Department of Microbiology, Kaohsiung Medical University, Kaohsiung, Taiwan.
Eur J Clin Invest. 2009 Sep;39(9):807-12. doi: 10.1111/j.1365-2362.2009.02166.x. Epub 2009 Jul 17.
Amoxicillin-resistant Helicobacter pylori with minimal inhibitory concentration (MIC) >or= 256 mg L(-1) was isolated from a gastritis patient. The aims were to investigate the mechanism of high-level amoxicillin resistance in H. pylori.
The beta-lactamase production was determined by means of nitrocefin sticks and the presence of gene encoding the beta-lactam antibiotic resistance enzyme TEM beta-lactamase was analysed by polymerase chain reaction (PCR), sequencing and dot-blot hybridization. Sequencing analysis of pbp1A gene was performed and amoxicillin-susceptible isolate was transformed with pbp1A PCR products from the resistant isolate. The expression of hefC efflux system was analysed using real-time quantitative PCR.
Activity of beta-lactamase was detected. Sequence analysis showed that the PCR product derived from H. pylori 3778 was identical to the bla(TEM-1) (GenBank accession EU726527). Dot-blot hybridization confirmed the presence of beta-lactamase gene bla(TEM-1.) By transformation of PCR product of mutated pbp1A gene from H. pylori 3778 into amoxicillin-susceptible strain showed that substitutions in Thr(556)-->Ser, Lys(648)-->Gln, Arg(649)-->Lys and Arg(656)-->Pro contribute to low-level amoxicillin resistance. The MIC of amoxicillin for the transformants was 0.75 mg L(-1). Over-expression of hefC was not found.
High-level amoxicillin resistance is associated with beta-lactamase production in H. pylori. Low-level amoxicillin resistance is linked to a point mutation on pbp1A. Because H. pylori can exchange DNA through natural transformation, spreading of bla(TEM-1) amoxicillin resistance gene among H. pylori is a potential threat when treating H. pylori infection.
从一位胃炎患者中分离出对最小抑菌浓度（MIC）≥256 毫克/升的阿莫西林耐药幽门螺杆菌。目的是研究幽门螺杆菌高水平阿莫西林耐药的机制。
采用硝基头孢菌素棒法检测β-内酰胺酶的产生，并通过聚合酶链反应（PCR）、测序和点印迹杂交分析编码β-内酰胺抗生素耐药酶 TEM β-内酰胺酶的基因。对 pbp1A 基因进行测序分析，并将耐药分离株的 pbp1A PCR 产物转化为阿莫西林敏感分离株。使用实时定量 PCR 分析 hefC 外排系统的表达。
检测到β-内酰胺酶的活性。序列分析表明，从幽门螺杆菌 3778 中获得的 PCR 产物与 bla(TEM-1)（GenBank 登录号 EU726527）完全相同。点印迹杂交证实存在 bla(TEM-1)β-内酰胺酶基因。将突变的 pbp1A 基因的 PCR 产物从幽门螺杆菌 3778 转化为阿莫西林敏感株表明，Thr(556)→Ser、Lys(648)→Gln、Arg(649)→Lys 和 Arg(656)→Pro 的取代导致低水平阿莫西林耐药。转化子的阿莫西林 MIC 为 0.75 毫克/升。未发现 hefC 的过度表达。
幽门螺杆菌中产β-内酰胺酶与高水平阿莫西林耐药有关。低水平阿莫西林耐药与 pbp1A 上的点突变有关。由于幽门螺杆菌可以通过自然转化交换 DNA，因此在治疗幽门螺杆菌感染时，bla(TEM-1)阿莫西林耐药基因在幽门螺杆菌中的传播是一个潜在的威胁。
