Medical Microbiology, Department of Laboratory Medicine Malmö, Lund University, Malmö, Sweden.
J Antimicrob Chemother. 2014 Jan;69(1):117-20. doi: 10.1093/jac/dkt307. Epub 2013 Aug 2.
Group A streptococci (GAS) cause, among other infections, pharyngotonsillitis in children. The species is frequently localized with the Gram-negative respiratory pathogens non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis, which both produce outer membrane vesicles (OMVs). The aim of this study was to investigate whether OMVs isolated from NTHi contain functional β-lactamase and whether the OMVs hydrolyse amoxicillin and thus protect GAS from killing by the antibiotic.
The antibiotic susceptibility of isolates was determined using the Etest. The resistance genes blaTEM-1 (encoding NTHi β-lactamase), bro-1 (encoding M. catarrhalis β-lactamase) and ftsI (encoding NTHi penicillin-binding protein 3) were searched for by PCR, followed by sequencing. OMVs were isolated by ultracentrifugation and the presence of β-lactamase was detected by western blots including specific rabbit polyclonal antibodies. The chromogenic substrate nitrocefin was used to quantify and compare the β-lactamase enzyme activity in the OMVs. The hydrolysis of amoxicillin by β-lactamase was estimated by an agar diffusion method.
We showed that OMVs released from β-lactam-resistant M. catarrhalis and NTHi contain functional β-lactamase that hydrolyses amoxicillin and protects GAS from killing by amoxicillin.
This is the first report of the presence of β-lactamase in NTHi OMVs. We suggest that OMV-derived β-lactamase from coinfecting pathogens such as NTHi and M. catarrhalis may contribute to the occasional treatment failures seen in GAS tonsillitis.
A 群链球菌（GAS）可引起咽扁桃体炎等感染。该细菌常与革兰氏阴性呼吸道病原体无定型流感嗜血杆菌（NTHi）和卡他莫拉菌定植，两者均产生外膜囊泡（OMVs）。本研究旨在研究 NTHi 分离的 OMVs 是否含有功能性β-内酰胺酶，以及 OMVs 是否水解阿莫西林从而保护 GAS 免受抗生素杀伤。
使用 Etest 测定分离株的抗生素敏感性。通过 PCR 搜索 blaTEM-1（编码 NTHi β-内酰胺酶）、bro-1（编码 M. catarrhalis β-内酰胺酶）和 ftsI（编码 NTHi 青霉素结合蛋白 3）耐药基因，随后进行测序。通过超速离心分离 OMVs，并通过 Western blot 检测包括特异性兔多克隆抗体在内的β-内酰胺酶。使用显色底物硝头孢菌素定量和比较 OMVs 中的β-内酰胺酶活性。通过琼脂扩散法估计β-内酰胺酶对阿莫西林的水解作用。
我们表明，β-内酰胺耐药的 M. catarrhalis 和 NTHi 释放的 OMVs 含有可水解阿莫西林并保护 GAS 免受阿莫西林杀伤的功能性β-内酰胺酶。
这是首次报道 NTHi OMVs 中存在β-内酰胺酶。我们认为，来自共生病原体（如 NTHi 和 M. catarrhalis）的 OMV 衍生β-内酰胺酶可能导致 GAS 扁桃体炎治疗偶尔失败。
