Transcriptome analysis of the biofilm formed by methicillin-susceptible Staphylococcus aureus

Abstract

Biofilm formation is regarded as one of the major determinants in the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) as pathogens of medical device-related infection. However, methicillin-susceptible S. aureus (MSSA) can also form biofilm in vitro and such biofilms are resistant to vancomycin. Hence, researching the possible mechanisms of MSSA biofilm formation is urgent and necessary. Here, we used S. aureus ATCC25923 as the model strain, and studied gene expression profiles in biofilms after the treatment of ursolic acid and resveratrol using RNA-seq technology. The results showed that only ursolic acid could inhibit biofilm formation, which differed from their applied on the multiple clinical drugs resistant MRSA biofilm. RNA-seq data was validated by examining the expression of six genes involved in biofilm formation by qRT-PCR. These data analysis indicated that the mechanism of the MSSA biofilm formation was different from that of the MRSA, due to absence of accessory gene regulator (agr) function. These findings suggest that biofilms of S. aureus with agr dysfunction may be more resistant than those with agr function. Therefore, the infection from clinical MSSA may be recalcitrant once forming biofilm. Further study is necessary to uncover the mechanisms of biofilm formation in other clinical S. aureus.

Figure 1. Scanning electron microscopic images showing the structure of the biofilm of Staphylococcus aureus ATCC25923.

Magnifications, ×3000. (a) Control without ethanol (18-h incubation) (218), (b) 30 μg/mL ursolic acid (2U30), (c) 100 μg/mL resveratrol (2R100), (d) control without ethanol (36 h) (236), (e) 150 μg/mL resveratrol (2R150), (f) 8 μg/mL vancomycin (2V), and (g) the mixture of 8 μg/mL vancomycin and 150 μg/mL resveratrol.

Figure 2. Heatmap of differentially expressed genes associated with S. aureus ATCC25923 biofilm formation and virulence.

(a) ursolic acid inhibiting S. aureus ATCC25923 biofilm formation condition, (b) resveratrol inhibiting S. aureus ATCC25923 biofilm formation condition, (c) resveratrol removing established S. aureus ATCC25923 biofilm condition, and (d) the mixture of resveratrol and vancomycin removing established S. aureus ATCC25923 biofilm condition.

Figure 3. PCR product agarose gel electrophoresis for the identification of S. aureus ATCC25923 agr genotype.

Whole- cell PCR was performed with each of the four agr specificity primers. Lines 1 to 4, PCR product using the same forward primer pan-agr and different reverse primers (agrI, agrII, agrIII, and agrIV) with S. aureus ATCC25923 genomic DNA as templates (439 bp for agrI, 573 bp for agrII, 406 bp for agrIII, and 657 bp for agrIV); Lines 5 to 8, negative controls responsible to lines 1 to 4, respectively; Lines M, 100 bp for DNA ladder.

Figure 4. Expression ratio (log₂) obtained by qRT-PCR and RNA-seq of these six selected genes associated with S. aureus biofilm formation.

pyk was used as a reference gene for normalization of qRT-PCR data. All gene expression ratios from qRT-PCR between treatment groups and controls under different conditions were significantly different (P < 0.05). Bars represent the error standard (n = 3). The x-axis indicates the comparison between treatment groups and controls under different conditions. The y-axis shows the gene expression ratios.