Method to grow Actinobacillus pleuropneumoniae biofilm on a biotic surface

BMC Veterinary Research

Mario Jacques²  Ruud PAM Segers¹  Cynthia Lévesque²  Yannick DN Tremblay² 

[1] Microbiological R&D, MSD Animal Health, 5831 AN, Boxmeer, The Netherlands;Groupe de recherche sur les maladies infectieuses du porc, Faculté de médecine vétérinaire, Université de Montréal, 3200 rue Sicotte, St-Hyacinthe, Québec J2S 7C6, Canada

关键词: Host-pathogen interaction;    Confocal laser scanning microscopy;    Actinobacillus pleuropneumoniae;    SJPL cell line;    Biotic surface;    Biofilm;   

Others  :  1119425
DOI  :  10.1186/1746-6148-9-213

 received in 2013-07-05, accepted in 2013-10-16,  发布年份 2013

【 摘 要 】

Background

Actinobacillus pleuropneumoniae is a Gram-negative bacterium and a member of the Pasteurellaceae family. This bacterium is the causative agent of porcine pleuropneumonia, which is a highly contagious respiratory disease causing important economical losses to the worldwide pig industry. It has been shown that A. pleuropneumoniae can form biofilms on abiotic surfaces (plastic and glass). Although in vitro models are extremely useful to gain information on biofilm formation, these models may not be representative of the conditions found at the mucosal surface of the host, which is the natural niche of A. pleuropneumoniae.

Results

In this paper, we describe a method to grow A. pleuropneumoniae biofilms on the SJPL cell line, which represents a biotic surface. A non-hemolytic, non-cytotoxic mutant of A. pleuropneumoniae was used in our assays and this allowed the SJPL cell monolayers to be exposed to A. pleuropneumoniae for longer periods. This resulted in the formation of biofilms on the cell monolayer after incubations of 24 and 48 h. The biofilms can be stained with fluorescent probes, such as a lectin against the polymer of N-acetyl-D-glucosamine present in the biofilm matrix, and easily observed by confocal laser scanning microscopy.

Conclusions

This is the first protocol that describes the formation of an A. pleuropneumoniae biofilm on a biotic surface. The advantage of this protocol is that it can be used to study biofilm formation in a context of host-pathogen interactions. The protocol could also be adapted to evaluate biofilm inhibitors or the efficacy of antibiotics in the presence of biofilms.

【 授权许可】

   
2013 Tremblay et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Bossé JT, Janson H, Sheehan BJ, Beddek AJ, Rycroft AN, Kroll JS, Langford PR: Actinobacillus pleuropneumoniae: pathobiology and pathogenesis of infection. Microbes Infect 2002, 4:225-235.
• [2]Chiers K, De Waele T, Pasmans F, Ducatelle R, Haesebrouck F: Virulence factors of Actinobacillus pleuropneumoniae involved in colonization, persistence and induction of lesions in its porcine host. Vet Res 2010, 41:65.
• [3]Costerton JW, Stewart PS, Greenberg EP: Bacterial biofilms: a common cause of persistent infections. Science 1999, 284:1318-1322.
• [4]Hall-Stoodley L, Costerton JW, Stoodley P: Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol 2004, 2:95-108.
• [5]Jacques M, Aragon V, Tremblay YDN: Biofilm formation in bacterial pathogens of veterinary importance. Anim Health Res Rev 2010, 11:97-121.
• [6]Kaplan JB, Mulks MH: Biofilm formation is prevalent among field isolates of Actinobacillus pleuropneumoniae. Vet Microbiol 2005, 108:89-94.
• [7]Labrie J, Pelletier-Jacques G, Deslandes V, Ramjeet M, Auger E, Nash JH, Jacques M: Effects of growth conditions on biofilm formation by Actinobacillus pleuropneumoniae. Vet Res 2010, 41:3.
• [8]Grasteau A, Tremblay YDN, Labrie J, Jacques M: Novel genes associated with biofilm formation of Actinobacillus pleuropneumoniae. Vet Microbiol 2011, 153:134-143.
• [9]Archambault M, Harel J, Gouré J, Tremblay YDN, Jacques M: Antimicrobial susceptibilities and resistance genes of Canadian isolates of Actinobacillus pleuropneumoniae. Microb Drug Res 2012, 18:198-206.
• [10]Tremblay YDN, Deslandes V, Jacques M: Actinobacillus pleuropneumoniae genes expression in biofilms cultured under static conditions and in a drip-flow apparatus. BMC Genomics 2013, 14:364.
• [11]Izano EA, Wang H, Ragunath C, Ramasubbu N, Kaplan JB: Detachment and killing of Aggregatibacter actinomycetemcomitans biofilms by dispersin B and SDS. J Dent Res 2007, 86:618-622.
• [12]Kaplan JB, Velliyagounder K, Ragunath C, Rohde H, Mack D, Knobloch JK, Ramasubbu N: Genes involved in the synthesis and degradation of matrix polysaccharide in Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae biofilms. J Bacteriol 2004, 186:8213-8220.
• [13]Auger E, Deslandes V, Ramjeet M, Contreras I, Nash JH, Harel J, Gottschalk M, Olivier M, Jacques M: Host-pathogen interactions of Actinobacillus pleuropneumoniae with porcine lung and tracheal epithelial cells. Infect Immun 2009, 77:1426-1441.
• [14]Provost C, Jia JJ, Music N, Lévesque C, Lebel ME, Del Castillo JR, Jacques M, Gagnon CA: Identification of a new cell line permissive to porcine reproductive and respiratory syndrome virus infection and replication which is phenotypically distinct from MARC-145 cell line. Virol J 2012, 9:267.
• [15]Bossé JT, Durham AL, Rycroft AN, Kroll JS, Langford PR: New plasmid tool for genetic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae. Appl Environ Microbiol 2009, 75:6124-6131.
• [16]Parker D, Soong G, Planet P, Brower J, Ratner AJ, Prince A: The NanA neuraminidase of Streptococcus pneumoniae is involved in biofilm formation. Infect Immun 2009, 77:3722-3730.
• [17]Marks LR, Parameswaran GL, Hakansson AP: Pneumococcal interactions with epithelial cells are crucial for optimal biofilm formation and colonization in vitro and in vivo. Infect Immun 2012, 80:2744-2760.
• [18]Vidal JE, Howery KE, Ludewick HP, Nava P, Klugman KP: Quorum sensing systems LuxS/AI-2 and Com regulate Streptococcus pneumoniae biofilms in a bioreactor with living cultures of human respiratory cells. Infect Immun 2013, 81:1341-1353.
• [19]Yu Q, Griffin EF, Moreau-Marquis S, Schwartzman JD, Stanton BA, O’Toole GA: In vitro evaluation of tobramycin and aztreonam versus Pseudomonas aeruginosa biofilms on cystic fibrosis-derived human airway epithelial cells. J Antimicrob Chemother 2012, 67:2673-2681.
• [20]Jansen R, Biaire J, Smith HE, Dom P, Haesebrouk F, Kamp EM, Gielkens AL, Smits MA: Knockout mutants of Actinobacillus pleuropneumoniae serotype 1 that are devoid of RTX toxins do not activate or kill porcine neutrophils. Infect Immun 1995, 63:27-37.
• [21]Seo SH, Goloubeva O, Webby R, Webster RG: Characterization of a porcine lung epithelial cell line suitable for influenza virus studies. J Virol 2001, 75:9517-9525.
• [22]Silversides DW, Music N, Jacques M, Gagnon CA, Webby R: Investigation of the species origin of the St. Jude Porcine Lung epithelial cell line (SJPL) made available to researchers. J Virol 2010, 84:5454-5455.