Elsevier

Microbial Pathogenesis

Volume 119, June 2018, Pages 54-59
Microbial Pathogenesis

Rutin inhibits quorum sensing, biofilm formation and virulence genes in avian pathogenic Escherichia coli

https://doi.org/10.1016/j.micpath.2018.04.007Get rights and content

Highlights

  • Rutin significantly reduced the secretion of AI-2 to interfere with quorum sensing.

  • Rutin markedly inhibited the biofilm formation of APEC.

  • Rutin remarkably reduced the expression of virulence genes of APEC.

  • Rutin markedly decreased adhesion and damage of APEC to chicken type II pneumocytes.

Abstract

The study aimed to investigate whether rutin affects the quorum sensing (QS) of avian pathogenic Escherichia coli (APEC). In this study, APEC-O78 was selected as the test strain. We mainly examined the effects of rutin on the AI-2 secretion by bioluminescence assay, biofilm formation through a crystal violet staining method, and expression of virulence genes of APEC by qRT-PCR. We found that rutin can significantly interfering with QS through reducing the secretion of AI-2, inhibited the biofilm formation, and reduced the expression of virulence genes of APEC. Moreover, rutin markedly decreased adhesion and damage of APEC to chicken type II pneumocytes. These results suggested rutin reduces cell damage of APEC-infected chicken type II pneumocytes through interfering with QS via decreasing AI-2 production, biofilm formation, and the expression of virulence genes. This paper may provide a new evidence for colibacillosis prevention in chicken.

Introduction

Quorum sensing (QS) is a widespread signaling system used by bacteria for cell-to-cell communication, in which bacterial cells communicate with each other by releasing, sensing and responding to small diffusible signal molecules called autoinducers (AIs) in response to environmental signs. Autoinducer-1 (AI-1) QS system is referred to as an intra-species signaling feature, with the autoinducer-2 (AI-2) QS system being proposed to be an inter-species signaling system [1], which can regulate a variety of pathological behaviors such as bioluminescence, biofilm formation, bacterial virulence, conjugation, sporulation, swarming motility and antibiotic production [2,3]. The gene responsible for the production of AI-2 is LuxS [4], which is present in both Gram-positive and Gram-negative bacteria [5]. Ronghua et al. [6] demonstrated that inactivation of LuxS , which encodes AI-2 synthase, resulted in increased biofilm formation. Many studies have highlighted the significance of AI-2/luxS in the biological processes of various bacterial species including antibiotic production, biofilm formation, carbohydrate metabolism, virulence expression and so on, although the mechanisms of signal transduction and gene regulation still remain to be studied [[7], [8], [9], [10], [11]]. QS in E. coli has been demonstrated to act as key player in the expression of virulence genes at stationary phase [12]. Avian pathogenic Escherichia coli (APEC), which has AI-2/LuxS quorum sensing system, causes major economic losses to the poultry industry [13]. Some reports [14] revealed that most (55.2%) APEC were able to form a moderate or strong biofilm. The biofilm aggregations of APEC are involved in persistent colonization of a new host and biofilm infections often resist to the highest deliverable levels of antibiotics and threatening the host immune response [15]. The AI-2/LuxS quorum sensing which acts as a key player in the expression of virulence genes associated with bacterial iron acquisition, biofilm formation, adhesion and invasion, which can regulate the physiological functions of avian pathogenic Escherichia coli (APEC) [16]. Some report has suggested that biofilm formation was a common phenomenon among APEC and there seemed a positive correlation between adhesion-associated genes and the phenotype of biofilms [17]. Our previous study illustrated that AI-2 activity and the mRNA expression of the virulence genes of APEC. As both of them were controlled by QS, we hypothesized that the virulence gene expression of APEC were also medicated by AI-2 of QS [18].

Given that QS is an important process in bacterial survival, pathogenicity and virulence, the development of therapeutic drugs which prevent or manage bacterial pathogenesis by inhibiting bacterial QS is critical [19]. Therefore, interfering with QS and seeking effective inhibitors of the bacterial could lead to a valuable beneficial anti-pathogenic strategy for the development of antimicrobial agents. There is thus an increasing need for the identification of novel non-toxic QS antimicrobial drugs for treating bacterial diseases in humans, in agriculture, aquaculture and animal husbandry.

Flavonoids are widely found in medicinal plants, with the lipid-lowering, expansion of coronary artery, stopping bleeding, antitussive, expectorant, reducing vascular fragility and other pharmacological effects. Rutin (Fig. 1) is a flavonoid that ubiquitously present in the plant kingdom [20,21], it is used as an antimicrobial, anti-viral agent, anti-inflammatory, anti-oxidant and anti-virulence drug [22]. It has recently been reported that rutin inhibits QS and biofilm formation in Vibrio harveyi (V. harveyi) and E. coli O157:H7 [23]. However, whether rutin can interfere the QS in APEC has not been reported. Therefore, we investigated the effect of rutin on the QS system and virulence of APEC.

Section snippets

Reagents

Rutin (purity > 99%) was purchased from Chengdu Must Biotechnology Co., Ltd. (Chengdu, China). Luria-Bertani (LB) medium was obtained from Sigma-Aldrich (St.Louis, MO, USA). Fetal bovine serum (FBS) and DMEM were obtained from Gibco (Invitrogen S. r.l., Milan, Italy). TRIzol reagent and PrimeScriptTM RT Reagent Kit with gDNA Eraser were purchased from TaKaRa (Da Lian, Liaoning, China).

Bacterial strain and growth conditions

APEC-O78 strain (CVCC1418) was purchased from the Chinese Veterinary Culture Collection Center. Diluted

MIC of rutin against APEC and response of APEC growth to rutin

We used a serial 2-fold dilution method to assess the antibacterial effect of rutin on APEC-O78. As shown in Fig. 2A, rutin (1.56–50 μg/mL) had no effect on the growth of APEC-O78. Therefore, the MIC of rutin against APEC-O78 was >50 μg/mL. Furthermore, Fig. 2B showed that rutin (12.5–50 μg/mL) had no effect on the growth curve of APEC-O78.

Effect of rutin on AI-2 activity produced by APEC

To determine whether rutin regulated the quorum sensing of APEC-O78, we examined AI-2 activity produced by APEC-O78 in the presence or absence of rutin.

Discussion

AI-2 of QS is a global regulatory factor and plays a decisive role on the cell damage caused by E. coli. In the current study, the sub-MIC of rutin inhibited the release of AI-2, not affecting the growth of APEC. These indicate that rutin is an antagonist of AI-2 mediated cell-cell signaling. In order to analyze how rutin interferes with the QS system of APEC, we investigated the QS signal molecule synthesis genes, such as LuxS and Pfs, and transported and phosphorylated genes, such as LsrB and

Conflicts of interest

The authors have declared no conflict of interest.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 31372470).

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