Actinobacillus pleuropneumoniae grows as aggregates in the lung of pigs: is it time to refine our in vitro biofilm assays?

Abstract

Actinobacillus pleuropneumoniae causes porcine pleuropneumonia and forms biofilms in vitro on abiotic surfaces; however, presence of biofilms during infections has not been documented. The aim of this study was to use a species-specific fluorescent oligonucleotide probe and confocal microscopy to localize A. pleuropneumoniae in the lungs of two naturally infected pigs. Actinobacillus pleuropneumoniae was detected by fluorescence in situ hybridization and observed to grow as aggregates (~30-45 μm) during a natural infection. As the A. pleuropneumoniae aggregates observed in porcine lungs differed from the biofilms grown on a solid surface obtained in vitro, we designed a new biofilm assay using agarose, a porous substrate, favouring the formation of aggregates. In this study, we described for the first time the mode of growth of A. pleuropneumoniae during a natural infection in pigs. We also propose an in vitro biofilm assay for A. pleuropneumoniae using a porous substrate which allows the formation of aggregates. This assay might be more representative of the in vivo situation, at least in terms of the size of the bacterial aggregates and the presence of a porous matrix, and could potentially be used to test the susceptibility of A. pleuropneumoniae aggregates to antibiotics and disinfectants.

Figure 1

Swine lung, case SHY13‐04294. A large area of coagulation necrosis (N) that spares the interlobular septa, large vessels and airways (S) is present in the parenchyma, associated with oedema, congestion and fibrin exudation. It is surrounded by a rim of neutrophils and degenerated macrophages (L). Fibrin, oedema and leucocytes moderately distend the interlobular septa and pleura. The pleura (P) is covered by a thin layer of fibrin. Haematoxylin‐eosin‐phloxin‐saffron (HEPS), 25× magnification.

Figure 2

Biofilm formation by Actinobacillus pleuropneumoniae isolates APP286 and APP4294 in microtiter plates after 24 h of incubation. (A) OD _590 nm after crystal violet staining. (B) Confocal laser scanning microscopic image of biofilm of isolate APP286 stained with FilmTracer FM 1‐43. Stack of sections through the X‐Z plane is shown.

Figure 3

Fluorescence in situ hybridization (FISH) with an apxIV probe on the Actinobacillus pleuropneumoniae‐infected lung samples. CLSM images of A. pleuropneumoniae aggregates (red) within the lung tissue (blue) obtained from pigs of clinical cases SHY14‐286 (A) and SHY13‐04294 (B). (C) FISH on a lung sample from a control pig that was negative for A. pleuropneumoniae.

Figure 4

CLSM images of Actinobacillus pleuropneumoniae embedded in agarose after 24 h of incubation. (A) Agarose control, not inoculated with bacteria observed in the differential interference contrast mode. (B) A. pleuropneumoniae isolate APP286 aggregates as observed in the differential interference contrast mode, stack of ~10 images. (C) A. pleuropneumoniae isolate APP286 aggregates stained with WGA‐Oregon Green and observed in the fluorescence mode, stack of ~10 images. Bars = 30 μm.