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

Chitchanok Anutrakunchai,Bastiaan P Krom,Sakawrat Kanthawong,Suwimol Taweechaisupapong,Sorujsiri Chareonsudjai,Jan G M Bolscher,William C Nierman

doi:10.1371/journal.pone.0194946

Abstract

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.

Highlights

Burkholderia pseudomallei is the etiological agent of melioidosis, an emerging infectious disease associated with high morbidity and mortality [1,2]
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determinations of planktonic B. thailandensis E264 and B. pseudomallei K96243 revealed that IMI and MEM exhibited higher killing activities than CAZ and DOX to both bacterial strains whereas DOX exhibited the lowest killing activity (Table 1)
confocal laser scanning microscopy (CLSM) was used to characterize and compare B. thailandensis E264 and B. pseudomallei K96243 biofilms grown under different drugs and nutrient concentrations

Summary

Introduction

Burkholderia pseudomallei is the etiological agent of melioidosis, an emerging infectious disease associated with high morbidity and mortality [1,2]. B. pseudomallei exhibits intrinsic resistance to diverse groups of antibiotics and is even more resistant when growing in biofilms, leading to treatment difficulties [3,4,5]. The disease has a high relapse rate despite appropriate antibiotic therapy [6]. Relapsing melioidosis correlates with biofilm formation [7]. Treatment recommendations are derived from the outcomes of a series of clinical trials conducted in endemic regions, mostly Thailand and northern Australia, and is summarized in several studies [8,9,10]. Different antibiotic regimens have been used by different groups to treat melioidosis, and many unanswered questions remain

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