，每年导致数千人死亡。在关键的临床情况下，应该受益于一种快速评估抗生素对细胞壁敏感性或耐药性的方法。我们已经适应了一种快速测定细菌 DNA 片段化的试剂盒，以评估细胞壁的完整性。
将抗生素孵育的细胞嵌入载玻片上的琼脂糖微凝胶中，在改良的裂解缓冲液中孵育，用 DNA 荧光染料 SYBR Gold 染色，在荧光显微镜下观察。根据细胞壁的完整性，裂解液对细胞的影响不同。如果细菌对抗生素敏感，那么较弱的细胞壁会受到裂解液的影响，因此细菌内部的 DNA 核体被释放并扩散。相反，如果细菌对抗生素耐药，它实际上不受裂解液的影响，不会释放核体，保持其正常的形态外观。在初步研究中，该方法能够准确区分对氨苄西林/克拉维酸敏感、中介和耐药的大肠杆菌菌株。当细菌来自指数生长期的液体培养物时，β-内酰胺对细胞壁的作用比来自琼脂平板的细菌更早明显。对氨苄西林敏感的大肠杆菌菌株进行剂量反应实验表明，在 MIC 剂量之前就有微弱的作用。细胞壁损伤在不同细胞之间不均匀，但随着剂量的增加，损伤程度增加，每个剂量的主要影响程度增加。在β-内酰胺处理后，革兰氏阴性敏感菌株的细胞外出现微颗粒状纤维背景。该物质被 DNA 酶 I 消化，与特异性全基因组探针杂交，并被识别为细菌释放的 DNA 片段。最后，对来自 8 种革兰氏阴性和 4 种革兰氏阳性种的 46 株临床菌株进行了盲法评估，以确定其对 4 种不同β-内酰胺和万古霉素的敏感性或耐药性，证实了该方法的适用性。
评估细胞壁完整性的技术似乎是一种快速、简单的方法，可以识别干扰肽聚糖生物合成的抗生素的耐药和敏感菌株。
