Streptococcus pluranimalium 2N12 Exerts an Antagonistic Effect Against the Swine Pathogen Actinobacillus pleuropneumoniae by Producing Hydrogen Peroxide

Actinobacillus pleuropneumoniae is the causal agent of porcine pleuropneumonia, a highly contagious and often deadly respiratory disease that causes major economic losses in the swine industry worldwide. The aim of the present study was to investigate the hydrogen peroxide (H2O2)-dependent antagonistic activity of Streptococcus pluranimalium 2N12 (pig nasal isolate) against A. pleuropneumoniae. A fluorimetric assay showed that S. pluranimalium produces H2O2 dose- and time-dependently. The production of H2O2 increased in the presence of exogenous lactate, suggesting the involvement of lactate oxidase. All 20 strains of A. pleuropneumoniae tested, belonging to 18 different serovars, were susceptible to H2O2, with minimal inhibitory concentrations and minimal bactericidal concentrations ranging from 0.57 to 2.3 mM. H2O2, as well as a culture supernatant of S. pluranimalium, killed planktonic cells of A. pleuropneumoniae. Treating the culture supernatant with catalase abolished its bactericidal property. H2O2 was also active against a pre-formed biofilm-like structure of A. pleuropneumoniae albeit to a lesser extent. A checkerboard assay was used to show that there were antibacterial synergistic interactions between H2O2 and conventional antibiotics, more particularly ceftiofur. Based on our results and within the limitations of this in vitro study, 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.


INTRODUCTION
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)(5)(6)(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 (H 2 O 2 ) (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 H 2 O 2mediated antagonistic activity of S. pluranimalium 2N12 against A. pleuropneumoniae.

Bacteria and Growth Conditions
The A. pleuropneumoniae strains used in this study ( Table 1) were grown in Todd Hewitt Broth (THB; BD-Canada, Mississauga, ON, Canada) supplemented with NAD (20 µg/mL; THB-NAD) and incubated in an anaerobic chamber (80% N 2 , 10% CO 2 , 10% H 2 ) at 37 • C. S. pluranimalium 2N12, which was isolated by our laboratory from the nasal cavity of a healthy pig, was identified by 16S rRNA gene sequencing. It was cultivated in THB and was incubated at 37 • C in aerobic conditions. Two-µL aliquots of an overnight broth culture of S. pluranimalium 2N12 were applied on the surface of THB agar plates. After a 24-h incubation at 37 • C to allow bacterial growth, the plates were overlaid with soft THB-NAD agar (0.75%, w/v) that had been inoculated with an overnight culture of either A. pleuropneumoniae K17 (biotype 1, serovar 5a), 81750 (biotype 1, serovar 5b), or L20 (biotype 1, serovar 5b) (700 µL of bacterial culture in 7 mL of soft THB-NAD agar), and were incubated for a further 24 h at 37 • C. The inhibitory zones (in mm) were measured from the edge of the S. pluranimalium growth to the margin of the inhibition area. The effect of adding catalase (100 units/mL) to the THB agar plates when growing S. pluranimalium was evaluated. Assays were performed in triplicate, and the means ± standard deviations (SD) were calculated.

Effect of Catalase on the Growth of and Biofilm Formation by S. pluranimalium
The effect of catalase (   triplicate in three independent experiments, and the means ± SD were calculated.

Statistical Analysis
Statistical analyses were performed using a one-way ANOVA analysis of variance with a post-hoc Bonferroni multiple comparison test (GraphPad Software Inc., San Diego, CA, USA). All results were considered statistically significant at p < 0.01.

RESULTS
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 H 2 O 2 by S. pluranimalium. Adding catalase (100 units/mL) resulted in a significant reduction or complete abolition of the inhibitory zones ( Table 2). As H 2 O 2 is a potential inhibitory compound that is active against A. pleuropneumoniae, its production by S. pluranimalium 2N12 was confirmed by quantifying H 2 O 2 levels in a culture supernatant (LAPTg medium) using a fluorimetric assay. As shown in Figure 1A, S. pluranimalium dose-and timedependently produced H 2 O 2 . A low bacterial inoculum (OD 660 of 0.1) resulted in the production of 56.3 µM of H 2 O 2 after a 2-h incubation, while the use of a higher bacterial inoculum (OD 660 of 0.6) was associated with the production of 845.5 µM of H 2 O 2 , 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 (OD 660 of 0.1) resulted in the production of higher amounts of H 2 O 2 . The results reported in Figure 1B show that extending the incubation to 24 h was associated with lower amounts of H 2 O 2 compared to the 6-h incubation time.
Lactate oxidase is a bacterial enzyme that converts lactate into pyruvate and H 2 O 2 . Figure 2 indicates that exogenous lactate added to the LAPTg culture medium dose-dependently increased the amount of H 2 O 2 produced by S. pluranimalium. More specifically, the presence of 25 mM lactate increased the production of H 2 O 2 1.57-fold compared to the control (no lactate), suggesting that the production of H 2 O 2 by The killing of planktonic A. pleuropneumoniae (strains 81750 and K17) caused by either H 2 O 2 or a culture supernatant of S. pluranimalium (treated or not with catalase) after a 4-h incubation was monitored. As reported in Figure 3A, H 2 O 2 FIGURE 6 | Dose-dependent effect of catalase on the growth (A) and biofilm formation (B) of S. pluranimalium 2N12. Bacterial growth was monitored by recording the optical density at 660 nm (OD 660 ). Biofilm biomass was quantified by crystal violet staining. Assays were performed in triplicate in three independent experiments, and the means ± SD were calculated. *Significantly different at p < 0.01 compared to the control (no catalase).
used at a concentration of 2.3 mM, which corresponded to the MBC, reduced the viability of A. pleuropneumoniae 81750 (initial concentration: 5 × 10 6 CFU/mL) and K17 (initial concentration: 4.45 × 10 6 CFU/mL) below the minimum detection limit (10 1 CFU/mL). On the other hand, in the control assay (no H 2 O 2 ), the bacterial concentrations increased slightly. Similarly, a culture supernatant of S. pluranimalium (containing 0.5 mM H 2 O 2 ) significantly reduced the number of planktonic A. pleuropneumoniae cells. Figure 3B shows that the CFUs for A. pleuropneumoniae 81750 dropped from 4.45 × 10 6 CFU/mL to 2 ± 0.6 × 10 3 CFU/mL, and from 3.4 ± 0.4 × 10 7 CFU/mL to below the minimum detection limit (10 1 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 H 2 O 2, completely abolished its bactericidal activity.
The ability of H 2 O 2 and the culture supernatant of S. pluranimalium to cause the killing and desorption of biofilmlike 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), H 2 O 2 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 H 2 O 2 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 H 2 O 2 . As reported in Figure 5, no significant effects were observed.
The effect of H 2 O 2 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 H 2 O 2 was used in combination with ceftiofur. Synergistic interactions were only observed for strain K17 with the H 2 O 2 /penicillin G and H 2 O 2 /tetracycline combinations.
Preliminary assays showed that S. pluranimalium has the ability to form a biofilm. We thus investigated whether H 2 O 2 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 OD 660 (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%.

DISCUSSION
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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 -producing enzyme in bacteria that catalyzes the formation of pyruvate and H 2 O 2 from lactate and oxygen (17). In the present study, the addition of exogenous lactate increased the production of H 2 O 2 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 H 2 O 2 , should not be excluded. Studies are currently in progress in our laboratory to investigate how environmental parameters modulate H 2 O 2 production by S. pluranimalium.
As H 2 O 2 is a harmful byproduct of aerobic metabolism and as S. pluranimalium does not produce catalase, it must possess a molecular mechanism to neutralize H 2 O 2 . Similarly to S. pluranimalium, Streptococcus pneumoniae produces large amounts of H 2 O 2 as a byproduct of its metabolism (18). Several mechanisms have been suggested to explain how S. pneumoniae defends against the H 2 O 2 -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 H 2 O 2 needs to be characterized.
Evidence was brought that H 2 O 2 production by S. pluranimalium is involved in its ability to form a biofilm given that the presence of catalase dose-dependently prevented biofilm formation. H 2 O 2 -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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 (0.5 mM). Additional studies are required to demonstrate the killing effect of H 2 O 2 using more relevant biofilm models of A. pleuropneumoniae.
The effect of H 2 O 2 on the activity of conventional antibiotics used to treat A. pleuropneumoniae infections was assessed using the checkerboard technique. Synergistic interactions between H 2 O 2 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 H 2 O 2 was the most effective bacterial metabolite for increasing the sensitivity of both Gram-positive and Gram-negative bacteria to several antibiotics. They proposed that H 2 O 2 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 H 2 O 2 by S. pluranimalium could be regarded as a potential protection mechanism of the upper respiratory tract against H 2 O 2 -sensitive pathogens such as A. pleuropneumoniae. Interestingly, previous studies have documented the role of H 2 O 2 produced by commensal streptococci colonizing the oral cavity in controlling cariogenic bacteria (mainly Streptococcus mutans) (25)(26)(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 H 2 O 2 .
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.

CONCLUSIONS
Based on our results, the production of H 2 O 2 by S. pluranimalium could be regarded as a potential protective mechanism of the upper respiratory tract against H 2 O 2 -sensitive pathogens such as A. pleuropneumoniae.

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