1. Culturable aerobic bacteria from porcine respiratory tracts
One hundred and sixteen aerobic bacterial isolates from five porcine respiratory tracts were successfully cultured from the trachea, tracheobronchial lymph node, epical lobe, cardiac lobe, and diaphragmatic lobe of both the left and right lungs. An average of 23 ± 10 isolates was obtained from each lung. The L1 and L4 lungs had the highest numbers of 31 and 36 isolates, respectively. The majority of the isolates (56%) were from the epical and diaphragmatic lobes of the lungs. Almost 90% of these isolates were gram-negative rod-shaped bacteria that had different colony characteristics, i.e., colony forms (63% circular and 37% irregular), margins (55% undulate, 26% entire, 18% curled, and 1% lobate), and mucosity (66% nonmucoid and 34% mucoid). These culturable isolates represented a proportion of aerobic commensal bacteria inside the porcine respiratory tracts in this study. Ninety-three (80%) of these aerobic culturable bacteria were successfully identified and classified into 14 genera and 21 species (Additional file 1) from seven families of three bacterial phyla (97% Proteobacteria, 17% Firmicutes, and 2% Bacteroidetes), i.e., Acidovorax, Acinetobacter, Aeromonas, Escherichia, Enterobacter, Hafnia, Klebsiella, Macrococcus, Proteus, Providencia, Shewanella, Shigella, Weeksella, and Wohlfahrtiimonas, based on 16S rRNA sequence analysis (Figs. 1 and 2). Phylogenetic analysis clustered these 93 isolates into seven major groups (Fig. 2). The first four groups (60%) were members of the family Enterobacteriaceae, including Escherichia, Shigella, Enterobacter, Klebsiella, Hafnia, Proteus, and a small cluster of Providencia. The fifth group contained Macrococcus, which was the only gram-positive bacterial genus belonging to the family Staphylococcaceae. The sixth and seventh groups consisted of the genera Acinetobacter (family Moraxellaceae) and Aeromonas (family Aeromonadaceae). The remaining isolates had only one or two members, including Acidovorax (family Comamonadaceae), Shewanella (family Shewanellaceae), Wohlfahrtiimonas (unclassified bacteria in the class Gammaproteobacteria), and Weeksella (family Flavobacteriaceae). One isolate was selected to represent each identified genus, except two isolates of Macrococcus, NS20 in the fifth group containing Macrococcus samples and NS108 in the seventh group together with Aeromonas.
2. Growth of the selected aerobic bacterial isolates from the porcine respiratory tracts in different conditioned media
Fifteen isolates from 14 genera of the isolated aerobic bacteria from the porcine respiratory tracts and two porcine strains of P. multocida with capsular types A and D (PM7 and PM2) were cocultured in the conditioned media (spent BHIB) and the unconditioned media (fresh BHIB), resulting in 289 interacting pairs (17 × 17) as shown in Fig. 3. Nearly all conditioned media could inhibit the growth of these two P. multocida strains, except that of Acinetobacter. The conditioned medium of Acinetobacter supported or slightly slowed the growth of all tested bacteria. The conditioned medium of Providencia inhibited the growth of every isolate, including itself. The media of Shigella and Macrococcus Group 7 (G7) had a lower inhibitory effect on Klebsiella, Escherichia, Shigella, and Enterobacter. Conditioned media from Proteus and Escherichia only supported the growth of Klebsiella with a prolonged lag phase. The media of five bacterial samples (Klebsiella, Shewanella, Acidovorax, Enterobacter, and Hafnia) only inhibited the growth of P. multocida. The media of both P. multocida strains similarly inhibited Aeromonas, Wohlfahrtiimonas, Shewanella, Acidovorax, Macrococcus, Acinetobacter, Providencia, and Weeksella as well as themselves. The media of the remaining four samples (Weeksella, Wohlfahrtiimonas, Aeromonas, and Macrococcus G5) had different effects on the tested bacteria. Some conditioned media could promote bacterial growth compared to the control. For example, Weeksella grew better in the conditioned media of five bacterial samples (Acinetobacter, Wohlfahrtiimonas, Shewanella, Acidovorax, and Enterobacter).
3. Interaction between the porcine strains of Pasteurella multocida and the selected aerobic bacteria from the porcine respiratory tracts
This study measured bacterial interactions using the interaction parameter ε, which was calculated from the log ratio of maximum growth yield in the conditioned medium compared with that in the unconditioned medium. Pairwise interactions between 17 bacterial isolates revealed that most of the interactions (220 interactions) were negative interactions (Fig. 4). All negative interactions (-/-) were observed when growing the isolates in the conditioned media from Escherichia, Macrococcus, Pasteurella, Proteus, Providencia, Shigella, and Weeksella. Strong negative interactions (59 interactions, ε < -1) were observed in the conditioned media of Providencia (17 interactions), Macrococcus G5 (12 interactions), Escherichia (11 interactions), Shigella (11 interactions), and Weeksella (3 interactions), and four interactions were observed in the media of Aeromonas, Klebsiella, and Wohlfahrtiimonas. Most of these spent media, except that of Wohlfahrtiimonas, had a pH between 5.4–6.5, which was lower than the pH of the reference medium BHIB (7.4) (top dendrogram in Fig. 4). Conditioned media from four bacteria (Aeromonas, Klebsiella, Macrococcus G5, and Providencia) had a strong negative effect on P. multocida growth. Notably, the medium of Providencia (pH 5.5) had a strong negative interaction with all tested isolates, including itself. The low pH (5.4) of the media from the two P. multocida strains resulted in mild to moderate negative interactions with the other tested bacteria. The interaction patterns of P. multocida with these 17 conditioned media were separated from those of other bacterial samples (as shown in the right dendrogram) similar to the second cluster of four isolates from the Enterobacteriaceae family and the third cluster of ten samples. Seventeen mild positive interactions (+/+, 0 < ε < 0.1) were observed with the media of Acidovorax, Acinetobacter, Aeromonas, Enterobacter, Hafnia, Klebsiella, Shewanella, and Wohlfahrtiimonas. Six of these interactions observed in spent media (Acidovorax, Acinetobacter, Enterobacter, Hafnia, Shewanella, and Wohlfahrtiimonas) had pH values between 6.5–7.3, which were close to the pH of BHIB. Weeksella had positive interactions in five conditioned media, Acidovorax, Acinetobacter, Enterobacter, Shewanella, and Wohlfahrtiimonas, which was the highest number among the media (highlighted in red in Fig. 4). The conditioned medium of Enterobacter supported the highest number of positive interactions with five bacteria, which included Acinetobacter, Acidovorax, Macrococcus G7, Wohlfahrtiimonas, and Weeksella. A high proportion of mild negative interactions (88 interactions, 0 > ε > -0.1) was observed from bacteria grown in nearly all media, except Providencia and Shigella. These mild positive and negative interactions could be classified as neutral interactions. However, strong positive interactions were not observed in this study.
By focusing on the locations of the porcine respiratory tract in Fig. 5, the trachea (T) had the least number of bacterial genera (5) and had Providencia and Shigella as strong negative influencers. Macrococcus G7 and Shigella had substantial negative impacts on the others in the tracheobronchial lymph node (TN), while Acidovorax, Acinetobacter, Enterobacter, and Klebsiella provided positive support to some bacteria in this community. For the epical, cardiac, and diaphragmatic lobes of the porcine lung, Macrococcus G7 was the major bacteria that had a strong negative interaction with the others, except for the epical lobe, in which Providencia also exerted a negative effect. These three lobes of the lung shared six common bacteria with mild negative or positive interactions (Acinetobacter, Escherichia, Enterobacter, Klebsiella, Macrococcus G5, and Proteus), whereas Shewanella and Wohlfahrtiimonas were unique to the epical lobe, Hafnia was unique to the cardiac lobe and Weeksella was unique to the diaphragmatic lobe.