Chávez-Jacobo Víctor M, Hernández-Ramírez Karen C, Silva-Sánchez Jesus, Garza-Ramos Ulises, Barrios-Camacho Humberto, Ortiz-Alvarado Rafael, Cervantes Carlos, Meza-Carmen Víctor, Ramírez-Díaz Martha I
Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, México.
Centro de Investigación Sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Cuernavaca, Morelos, México.
J Antimicrob Chemother. 2019 May 1;74(5):1253-1259. doi: 10.1093/jac/dky562.
This study investigated the presence of the crpP gene, which encodes an enzymatic mechanism of antibiotic phosphorylation that decreases ciprofloxacin susceptibility, in ESBL-producing clinical isolates and its effect in transconjugants.
A collection of 77 ESBL-producing clinical isolates of Enterobacteriaceae and 68 ESBL-producing transconjugants that had acquired plasmids from clinical isolates from hospitals in Mexico obtained from 1988 to 2012 was employed. The crpP homologue genes were identified by dot-blot and PCR assays; five of them were sequenced and an in silico analysis was conducted. Expression of CrpP proteins was determined by western blot assays using antibodies against CrpP from plasmid pUM505. Three crpP homologue genes were cloned and transferred to Escherichia coli J53-3 as recipient strain.
The crpP gene was identified in four (5.19%) ESBL-producing isolates and five (7.35%) ESBL-producing transconjugants with plasmids from clinical isolates. Analysis of the deduced amino acid (aa) sequence of the CrpP protein homologues revealed that they all corresponded to small proteins (63-70 aa) with an identity of 10.1%-43.7% with respect to the pUM505 CrpP sequence. In addition, all crpP-positive transconjugants expressed a CrpP protein. Finally, transfer of crpP homologues conferred lower ciprofloxacin susceptibility to E. coli.
These findings indicate the presence of crpP genes among ESBL-producing isolates from Mexican hospitals and point to widespread crpP-type genes in old Enterobacteriaceae clinical isolates (from 1994). CrpP probably confers resistance by means of the phosphorylation of ciprofloxacin.
本研究调查了产超广谱β-内酰胺酶（ESBL）的临床分离株中编码抗生素磷酸化酶机制（该机制会降低环丙沙星敏感性）的crpP基因的存在情况及其在接合子中的作用。
使用了一组77株产ESBL的肠杆菌科临床分离株和68株从1988年至2012年从墨西哥医院的临床分离株获得质粒的产ESBL接合子。通过斑点印迹和PCR检测鉴定crpP同源基因；对其中5个进行测序并进行了电子分析。使用针对质粒pUM505的CrpP抗体通过蛋白质印迹检测确定CrpP蛋白的表达。克隆了3个crpP同源基因并将其转移至大肠埃希菌J53-3作为受体菌株。
在4株（5.19%）产ESBL的临床分离株和5株（7.35%）带有临床分离株质粒的产ESBL接合子中鉴定出crpP基因。对CrpP蛋白同源物推导的氨基酸序列分析表明，它们均对应于小蛋白（63 - 70个氨基酸），与pUM505 CrpP序列的同一性为10.1% - 43.7%。此外，所有crpP阳性接合子均表达CrpP蛋白。最后，crpP同源物的转移使大肠埃希菌对环丙沙星的敏感性降低。
这些发现表明墨西哥医院产ESBL的分离株中存在crpP基因，并指出在旧的肠杆菌科临床分离株（自1994年起）中crpP型基因广泛存在。CrpP可能通过环丙沙星的磷酸化赋予耐药性。
