Auxotrophic Actinobacillus pleurpneumoniae grows in multispecies biofilms without the need for nicotinamide-adenine dinucleotide (NAD) supplementation

Abstract

Background: Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, which causes important worldwide economic losses in the swine industry. Several respiratory tract infections are associated with biofilm formation, and A. pleuropneumoniae has the ability to form biofilms in vitro. Biofilms are structured communities of bacterial cells enclosed in a self-produced polymer matrix that are attached to an abiotic or biotic surface. Virtually all bacteria can grow as a biofilm, and multi-species biofilms are the most common form of microbial growth in nature. The goal of this study was to determine the ability of A. pleuropneumoniae to form multi-species biofilms with other bacteria frequently founded in pig farms, in the absence of pyridine compounds (nicotinamide mononucleotide [NMN], nicotinamide riboside [NR] or nicotinamide adenine dinucleotide [NAD]) that are essential for the growth of A. pleuropneumoniae.

Results: For the biofilm assay, strain 719, a field isolate of A. pleuropneumoniae serovar 1, was mixed with swine isolates of Streptococcus suis, Bordetella bronchiseptica, Pasteurella multocida, Staphylococcus aureus or Escherichia coli, and deposited in 96-well microtiter plates. Based on the CFU results, A. pleuropneumoniae was able to grow with every species tested in the absence of pyridine compounds in the culture media. Interestingly, A. pleuropneumoniae was also able to form strong biofilms when mixed with S. suis, B. bronchiseptica or S. aureus. In the presence of E. coli, A. pleuropneumoniae only formed a weak biofilm. The live and dead populations, and the matrix composition of multi-species biofilms were also characterized using fluorescent markers and enzyme treatments. The results indicated that poly-N-acetyl-glucosamine remains the primary component responsible for the biofilm structure.

Conclusions: In conclusion, A. pleuropneumoniae apparently is able to satisfy the requirement of pyridine compounds through of other swine pathogens by cross-feeding, which enables A. pleuropneumoniae to grow and form multi-species biofilms.

Keywords: Actinobacillus pleuropneumoniae; Biofilms; Bordetella bronchiseptica; Escherichia coli; Pasteurella multocida; Pyridine compounds; Staphylococcus aureus; Streptococcus suis.

Fig. 1

Multi-species biofilms formation by A. pleuropneumoniae with other swine pathogens. A. pleuropneumoniae, S. suis, B. bronchiseptica, P. multocida, S. aureus and E. coli. The biofilms grown as (a) single, (b) dual or (c) triple-species biofilms in BHI media with or without NAD, they were stained with crystal violet staining. The colony forming units (CFU) of A. pleuropneumoniae and the other bacteria were enumerated from multi-species biofilms grown in BHI media with or without NAD grown as (d) single, (e) two or (f) three-species biofilms. App: A. pleuropneumoniae; Ss: S. suis; Bb: B. bronchiseptica; Pm: P. multocida; Sa: S. aureus; Ec: E. coli

Fig. 2

Effect of enzymatic treatment on multi-species biofilms. Dispersion by (a) proteinase K, (b) DNase I, and (c) dispersin B of multi-species biofilms formed by A. pleuropneumoniae, S. suis, B. bronchiseptica, P. multocida, S. aureus and E. coli grown in BHI media with or without NAD. App: A. pleuropneumoniae; Ss: S. suis; Bb: B. bronchiseptica; Pm: P. multocida; Sa: S. aureus; Ec: E. coli. * p < 0.05

Fig. 3

CLSM of multi-species biofilms of A. pleuropneumoniae with other swine pathogens without NAD supplementation. A. pleuropneumoniae, S. suis, B. bronchiseptica, P. multocida, S. aureus and E. coli grown as single, dual or triple-species biofilms in BHI media without NAD stained with FM 1-43, SYTO 9, propidium iodide, wheat-germ agglutinin (WGA)-Oregon green, BOBO-3, and SYPRO Ruby (all from Invitrogen, Eugene, OR). PGA: poly-N-acetylglucosamine; eDNA: extracellular DNA; App: A. pleuropneumoniae; Ss: S. suis; Bb: B. bronchiseptica; Pm: P. multocida; Sa: S. aureus; Ec: E. coli. Scale bar 30 μm

Fig. 4

Confirmation of the presence of A. pleuropneumoniae in multi-species biofilms. Confirmation of A. pleuropneumoniae in the dual-species biofilm of A. pleuropneumoniae 719 and S. suis 735 or B. bronchiseptica 276 by FISH with an ApxIVAN-AlexaFluor 633 probe (red). Images of the X-Z plane of biofilm of single and dual-species biofilms grown in BHI with or without NAD. Bacterial cell in the biofilms were stained with FilmTracer ™ FM ® 1-43 (Molecular Probes) which are represented in green. Yellow represent the co-localization of both the ApxIVAN probe and the stain FM 1-43. App: A. pleuropneumoniae; Ss: S. suis; Bb: B. bronchiseptica

Fig. 5

3D models of A. pleuropneumoniae in multi-species biofilms. Duals-species biofilms of (a) A. pleuropneumoniae 719 and S. suis 735 or (b) A. pleuropneumoniae 719 and B. bronchiseptica 276 detected with ApxIVAN-AlexaFluor 633 probe (red). Bacterial cell in the biofilms were stained with FilmTracer ™ FM ® 1-43 (Molecular Probes) which are represented in green. Yellow represent the co-localization of both the ApxIVAN probe and FM 1-43