Analysis of multidrug resistance in Streptococcus suis ATCC 700794 under tylosin stress

Abstract

Streptococcus suis is an important zoonotic pathogen. The massive use of tylosin and other antibiotics in swine production has led to the emergence of resistant phenotypes of S. suis. However, there are no adequate measures available to address the problem of bacterial resistance. This study involved the use of 1/4 MIC (0.125 µg/mL) of tylosin to investigate resistance-related proteins by S. suis ATCC 700794. Our results showed that 171 proteins were differentially expressed in S. suis tested with 1/4 MIC (0.125 µg/mL) of tylosin using iTRAQ-based quantitative proteomic methods. TCS, heat shock protein and elongation factors were differentially expressed at 1/4 MIC (0.125 µg/mL) of tylosin compared to non treated, control cells. Using quantitative RT-PCR analysis, we verified the relationship between the differentially expressed proteins in S. suis with different MIC values. The data showed that expression profile for elongation factor G (fusA), elongation factor Ts (tsf), elongation factor Tu (tuf), putative histidine kinase of the competence regulon, ComD (comD), putative competence-damage inducible protein (cinA) and protein GrpE (grpE), observed in tylosin-resistant S. suis, correlated with that of S. suis ATCC 700794 at 1/4 MIC (0.125 µg/mL). The MIC of tylosin-resistant showed high-level resistance in terramycin, chlortetracycline, ofloxacin and enrofloxacin. Our findings demonstrated the importance of elongation factors, TCS and heat shock protein during development of tylosin resistance in S. suis. Thus, our study will provide insight into new drug targets and help reduce bacterial multidrug resistance through development of corresponding inhibitors.

Keywords: Streptococcus suis; iTRAQ; multidrug resistance; tylosin.

Figure 1.

Growth curve of Streptococcus suis ATCC 700794 in the absence of tylosin and in the presence of 1/4 MIC of tylosin. Data are expressed as means ± standard deviations.

Figure 2.

K-means clustering representation of 171 profiles of Streptococcus suis with up-regulations (a) and down-regulations (b). The magnitude of the percentage is represented by a color scale (top right) going from low (green) to high (red).

Figure 3.

The network of significantly differentially expressed proteins (ratio>1.2 or <0.8 (p-value<0.05)) was analyzed by STRING. Small nodes represent proteins of unknown 3D structure; large nodes represent proteins with some 3D structure information or predicted. Coloured nodes represent query proteins and first shell of interactors; white nodes represent second shell of interactors. The blue lines represent database evidence; the purple lines represent experimental evidence; yellow lines represent text mining evidence; the black lines represent coexpression evidence; and green lines represent neighbourhood evidence.

Figure 4.

(a). Effect of 1/4 MIC of tylosin on mRNA expression of genes in Streptococcus suis ATCC 700794. Data are expressed as means ± standard deviations. The expression was normalized to 16S rRNA. Significantly different (*p < 0.05) compared to untreated control bacteria. (b). Effect on mRNA expression of genes in drug resistance level at 16 μg/mL of tylosin (S-t-R 16) Streptococcus suis ATCC 700794. Data are expressed as means ± standard deviations. The expression was normalized to 16S rRNA. Significantly different (*p < 0.05) compared to control bacteria. (c). Effect on mRNA expression of genes in drug resistance level at 128 μg/mL of tylosin (S-t-R 128) Streptococcus suis ATCC 700794. Significantly different (*p < 0.05) compared to untreated control bacteria. Data are expressed as means ± standard deviations. The expression was normalized to 16S rRNA. Significantly different (*p < 0.05) compared to control bacteria. (d). Effect on mRNA expression of fusA, tuf, tsf in Streptococcus suis ATCC 700794, drug resistance level at 16 μg/mL of tylosin (S-t-R 16) Streptococcus suis ATCC 700794 and drug resistance level at 128 μg/mL of tylosin (S-t-R 128) Streptococcus suis ATCC 700794. Data are expressed as means ± standard deviations. The expression was normalized to 16S rRNA. Significantly different (*p < 0.05) compared to control bacteria. (e). Effect on mRNA expression of comD, cinA, grpE in Streptococcus suis ATCC 700794, drug resistance level at 16 μg/mL of tylosin (S-t-R 16) Streptococcus suis ATCC 700794 and drug resistance level at 128 μg/mL of tylosin (S-t-R 128) Streptococcus suis ATCC 700794. Data are expressed as means ± standard deviations. The expression was normalized to 16S rRNA. Significantly different (*p < 0.05) compared to control bacteria.