Characterization of biofilm formation by Riemerella anatipestifer

https://doi.org/10.1016/j.vetmic.2010.02.023Get rights and content

Abstract

Riemerella anatipestifer (RA) causes epizootics of infectious disease in poultry and results in serious economic losses, especially for the duck industry. The present study focuses on understanding the biofilm-producing ability of RA strains in attempt to explain the intriguing persistence of RA post-infection on duck farms. Four RA serotype reference strains and 39 field RA isolates were measured for the biofilm formation by crystal violet staining. Eighteen out of the 43 RA strains produced biofilms. Furthermore, RA isolate CH3 was treated with carbohydrates (sucrose; glucose), disodium EDTA (EDTA), antibiotics (ampicillin; chloramphenicol) or detergent (Triton X-100) to determine the effect of the treatments on biofilm formation. Biofilm formation by RA isolate CH3 was independent of sucrose but significantly inhibited by 5% glucose and 0.1 mmol/L EDTA. Biofilmed CH3 culture (CH3 grown with a biofilm) was 5–31 times more resistant to the treatments of ampicillin, chloramphenicol or Triton X-100 than planktonic CH3 culture on the basis of minimal inhibitory concentration and minimal bactericidal concentration. The development and architecture of the biofilm formed by CH3 were also assessed using confocal laser scanning microscopy, scanning electron microscopy and fluorescence microscopy. In addition, animal experiment was performed to determine the median lethal doses (LD50) of three RA isolates with different biofilm formation abilities. Despite the result that virulence is strain-dependent as a result of various factors other than biofilm-producing ability, the fact that biofilmed isolate is more resistant to antibiotic and detergent treatments than planktonic isolate suggest that biofilm formation by RA may contribute to the persistent infections on duck farms.

Introduction

Riemerella anatipestifer (RA) is a Gram-negative, nonmotile, nonspore-forming, rod-shaped bacterium. It belongs to the family Flavobacteriaceae in rRNA superfamily V based on 16s rRNA gene sequence analyses (Subramaniam et al., 1997). Currently, there are at least 21 known serotypes of RA (Pathanasophon et al., 2002). RA causes the anatipestifer syndrome in ducks, characterized by diarrhea, lethargy, and respiratory and nervous symptoms, which can lead to high mortality and consequently great economic losses (Asplin, 1955, Glunder and Hinz, 1989). RA infection has been a continued problem in many duck farms (Subramaniam et al., 2000). The precise mechanisms of RA persistence in duck farms are still unknown. The facts that a number of bacteria achieve their persistence via formation of biofilm lead us to investigate whether this is a mechanism for RA persistence as well.

Biofilm is a microbial community that consists of microorganisms surrounded by an extracellular polymeric matrix. Biofilm formation has been demonstrated for numerous pathogens such as Escherichia coli, Streptococcus suis, and Haemophilus influenzae (Aparna and Yadav, 2008, Hall-Stoodley and Stoodley, 2009). A large amount of data suggested that the biofilm formation was relevant to bacterial persistence due to their recaltitrance to eradication by antibiotics (Jayaraman, 2008, Lewis, 2005, Lewis, 2007). In this study, we first investigated the ability of 43 RA strains to produce biofilm, tested the effect of various chemical treatments on biofilmed versus planktonic CH3 isolates, and challenged ducklings with RA isolates to elucidate whether the ability to form biofilm plays a role in the persistence of RA infections.

Section snippets

Bacterial strains and growth conditions

Thirty-nine isolates of RA were obtained from 39 outbreaks on duck farms in China during the period of 1997–2009. The isolates were identified by phenotypic characters (Pathanasophon et al., 1994) and by amplification of 16s ribosomal RNA (Huang et al., 1999) and the ompA gene (Qinghai Hu et al., 2002). The serotypes of RA isolates were identified by the slide agglutination test with anti-RA rabbit antisera (Bisgaard, 1982). Four RA serotype reference strains were donated generously by Dr.

Biofilm assay for RA isolates

The examination of biofilm formation with CV staining revealed that of the 43 isolates, 18 (41.86%) were biofilm producers (OD595 > 0.31) and 25 (including all four RA serotype reference strains) were non-biofilm producers (OD595 < 0.31). Ten out of the 18 biofilm producers were weak producers (0.31 < OD595 < 1) and 8 were strong producers (OD595 > 1).

The effects of sucrose, glucose or EDTA on biofilm formation by RA isolate CH3

To determine whether carbohydrates play a critical role in RA biofilm formation such as providing the material necessary to construct the extracellular

Discussion

Worldwide, infection with RA is one of the most economically important diseases of farmed ducks. The intriguing fact that once duck farms suffer RA infection, the infection tends to persist for a while before being eliminated leads us to understand such persistence. As literature suggests a connection between biofilm formation and bacterial persistence, we were interested in investigating whether such correlation also exists in RA to cause prolonged infections (Jayaraman, 2008, Lewis, 2005,

Acknowledgements

We thank Dr. Guoqiang Zhu from Yangzhou University and Dr. Zhizhong Cui from Shangdong Agriculture University for providing RA serotype reference strains. This work was funded by National Basic Funds for Research Institutes, which are supported by the Chinese Academy of Agricultural Sciences (2008JB16) and Shanghai Nature Science Fund (09ZR1438600).

References (22)

  • L. Hall-Stoodley et al.

    Evolving concepts in biofilm infections

    Cell Microbiol.

    (2009)
  • Cited by (53)

    View all citing articles on Scopus
    View full text