Discriminatory Detection of Inhibitor-Resistant β-Lactamases in Escherichia coli by Single-Strand Conformation Polymorphism-PCR

Plasmid-mediated mechanisms, comprising TEM hyperproduction, TEM derivative production, and OXA production, lead to amoxicillin-clavulanic acid resistance in enterobacteria. The ability of the single-strand conformation polymorphism (SSCP)-PCR method to differentiate the genes encoding inhibitor-resistant β-lactamases was evaluated with three bla(TEM) primer pairs. The bla(TEM) genes, which were known to be different on the basis of their nucleotide sequences (bla(TEM-1A), bla(TEM-1B), bla(TEM-2), bla(TEM-30), bla(TEM-32), and bla(TEM-35)), were identified as different by their electrophoretic mobilities. The bla(TEM-33), bla(TEM-34), bla(TEM-36), bla(TEM-37), bla(TEM-38), and bla(TEM-39) genes, whose sequence differences have been established by oligotyping, displayed different SSCP profiles for different fragments, suggesting genetic differences in addition to those defined by oligotyping. Confirmed by sequencing, these additional genetic events concerned silent mutations at certain positions and, notably, a G→T transversion at position 1 of the −10 consensus sequence in bla(TEM-34), bla(TEM-36), bla(TEM-37), and bla(TEM-39). Applied to eight clinical isolates of Escherichia coli resistant to amoxicillin-clavulanic acid, the SSCP method detected TEM-1 in three strains and TEM-30, TEM-32, and TEM-35 in three other strains, respectively. A novel TEM derivative (TEM-58) was detected in another strain, and the deduced amino acid sequence showed two substitutions: Arg244Ser, which is known to confer amoxicillin-clavulanic acid resistance in TEM-30, and Val261Ile, which has not been described previously. The eighth strain produced an OXA β-lactamase. Given the discriminatory power and the applicability of SSCP-PCR, this method can be proposed as a means of following the evolution of the frequencies of the different inhibitor-resistant β-lactamases.

质粒介导的机制包括TEM酶高产、TEM衍生物产生以及OXA酶产生，可导致肠杆菌科细菌对阿莫西林-克拉维酸产生耐药性。本研究采用3对bla(TEM)引物，评估了单链构象多态性-聚合酶链反应（single-strand conformation polymorphism-PCR，SSCP-PCR）区分编码抑制剂耐药β-内酰胺酶基因的能力。已知根据核苷酸序列可区分的bla(TEM)基因（包括bla(TEM-1A)、bla(TEM-1B)、bla(TEM-2)、bla(TEM-30)、bla(TEM-32)和bla(TEM-35)），可通过其电泳迁移率加以区分。经寡核苷酸分型明确序列差异的bla(TEM-33)、bla(TEM-34)、bla(TEM-36)、bla(TEM-37)、bla(TEM-38)及bla(TEM-39)基因，不同片段呈现出不同的SSCP图谱，提示除寡核苷酸分型所界定的遗传差异外，还存在其他遗传变异。经测序验证，这些额外的遗传事件涉及特定位置的沉默突变，尤其包括bla(TEM-34)、bla(TEM-36)、bla(TEM-37)及bla(TEM-39)中−10区保守序列第1位的G→T颠换。将该方法应用于8株阿莫西林-克拉维酸耐药的大肠埃希菌临床分离株时，SSCP-PCR分别在3株菌中检出TEM-1，在另外3株菌中检出TEM-30、TEM-32及TEM-35。另有1株菌检出新型TEM衍生物TEM-58，其推导的氨基酸序列存在两处替换：一处为已知可赋予TEM-30阿莫西林-克拉维酸耐药性的Arg244Ser，另一处为此前未见报道的Val261Ile。第8株菌产生OXA型β-内酰胺酶。鉴于SSCP-PCR的鉴别效力和适用性，该方法可作为追踪不同抑制剂耐药β-内酰胺酶的频率演化的手段予以推广应用。