Actinobacillus pleuropneumoniae is a Gram-negative facultative anaerobic encapsulated coccobacillus. It is classified into two biotypes: biotype 1 requires nicotinamide adenine dinucleotide (NAD) to grow while biotype 2 is NAD independent (1). Based on their antigenic composition and the properties of their lipopolysaccharides (LPS) and capsular polysaccharides, nineteen serovars of A. pleuropneumoniae have been described to date (2, 3). This bacterium is transmitted from pig to pig mainly by direct oral and nasal contact or by bioaerosols following the introduction of asymptomatic carrier pigs into a herd or piggery (2). A. pleuropneumoniae is a respiratory pathogen that colonizes the tonsils and nasal cavities of pigs and causes porcine pleuropneumonia, a highly contagious and often deadly respiratory disease responsible for significant economic losses in the swine industry worldwide (1). This disease, for which the clinical features range from peracute to chronic, is characterized by the presence of hemorrhagic, fibrinous, and necrotic lung lesions. The serovars most often isolated from infections differ depending on the region of the world. In North America, serovars 5 and 7 are predominant while serovars 2 and 9/11 are most often isolated in European countries (2, 4–7). The use of antibiotics at the onset of the disease is the most effective treatment for diminishing the severity of the clinical symptoms, the death rate, and the spread of infections (1, 2).

A. pleuropneumoniae produces a broad array of virulence factors that play crucial roles in the infectious process of porcine pleuropneumonia by contributing to colonization, evasion of the immune defense mechanisms, and induction of lung lesions (2, 8, 9). The most important and well-known virulence factors are the Apx toxins (I to III), which belong to the family of Repeats in ToXin (RTX) toxins (9, 10). They cause the lysis of different cell types, including red blood cells, alveolar epithelial cells, neutrophils, and macrophages (9, 10).

A wide variety of bacterial species are present in the upper respiratory tract of pigs (11), and are likely to have positive or negative interactions with A. pleuropneumoniae. To the best of our knowledge, the negative interactions, such as competition and antagonism, that occur in the porcine respiratory tract and that involve A. pleuropneumoniae have not been thoroughly investigated to date. Antagonism is related to the ability of microorganisms to produce antimicrobial substances, including bacteriocins, organic acids, and hydrogen peroxide (H₂O₂) (12, 13). Preliminary analyses in our laboratory showed that Streptococcus pluranimalium 2N12, which was isolated from a pig nasal sample, is able to antagonize the growth of A. pleuropneumoniae in a deferred growth inhibition assay. In the present study, we investigated the H₂O₂-mediated antagonistic activity of S. pluranimalium 2N12 against A. pleuropneumoniae.

The ability of S. pluranimalium 2N12 to exert an antagonistic effect on A. pleuropneumoniae was first examined using a deferred growth inhibition assay. As reported in Table 2, A. pleuropneumoniae 81750 and L20 strains, which belong to serovar 5b, displayed inhibitory zones of 4.0 mm and 6.3 mm, respectively. A. pleuropneumoniae K17 (serovar 5a) appeared less sensitive, showing an inhibitory zone of 1.3 mm. The deferred growth inhibition assay was then performed in the presence of catalase to determine whether the inhibition of A. pleuropneumoniae may have resulted from the production of H₂O₂ by S. pluranimalium. Adding catalase (100 units/mL) resulted in a significant reduction or complete abolition of the inhibitory zones (Table 2).

As H₂O₂ is a potential inhibitory compound that is active against A. pleuropneumoniae, its production by S. pluranimalium 2N12 was confirmed by quantifying H₂O₂ levels in a culture supernatant (LAPTg medium) using a fluorimetric assay. As shown in Figure 1A, S. pluranimalium dose- and time-dependently produced H₂O₂. A low bacterial inoculum (OD₆₆₀ of 0.1) resulted in the production of 56.3 μM of H₂O₂ after a 2-h incubation, while the use of a higher bacterial inoculum (OD₆₆₀ of 0.6) was associated with the production of 845.5 μM of H₂O₂, a 15-fold increase compared to that of the low bacterial inoculum. It was then determined whether a longer incubation time for the low bacterial inoculum (OD₆₆₀ of 0.1) resulted in the production of higher amounts of H₂O₂. The results reported in Figure 1B show that extending the incubation to 24 h was associated with lower amounts of H₂O₂ compared to the 6-h incubation time.

Lactate oxidase is a bacterial enzyme that converts lactate into pyruvate and H₂O₂. Figure 2 indicates that exogenous lactate added to the LAPTg culture medium dose-dependently increased the amount of H₂O₂ produced by S. pluranimalium. More specifically, the presence of 25 mM lactate increased the production of H₂O₂ 1.57-fold compared to the control (no lactate), suggesting that the production of H₂O₂ by S. pluranimalium relies, at least in part, on the activity of lactate oxidase.

The antibacterial activity of H₂O₂ against A. pleuropneumoniae strains belonging to eighteen different serovars was evaluated by determining the MIC and MBC values. As reported in Table 1, all the strains tested had MICs and MBCs ranging from 0.57 mM (corresponding to 0.002% [v/v]) to 2.3 mM (corresponding to 0.008% [v/v]) for H₂O₂.

The killing of planktonic A. pleuropneumoniae (strains 81750 and K17) caused by either H₂O₂ or a culture supernatant of S. pluranimalium (treated or not with catalase) after a 4-h incubation was monitored. As reported in Figure 3A, H₂O₂ used at a concentration of 2.3 mM, which corresponded to the MBC, reduced the viability of A. pleuropneumoniae 81750 (initial concentration: 5 × 10⁶ CFU/mL) and K17 (initial concentration: 4.45 × 10⁶ CFU/mL) below the minimum detection limit (10¹ CFU/mL). On the other hand, in the control assay (no H₂O₂), the bacterial concentrations increased slightly. Similarly, a culture supernatant of S. pluranimalium (containing 0.5 mM H₂O₂) significantly reduced the number of planktonic A. pleuropneumoniae cells. Figure 3B shows that the CFUs for A. pleuropneumoniae 81750 dropped from 4.45 × 10⁶ CFU/mL to 2 ± 0.6 × 10³ CFU/mL, and from 3.4 ± 0.4 × 10⁷ CFU/mL to below the minimum detection limit (10¹ CFU/mL) for A. pleuropneumoniae K17. Treating the culture supernatant of S. pluranimalium with catalase, which led to a residual concentration of 0.04 mM H₂O₂, completely abolished its bactericidal activity.

The ability of H₂O₂ and the culture supernatant of S. pluranimalium to cause the killing and desorption of biofilm-like structures of A. pleuropneumoniae 81750 and K17 following a 4-h treatment was then assessed. When used at a high concentration (MBC or two-fold MBC), H₂O₂ slightly but significantly decreased the viability of both A. pleuropneumoniae biofilm-like structures (Figure 4A). More specifically, at two-fold MBC (4.6 mM), viability was reduced by 11.5 ± 1.4% and 8.3 ± 2.7% for strains 81750 and K17, respectively. While H₂O₂ did not induce biofilm-like structure desorption for A. pleuropneumoniae 81750, it caused a dose-dependently desorption of the A. pleuropneumoniae K17 biofilm-like structure (Figure 4B). At two-fold MBC (4.6 mM), the biofilm-like biomass was reduced by 64.4 ± 1.3%. The same effects on A. pleuropneumoniae biofilm-like structure killing and desorption were investigated using the culture supernatant of S. pluranimalium containing 0.5 mM H₂O₂. As reported in Figure 5, no significant effects were observed.

The effect of H₂O₂ on the activity of conventional antibiotics, including ceftiofur, penicillin G, and tetracycline, against A. pleuropneumoniae 81750 and K17 was then evaluated. As reported in Table 3, synergistic effects were observed for both strains when H₂O₂ was used in combination with ceftiofur. Synergistic interactions were only observed for strain K17 with the H₂O₂/penicillin G and H₂O₂/tetracycline combinations.

Preliminary assays showed that S. pluranimalium has the ability to form a biofilm. We thus investigated whether H₂O₂ contributes to biofilm formation by growing S. pluranimalium in the presence of various amounts of catalase. On the one hand, the presence of catalase increased the bacterial growth rate, and the highest concentration tested (200 units/mL) also appeared to increase the final biomass as shown by the higher final OD₆₆₀ (Figure 6A). On the other hand, the presence of catalase dose-dependently reduced biofilm formation (Figure 6B). More specifically, when added at a concentration of 200 units/mL, catalase decreased the formation of a biofilm by 76.3 ± 1.3%.

A large number of different microorganisms reside in the upper respiratory tract of pigs (11). In the healthy state, these microbial communities live in homeostasis and it can be hypothesized that they protect the animals against external pathogens that may reach this site. This protective effect may rely on the ability of certain commensal bacteria to produce antimicrobial compounds such as bacteriocins, organic acids, and hydrogen peroxide that are active against pathogenic microorganisms (12, 13). In the present study, we investigated the antagonistic activity of S. pluranimalium 2N12 against A. pleuropneumoniae.

We first confirmed an antimicrobial activity of S. pluranimalium 2N12 against A. pleuropneumoniae, which was related to H₂O₂ production. This is supported by the fact that the ability of a culture supernatant of S. pluranimalium 2N12 to induce the killing of planktonic cells of A. pleuropneumoniae was totally eliminated following a treatment with catalase. Moreover, commercial H₂O₂ was highly bactericidal for all serovars of A. pleuropneumoniae tested, with MBC values ranging from 0.57 to 2.3 mM. The deleterious effects of H₂O₂ on A. pleuropneumoniae may be linked to the generation of hydroxyl radicals in the presence of Fe(II) once it enters the cells (16), which results in the oxidation of macromolecules such as DNA and proteins.

Lactate oxidase (LctO) is a common H₂O₂-producing enzyme in bacteria that catalyzes the formation of pyruvate and H₂O₂ from lactate and oxygen (17). In the present study, the addition of exogenous lactate increased the production of H₂O₂ by S. pluranimalium, providing support for the key role of LctO. However, other pathways such as those involving pyruvate oxidase and amino acid oxidase (17), which may also contribute to generating H₂O₂, should not be excluded. Studies are currently in progress in our laboratory to investigate how environmental parameters modulate H₂O₂ production by S. pluranimalium.

As H₂O₂ is a harmful byproduct of aerobic metabolism and as S. pluranimalium does not produce catalase, it must possess a molecular mechanism to neutralize H₂O₂. Similarly to S. pluranimalium, Streptococcus pneumoniae produces large amounts of H₂O₂ as a byproduct of its metabolism (18). Several mechanisms have been suggested to explain how S. pneumoniae defends against the H₂O₂-mediated oxidative stress, including (i) scarcity of proteins with iron-sulfur clusters which can be damaged by reactive oxygen species (ROS) in an iron-dependent manner, (ii) expression of ferritin-like proteins known as Dps (DNA-binding protein from starved cells) or Dpr (Dps-like peroxide resistance), and (iii) production of thiol peroxidase (TpxD) activity (18). The exact mechanism that allows protection of S. pluranimalium from H₂O₂ needs to be characterized.

Evidence was brought that H₂O₂ production by S. pluranimalium is involved in its ability to form a biofilm given that the presence of catalase dose-dependently prevented biofilm formation. H₂O₂-mediated biofilm formation has been previously reported for Streptococcus sanguinis and Streptococcus gordonii, which are primary colonizers of human dental biofilms (19, 20). It has been proposed that H₂O₂ induces the release of extracellular bacterial DNA, without autolysis, that promotes cell-to-cell adhesion and biofilm formation (20).

A. pleuropneumoniae has the ability to form a biofilm (21, 22) that enhances its resistance to antibiotics compared to planktonic cells (23). We showed that treating a pre-formed biofilm-like structure of A. pleuropneumoniae with H₂O₂ at a concentration corresponding to the MBC (2.3 mM) reduces its viability. No killing of the biofilm-like structure was obtained with a culture supernatant of S. pluranimalium, which is likely related to the fact that the supernatant contained a low amount of H₂O₂ (0.5 mM). Additional studies are required to demonstrate the killing effect of H₂O₂ using more relevant biofilm models of A. pleuropneumoniae.

The effect of H₂O₂ on the activity of conventional antibiotics used to treat A. pleuropneumoniae infections was assessed using the checkerboard technique. Synergistic interactions between H₂O₂ and some antibiotics, including ceftiofur, penicillin G, and tetracycline, were demonstrated, particularly against A. pleuropneumoniae K17. This observation was in agreement with the study of Sgibnev and Kremleva (24) who investigated the influence of various microbial metabolites on the antibiotic sensitivity of bacteria. The authors reported that H₂O₂ was the most effective bacterial metabolite for increasing the sensitivity of both Gram-positive and Gram-negative bacteria to several antibiotics. They proposed that H₂O₂ may cause a shift in the balance of pro-oxidants and antioxidants in bacteria and that the resulting oxidative stress enhances the effects of antibiotics on the target bacteria.

Based on our results, the production of H₂O₂ by S. pluranimalium could be regarded as a potential protection mechanism of the upper respiratory tract against H₂O₂-sensitive pathogens such as A. pleuropneumoniae. Interestingly, previous studies have documented the role of H₂O₂ produced by commensal streptococci colonizing the oral cavity in controlling cariogenic bacteria (mainly Streptococcus mutans) (25–27). It is worth mentioning that the presence of catalase positive staphylococci in the upper respiratory tract of pigs (11) may attenuate the beneficial impact of H₂O₂.

The resistance of A. pleuropneumoniae to various antibiotics, including tetracycline, ampicillin, and penicillin, is on the increase and is a growing concern (28–30). The identification of alternative strategies for controlling A pleuropneumoniae infections is thus of great interest. In this regard, further studies are required to explore the possibility of using S. pluranimalium as a probiotic to antagonize respiratory pathogens such as A. pleuropneumoniae.

Based on our results, the production of H₂O₂ by S. pluranimalium could be regarded as a potential protective mechanism of the upper respiratory tract against H₂O₂-sensitive pathogens such as A. pleuropneumoniae.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

MF, MG, and DG conceived and designed the experiments. KV performed the experimental assays and the statistical analysis. DG drafted and finalized the manuscript. All authors contributed to the article and approved the submitted version.

This study was supported by Fonds de recherche du Québec—Nature et technologies (2018-PR-205462).

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.

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