Impact of nutritional stress on drug susceptibility and biofilm structures of Burkholderia pseudomallei and Burkholderia thailandensis grown in static and microfluidic systems

Burkholderia pseudomallei is the causative agent of melioidosis and regarded as a bioterrorism threat. It can adapt to the nutrient-limited environment as the bacteria can survive in triple distilled water for 16 years. Moreover, B. pseudomallei exhibits intrinsic resistance to diverse groups of antibiotics in particular while growing in biofilms. Recently, nutrient-limited condition influenced both biofilm formation and ceftazidime (CAZ) tolerance of B. pseudomallei were found. However, there is no information about how nutrient-limitation together with antibiotics used in melioidosis treatment affects the structure of the biofilm produced by B. pseudomallei. Moreover, no comparative study to investigate the biofilm architectures of B. pseudomallei and the related B. thailandensis under different nutrient concentrations has been reported. Therefore, this study aims to provide new information on the effects of four antibiotics used in melioidosis treatment, viz. ceftazidime (CAZ), imipenem (IMI), meropenem (MEM) and doxycycline (DOX) on biofilm architecture of B. pseudomallei and B. thailandensis with different nutrient concentrations under static and flow conditions using confocal laser scanning microscopy. Impact of nutritional stress on drug susceptibility of B. pseudomallei and B. thailandensis grown planktonically or as biofilm was also evaluated. The findings of this study indicate that nutrient-limited environment enhanced survival of B. pseudomallei in biofilm after exposure to the tested antibiotics. The shedding planktonic B. pseudomallei and B. thailandensis were also found to have increased CAZ tolerance in nutrient-limited environment. However, killing activities of MEM and IMI were stronger than CAZ and DOX on B. pseudomallei and B. thailandensis both in planktonic cells and in 2-day old biofilm. In addition, MEM and IMI were able to inhibit B. pseudomallei and B. thailandensis biofilm formation to a larger extend compared to CAZ and DOX. Differences in biofilm architecture were observed for biofilms grown under static and flow conditions. Under static conditions, biofilms grown in full strength modified Vogel and Bonner’s medium (MVBM) showed honeycomb-like architecture while a knitted-like structure was observed under limited nutrient condition (0.1×MVBM). Under flow conditions, biofilms grown in MVBM showed a multilayer structure while merely dispersed bacteria were found when grown in 0.1×MVBM. Altogether, this study provides more insight on the effect of four antibiotics against B. pseudomallei and B. thailandensis in biofilm under different nutrient and flow conditions. Since biofilm formation is believed to be involved in disease relapse, MEM and IMI may be better therapeutic options than CAZ for melioidosis treatment.

类鼻疽伯克霍尔德菌（Burkholderia pseudomallei）是类鼻疽（melioidosis）的病原菌，且被视为生物恐怖主义威胁（bioterrorism threat）。该菌可适应营养受限环境，能够在三重蒸馏水中存活长达16年。此外，类鼻疽伯克霍尔德菌在生物膜（biofilm）状态下生长时，对多类抗生素具有固有耐药性。近期研究发现，营养受限条件会同时影响类鼻疽伯克霍尔德菌的生物膜形成与头孢他啶（ceftazidime, CAZ）耐受性。然而，目前尚无研究阐明营养限制联合类鼻疽治疗用抗生素，如何影响类鼻疽伯克霍尔德菌所形成生物膜的结构；同时也未有对比研究探讨不同营养浓度下，类鼻疽伯克霍尔德菌及其近缘种泰国伯克霍尔德菌（B. thailandensis）的生物膜结构差异。
因此，本研究旨在通过共聚焦激光扫描显微镜（confocal laser scanning microscopy），探究四种类鼻疽治疗用抗生素——即头孢他啶（CAZ）、亚胺培南（imipenem, IMI）、美罗培南（meropenem, MEM）与多西环素（doxycycline, DOX），在不同营养浓度、静态与流动培养条件下，对类鼻疽伯克霍尔德菌及泰国伯克霍尔德菌生物膜结构的影响；同时评估营养胁迫对浮游态或生物膜状态生长的类鼻疽伯克霍尔德菌与泰国伯克霍尔德菌药物敏感性的影响。
本研究结果显示：营养受限环境可提升类鼻疽伯克霍尔德菌生物膜在受试抗生素暴露后的存活率；脱落的浮游态类鼻疽伯克霍尔德菌与泰国伯克霍尔德菌，在营养受限环境中对CAZ的耐受性亦显著增强。不过，美罗培南（MEM）与亚胺培南（IMI）对浮游态及2日龄生物膜状态下的两类伯克霍尔德菌的杀菌活性，均强于头孢他啶（CAZ）与多西环素（DOX）。此外，相较于CAZ与DOX，MEM与IMI可更显著地抑制两类菌株的生物膜形成。
静态与流动培养条件下的生物膜结构存在明显差异：静态培养时，于全浓度改良Vogel-Bonner培养基（modified Vogel and Bonner’s medium, MVBM）中生长的生物膜呈现蜂窝状结构，而在营养受限条件（0.1×MVBM）下则观察到针织状结构；流动培养时，MVBM中生长的生物膜呈现多层结构，而在0.1×MVBM中仅可见分散的菌体。
综上，本研究进一步阐明了四种抗生素在不同营养与流动条件下，对生物膜状态下的类鼻疽伯克霍尔德菌与泰国伯克霍尔德菌的作用效果。鉴于生物膜形成被认为与疾病复发密切相关，相较于CAZ，MEM与IMI或许是更优的类鼻疽治疗选择。