Analysis of Morphologically Similar Staphylococcus aureus Colonies for Assessment of Phenotypic and Genotypic Correlation.
Analysis of Morphologically Similar Staphylococcus aureus Colonies for Assessment of Phenotypic and Genotypic Correlation.
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分析形态相似的金黄色葡萄球菌菌落以评估表型和基因型相关性。
DOI：
10.1128/jcm.00402-17
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发表时间：
2017
影响因子：
9.4
通讯作者：
Carroll,KarenC
中科院分区：
文献类型：
--
作者：
Totten,Marissa;Ross,Tracy;Voskertchian,Annie;Colantuoni,Elizabeth;Milstone,AaronM;Carroll,KarenC
为了追踪和控制金黄色葡萄球菌的传播，保存患者分离株用于流行病学研究 (1-3)。研究人员经常假设原始培养板上具有相同形态的菌落代表相同的克隆。然而，支持这一假设的文献非常有限。许多研究没有解决如何从培养物中选择分离株 (3-5)，并指出选择一个菌落作为代表性样本 (6)。目前，治疗父母以减少新生儿重症监护室金黄色葡萄球菌传播 (TREAT PARENTS) 试验（注册号 NCT02223520）正在评估定植父母及其新生儿的金黄色葡萄球菌菌株的一致性 (7)。为了测试上述假设，从单个培养板中保存多个金黄色葡萄球菌菌落并进行测试以确定其基因型和敏感性概况。一旦父母或监护人同意 TREAT PARENTS 试验，就从鼻孔、喉咙、腹股沟和肛周区域收集拭子样本，以筛查金黄色葡萄球菌的存在。使用 Copan Eswab 传输系统（Copan，Murrieta，CA）收集样本。对于每个样品，将 10 μl 等分到四分之一的金黄色葡萄球菌选择性显色琼脂 (SASelect; Bio-Rad, Hercules, CA) 平板和 5% 羊血琼脂 (SBA; Remel, Lenexa, KS) 平板上，并在 37°C 下孵育 16 至 24 小时。同时，将 100 μl 每种样品等分到含有 6.5% 氯化钠的胰蛋白酶大豆肉汤（Bio-Rad，Hercules，CA）中，并在 37°C 下孵育 16 至 24 小时。孵育后，将 10 μl 每种肉汤铺在 SASelect 培养基和 5% SBA 上，划线进行分离，并在 37°C 下孵育 16 至 24 小时。菌落必须具有相同的颜色、大小、稠度和完整性才能被称为相同。两名医学技术人员独立读取平板，根据所描述的标准确定相同与不同形态时，他们之间没有不一致之处。对于每个阳性平板上的每个金黄色葡萄球菌形态，将五个单独的菌落分别在5％SBA上继代培养，然后冷冻。通过脉冲场凝胶电泳 (PFGE) 和标准抗葡萄球菌药物的抗菌敏感性测试 (AST) 对这些分离株进行了分析。使用 SmaI 作为限制酶，根据标准方法进行 PFGE (8)。使用 Fingerprinting 2 软件 (Bio-Rad) 分析限制性消化模式。 PFGE 结果使用修改后的 Tenover 标准进行解释，如果分离株的模式具有三个或更少的条带差异，则认为它们是相关的 (9)。
In an effort to track and control the transmission of Staphylococcus aureus, patient isolates are saved for epidemiological studies (1–3). Study investigators often assume that colonies with the same morphology on the original culture plate represent the same clone. However, there is very limited literature to support this assumption. Many studies do not address how isolates are selected from a culture (3–5) and state that one colony is chosen as a representative sample (6). Currently, the Treating Parents to Reduce NICU Transmission of Staphylococcus aureus (TREAT PARENTS) trial (registration no. NCT02223520) is assessing the concordance of S. aureus strains colonizing parents and their neonates (7). To test the aforementioned assumption, multiple S. aureus colonies were saved from a single culture plate and tested to determine their genotypes and susceptibility profiles.Once parents or guardians consented to the TREAT PARENTS trial, swab samples were collected from the nares, throat, groin, and perianal region to screen for the presence of S. aureus. The samples were collected with the Copan Eswab transport system (Copan, Murrieta, CA). For each sample, 10 μl was aliquoted onto a quarter of one S. aureus selective chromogenic agar (SASelect; Bio-Rad, Hercules, CA) plate and one 5% sheep blood agar (SBA; Remel, Lenexa, KS) plate and incubated at 37 C for 16 to 24 h. At the same time, 100 μl of each sample was aliquoted into tryptic soy broth containing 6.5% sodium chloride (Bio-Rad, Hercules, CA) and incubated at 37 C for 16 to 24 h. After incubation, 10 μl of each broth was plated on SASelect medium and 5% SBA, streaked for isolation, and incubated at 37 C for 16 to 24 h. The colonies had to have the same color, size, consistency, and entirety to be called identical. Two medical technologists independently read the plates, and there were no inconsistencies between them in the determination of identical versus different morphologies on the basis of the criteria described. For each S. aureus morphology on every positive plate, five separate colonies were individually subcultured on 5% SBA and then frozen. These isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing (AST) with standard antistaphylococcal agents. PFGE was performed in accordance with standard methods by using SmaI as the restriction enzyme (8). Restriction digestion patterns were analyzed with the Fingerprinting 2 software (Bio-Rad). PFGE results were interpreted by using modified Tenover criteria, and isolates were considered related if their patterns had three or fewer band differences (9).