Antagonistic Effects of Corynebacterium pseudodiphtheriticum 090104 on Respiratory Pathogens

Abstract

In previous studies, it was demonstrated that Corynebacterium pseudodiphtheriticum 090104, isolated from the human nasopharynx, modulates respiratory immunity, improving protection against infections. Here, the antagonistic effect of the 090104 strain on respiratory pathogens, including Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii, was explored. In a series of in vitro studies, the capacity of C. pseudodiphtheriticum 090104, its bacterium-like particles, and its culture supernatants to coaggregate, inhibit the growth, and change the virulent phenotype of pathogenic bacteria was evaluated. The results showed that the 090104 strain was able to exert a bacteriostatic effect on K. pneumoniae and S. pneumoniae growth. In addition, C. pseudodiphtheriticum 090104 coaggregated, inhibited biofilm formation, and induced phenotypic changes in all the respiratory pathogens evaluated. In conclusion, this work demonstrated that, in addition to its beneficial effects exerted by host-microbe interactions, C. pseudodiphtheriticum 090104 can enhance protection against respiratory pathogens through its microbe-microbe interactions. The mechanisms involved in such interactions should be evaluated in future research.

Keywords: Corynebacterium pseudodiphtheriticum; microbe–microbe interaction; respiratory pathogens.

Figure 1

Autoaggregation assay. The autoaggregation was evaluated in distinct respiratory pathogens and C. pseudodiphtheriticum 090104 (Cp090104) and its BLPs (PCp090104). The cutoff value for bacterial aggregation is indicated by a solid line. Each bar corresponds to a time point of the assay, depicting the percentage of autoaggregation at 6 and 12 h. All tests were performed in triplicate and in three independent experiments (n = 9). Means with standard deviations are presented for each measurement.

Figure 2

Coaggregation assay. The coaggregation assay was performed with C. pseudodiphtheriticum 090104 (Cp090104) and its BLPs (PCp090104) and the distinct respiratory pathogens. Bars for each respiratory pathogen represent the sampling time at 10 h (endpoint). All tests were performed in triplicate and in three independent experiments (n = 9). Means with standard deviations are presented for each measurement.

Figure 3

Inhibitory activity assay. Inhibition with spot inoculation of two respiratory pathogens on C. pseudodiphtheriticum 090104 (Cp090104) lawn. Dilutions made for both pathogens are indicated. Photos on the right show the passage of residual inoculum after contact with Cp090104. All assays were performed in duplicates.

Figure 4

Inhibitory activity assay. Inhibition with spot inoculation of C. pseudodiphtheriticum 090104 (Cp090104) on A. baumannii lawn. Dilutions made for the respiratory commensal bacterium are indicated. Photos on the right show the passage of residual inoculum after contact with A. baumannii. All assays were performed in duplicates.

Figure 5

Biofilm formation assay. Biofilm formation assay of distinct respiratory pathogens and C. pseudodiphtheriticum 090104 (Cp090104). Two sampling times are presented, 24 and 48 h. All tests were performed in triplicate and in three independent experiments (n = 9). Means with standard deviations are presented for each measurement.

Figure 6

Inhibition of biofilm formation assay. Study of the effect of the cell-free supernatant of C. pseudodiphtheriticum 090104 (Cp090104) on biofilm formation by the respiratory pathogens. All tests were performed in triplicate and in three independent experiments (n = 9). Means with standard deviations are presented for each measurement. Significant differences between the indicated groups: (*) p < 0.05; (**) p < 0.01.

Figure 7

Hemolytic activity assay. Characterization of the effect of C. pseudodiphtheriticum 090104 (Cp090104) and its cell-free supernatant on the hemolytic activity of respiratory pathogens when cultured on BHI agar–blood medium. Tests were performed in triplicate and in three independent experiments.

Figure 8

Colony morphology assay. Characterization of the effect of C. pseudodiphtheriticum 090104 (Cp090104) on the colony morphology of respiratory pathogens when cultured on BHI agar medium. The diameter of colony was measured in the presence and absence of Cp090104. All tests were performed in triplicate; means with standard deviations are presented for each measurement. Significant differences between the indicated groups: (**) p < 0.01.