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. 2021 Sep 30:8:722683.
doi: 10.3389/fvets.2021.722683. eCollection 2021.

Actinobacillus pleuropneumoniae Surviving on Environmental Multi-Species Biofilms in Swine Farms

Abraham Loera-Muro  1 , Flor Y Ramírez-Castillo  2 , Adriana C Moreno-Flores  2 , Eduardo M Martin  2 , Francisco J Avelar-González  3 , Alma L Guerrero-Barrera  2
Affiliations

Actinobacillus pleuropneumoniae Surviving on Environmental Multi-Species Biofilms in Swine Farms

Abraham Loera-Muro et al. Front Vet Sci. .

Abstract

Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, an important respiratory disease for the pig industry. A. pleuropneumoniae has traditionally been considered an obligate pig pathogen. However, its presence in the environment is starting to be known. Here, we report the A. pleuropneumoniae surviving in biofilms in samples of drinking water of swine farms from Mexico. Fourteen farms were studied. Twenty drinking water samples were positive to A. pleuropneumoniae distributed on three different farms. The bacteria in the drinking water samples showed the ability to form biofilms in vitro. Likewise, A. pleuropneumoniae biofilm formation in situ was observed on farm drinkers, where the biofilm formation was in the presence of other bacteria such as Escherichia coli, Stenotrophomonas maltophilia, and Acinetobacter schindleri. Our data suggest that A. pleuropneumoniae can inhabit aquatic environments using multi-species biofilms as a strategy to survive outside of their host.

Keywords: Actinobacillus pleuropneumoniae (APP); biofilms; drinking water; environmental multi-species biofilms; swine farms.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Alignment of sequences with the program Jalview 2.9 of A. pleuropneumoniae specific gene apxIV obtained from samples of drinking water from pig farms. Samples randomly selected and positive for alignment with the sequences deposited in GenBank of apxIV gene are ApxIVA_Ags5-I (ApxIVA_Ags5-I/gb/KU169148/1-333), ApxIVA_Ags8-V (ApxIVA_Ags8-V/gb/KU169146/1-250), and ApxIVA_Ags12-II (ApxIVA_Ags12-II/gb/KU169147/1-335).
Figure 2
Figure 2
Biofilm formation by positive samples for A. pleuropneumoniae from drinking water. (a–c) Biofilms in the liquid–air interface formed in vitro from drinking water of one Mexican farm in three different samples (arrows show the biofilms). (d,e) FISH technique to detect A. pleuropneumoniae in biofilms from samples of drinking water from swine farms; A. pleuropneumoniae was detected with probes labeled with fluorescein (green) and other bacteria were labeled with ethidium bromide (red) (arrows show A. pleuropneumoniae label). (a,d,e) Ags5-I, (b) Ags5-II, and (c) Ags5-III.
Figure 3
Figure 3
Detection of A. pleuropneumoniae in biofilms produced from drinking water by fluorescent in situ hybridization. Micrographs were taken with confocal laser scanning microscopy (a–c) or epifluorescence microscopy (d–f). (a) A. pleuropneumoniae 1-4074 and in the box at the upper left corner (–) negative control (E. coli ATCC 25922). (b,c) A. pleuropneumoniae detection by CLSM. (d–f) A. pleuropneumoniae detection by EM (seen at 100×).
Figure 4
Figure 4
FISH of A. pleuropneumoniae in biofilms detected in situ at drinkers in swine farms. (a–d) Epifluorescence microscopy pictures (seen at 60×). (a) A. pleuropneumoniae 1-4074 and in the box in the lower right corner (–) specific control (E. coli ATCC 25922). (b) A. pleuropneumoniae positive samples from drinkers. (c) General DAPI staining of microorganisms in the sample. (d) Merge of (b,c).
Figure 5
Figure 5
Scanning electron microscopy (SEM) of biofilms produced by positive samples to A. pleuropneumoniae from drinking water in swine farms. (a) Biofilms of A. pleuropneumoniae 4074. (b,c) Biofilms of positive samples to A. pleuropneumoniae from drinking water collected at swine farms.
See this image and copyright information in PMC

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