Department of Microbiology, Aggeu Magalhães Institute - FIOCRUZ PE, Av. Prof. Moraes Rego, S/N Cidade Universitária, Recife, PE, Brazil.
Department of Bacteriology, Hospital das Clínicas da Universidade Federal de Pernambuco - HC-UFPE, Recife, PE, Brazil.
Appl Microbiol Biotechnol. 2023 Apr;107(7-8):2653-2660. doi: 10.1007/s00253-023-12452-0. Epub 2023 Mar 10.
We developed a simple new selective LB-based medium, named CYP broth, suitable for recovering long-term stored Y. pestis subcultures and for isolation of Y. pestis strains from field-caught samples for the Plague surveillance. It aimed to inhibit the growth contaminating microorganisms and enrich Y. pestis growth through iron supplementation. The performance of CYP broth on microbial growth from different gram-negative and gram-positive strains from American Type Culture Collection (ATCC®) and other clinical isolates, field-caught rodent samples, and more importantly, on several vials of ancient Y. pestis subcultures was evaluated. Additionally, other pathogenic Yersinia species such as Y. pseudotuberculosis and Y. enterocolitica were also successfully isolated with CYP broth. Selectivity tests and bacterial growth performance on CYP broth (LB broth supplemented with Cefsulodine, Irgasan, Novobiocin, nystatin and ferrioxamine E) were evaluated in comparison with LB broth without additive; LB broth/CIN, LB broth/nystatin and with traditional agar media including LB agar without additive, and LB agar and Cefsulodin-Irgasan-Novobiocin Agar (CIN agar) supplemented with 50 µg/mL of nystatin. Of note, the CYP broth had a recovery twofold higher than those of the CIN supplemented media or other regular media. Additionally, selectivity tests and bacterial growth performance were also evaluated on CYP broth in the absence of ferrioxamine E. The cultures were incubated at 28 °C and visually inspected for microbiological growth analysis and O.D. measurement between 0 and 120 h. The presence and purity of Y. pestis growth were confirmed by bacteriophage and multiplex PCR tests. Altogether, CYP broth provides an enhanced growth of Y. pestis at 28 °C, while inhibiting contaminant microorganisms. The media is a simple, but powerful tool to improve the reactivation and decontamination of ancient Y. pestis culture collections and for the isolation of Y. pestis strains for the Plague surveillance from various backgrounds. KEY POINTS: • The newly described CYP broth improves the recuperation of ancient/contaminated Yersinia pestis culture collections • CYP broth was also efficient in reducing environmental contamination in field-capture samples, improving Y. pestis isolation • CYP broth can also be used for the isolation of Y. enterocolitica and Y. pseudotuberculosis.
我们开发了一种简单的新型基于 LB 的选择性培养基，命名为 CYP 肉汤，适用于恢复长期储存的鼠疫耶尔森氏菌亚培养物，并从野外捕获的样本中分离鼠疫耶尔森氏菌菌株，用于鼠疫监测。它旨在通过补充铁来抑制生长的污染微生物并富集鼠疫耶尔森氏菌的生长。评估了 CYP 肉汤对来自美国典型培养物保藏中心 (ATCC®) 和其他临床分离株的不同革兰氏阴性和革兰氏阳性菌株、野外捕获的啮齿动物样本以及更重要的几个古老鼠疫耶尔森氏菌亚培养物的微生物生长的性能。此外，CYP 肉汤还成功分离出了其他致病性耶尔森氏菌，如假结核耶尔森氏菌和肠杆菌耶尔森氏菌。选择性试验和 CYP 肉汤（LB 肉汤中补充头孢磺啶、irgasan、新生霉素、制霉菌素和高铁血红素 E）上的细菌生长性能与无添加剂的 LB 肉汤进行了比较；LB 肉汤/CIN、LB 肉汤/制霉菌素和传统琼脂培养基，包括无添加剂的 LB 琼脂和补充有 50μg/mL 制霉菌素的头孢磺啶-irgasan-新生霉素琼脂（CIN 琼脂）。值得注意的是，CYP 肉汤的回收率比补充 CIN 的培养基或其他常规培养基高两倍。此外，还在没有高铁血红素 E 的情况下评估了 CYP 肉汤的选择性试验和细菌生长性能。将培养物在 28°C 下孵育，并在 0 至 120 小时之间进行微生物生长分析和 OD 测量的目视检查。通过噬菌体和多重 PCR 试验确认鼠疫耶尔森氏菌生长的存在和纯度。总的来说，CYP 肉汤在 28°C 下提供了鼠疫耶尔森氏菌的增强生长，同时抑制了污染微生物。该培养基是一种简单但功能强大的工具，可用于改善古老鼠疫耶尔森氏菌培养物的复活和去污，以及从各种背景中分离鼠疫耶尔森氏菌菌株进行鼠疫监测。要点：• 新描述的 CYP 肉汤可提高古代/污染鼠疫耶尔森氏菌培养物的恢复能力• CYP 肉汤还可有效减少野外捕获样本中的环境污染，提高鼠疫耶尔森氏菌的分离率• CYP 肉汤也可用于分离肠杆菌和假结核耶尔森氏菌。
