García Vanesa, Montero Ignacio, Bances Margarita, Rodicio Rosaura, Rodicio M Rosario
1 Área de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, Universidad de Oviedo , Oviedo, Asturias, Spain .
2 Laboratorio de Salud Pública, Consejería de Sanidad , Oviedo, Asturias, Spain .
Microb Drug Resist. 2017 Jun;23(4):405-412. doi: 10.1089/mdr.2016.0227. Epub 2016 Nov 3.
In this study, the incidence and genetic bases of nitrofurantoin resistance were established for clinical isolates of two successful clones of Salmonella enterica serovar Typhimurium, the pandemic "DT 104" and the pUO-StVR2 clone. A total of 61 "DT 104" and 40 pUO-StVR2 isolates recovered from clinical samples during 2008-2014 and assigned to different phage types, were tested for nitrofurantoin susceptibility. As previously shown for older isolates, all newly tested pUO-StVR2 isolates were highly resistant to nitrofurantoin (minimal inhibitory concentration [MIC] of 128 μg/ml), while 42.6%, 24.6%, and 32.8% of the "DT 104" isolates were susceptible, showed intermediate resistance or were highly resistant, with MICs of 8, 64, and 128 μg/ml, respectively. The genetic bases of nitrofurantoin resistance were established by PCR amplification and sequencing of the nfsA and nfsB genes encoding oxygen-insensitive nitroreductases. pUO-StVR2 isolates shared identical alterations in both nfsA (IS1 inserted into the coding region) and nfsB (in frame duplication of two codons). "DT 104" isolates with intermediate or high resistance had a missense mutation affecting the start codon of nfsA, while a single resistant isolate carried an additional frameshift mutation affecting nfsB. Complementation studies, performed with wild-type nfsA and nfsB, cloned independently and together into low and high copy-number vectors, confirmed NfsA and NfsB as responsible for nitrofurantoin toxicity. The same alterations persisted along time in isolates of each clone belonging to different phage types. Accordingly, changes leading to nitrofurantoin resistance have probably occurred before phage type diversification.
在本研究中，确定了肠炎沙门氏菌鼠伤寒血清型两个成功克隆株（大流行的“DT 104”和pUO-StVR2克隆株）临床分离株对呋喃妥因耐药的发生率和遗传基础。对2008 - 2014年期间从临床样本中分离出的、分属于不同噬菌体类型的61株“DT 104”和40株pUO-StVR2分离株进行了呋喃妥因敏感性测试。如先前对较老分离株的研究所示，所有新测试的pUO-StVR2分离株对呋喃妥因高度耐药（最低抑菌浓度[MIC]为128μg/ml），而“DT 104”分离株中分别有42.6%、24.6%和32.8%对呋喃妥因敏感、表现为中度耐药或高度耐药，其MIC分别为8、64和128μg/ml。通过对编码氧不敏感硝基还原酶的nfsA和nfsB基因进行PCR扩增和测序，确定了呋喃妥因耐药的遗传基础。pUO-StVR2分离株在nfsA（IS1插入编码区）和nfsB（两个密码子的框内重复）中存在相同的改变。具有中度或高度耐药性的“DT 104”分离株有一个影响nfsA起始密码子的错义突变，而一个耐药分离株还携带一个影响nfsB的额外移码突变。用野生型nfsA和nfsB进行的互补研究，将它们分别或一起克隆到低拷贝数和高拷贝数载体中，证实NfsA和NfsB是导致呋喃妥因毒性的原因。在属于不同噬菌体类型的每个克隆株的分离株中，相同的改变随时间持续存在。因此，导致呋喃妥因耐药的变化可能在噬菌体类型多样化之前就已发生。
