Norfloxacin Sub-Inhibitory Concentration Affects Streptococcus suis Biofilm Formation and Virulence Gene Expression

Streptococcus suis (S. suis) is a Gram-positive pathogen result in large economic losses to the swine industry worldwide (Wertheim et al., 2009). One of the main reason is the appearance of multi-drug resistant strains (Devi et al., 2017), which show resistance to almost all available antibiotics, including β-lactams, tetracyclines and fluoroquinolones (Huang et al., 2016).Norfloxacin now remain the only effective antibiotics for the treatment of MDR S. suis (Olson et al., 2002), although there have been worldwide reports of resistance against these antibiotics as well (Soares et al., 2014; Hernandez-Garcia et al., 2017).
       
Recently, numerous reports have demonstrated that biofilms also contribute to antibiotic resistance (Wang et al., 2018b; Wang et al., 2018a; Seitz et al., 2016; Chakraborty, 2019). Bacterial biofilm consists of a microbial community that grows on a surface is extracellular and self-secretes a matrix (Pandey, 2014). Biofilm formation is considered to be a protective mode of bacteria adapted to harsh environments, which is not sensitive to antibiotics and host immune responses (Leonal Rabins, 2018; Olwal et al., 2018; Q.J. Wu, 2019; Wang et al., 2014b). Our research group reviewed the current literature on S. suis biofilm formation, regulatory mechanisms, drug resistance mechanisms and disinfection strategies (Wang et al., 2018b; Wang et al., 2014a). Some reports have demonstrated that sub-inhibitory concentrations of antibiotics may influence bacterial virulence gene such as Capsular polysaccharide (CPS), extracellular factor (EF), muramidase-released protein(MRP), suilysin (SLY), Fibronectin and fibrinogen-bining protein (FBPS),Glutamate dehydrogenage (GDH) and Glycerol phosphate deoxygenase (GAPDH) (Wang et al., 2014a; Xiao et al., 2017).
       
Antibiotics are still the most effective measures in controlling bacterial pathogens. However, antibiotics are usually used at sub-MIC (Minimum Inhibitory Concentration) doses to prevent infection (Yuksel et al., 2018). The concentration of antibiotics in feed are often lower than the MIC in the treatment of infections (Adamowicz et al., 2018; Yang et al., 2013). Recent evidence suggests that antimicrobial agent have an role in the induction of biofilm formation (Vinod Kumar et al., 2018), which are very important gene in causing bacterial resistance in the clinic (Krcmery, 2011). Thus, this study is to determine the norfloxacin sub-MICs on biofilm formation in S. suis strains and to quantify the expression of virulence gene.

Bacterial strains and antibiotics susceptibility testing
 
Five S. suis strains were isolated from swine in Jiangsu Province and confirmed to be a virulent strain (Wang et al., 2014a). Norfloxacin bulk drugs were purchased from the China Institute of Veterinary Drugs Control (Beijing, China), and prepared to be 1280 μg mL^-1. S. suis strains were grown in Tryptone soya broth (TSB) (Soybean-Casein Digest Medium U.S.P). MICs of norfloxacin were determined with a microbroth dilution, according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2015). MICs were the lowest antibiotic concentration that inhibited growth. MBCs were determined the dilution yielding three colonies or less by subculturing 10 µl from each well without visible bacterial growth on TSA (Soybean-Casein Digest Medium U.S.P). For next-step analysis, the bacteria, growing in sub-MIC concentration were selected.
 
Biofilm formation assay
 
The biofilm production of S. suis as described in our previous report In brief, A single colony was placed into 3 ml of Tryptone soya broth (TSB) and incubated density to 10⁶ CFU/mL. The 96-well plate (Costar, Corning, USA) was inoculated with or without sub-MIC norfloxacin and incubated for 24 h at 37oC. Planktonic bacteria were aspirated and the residual adhesive cells in each well was rinsed three-times with Phosphate buffered saline(PBS) and added 200 µL 100% methanol incubated at 37oC for 20 min. The each well was washed twice with distilled water, stained with 200 mL crystal violet solution for 10 min and added 200 mL crystal violet 10% w/w to each well and incubated at 37oC for 10 min. The crystal violet was suctioned and each well was washed twice in distilled water allowed to air dry provided 200 µL 95 % ethanol (w/w) and incubated at 37oC for 5 min and the OD value was determined at 570 nm using spectrophotometer (Multiskan FC, Thermo, China).
 
Growth curves
 
The growth curve of bacteria was determined in the presence of different antibiotic conentrations. Briefly, a single colony was used for sub-cultivation in TSB overnight at 37oC. The next day, the initial culture was diluted 100-fold in fresh TSB with sub-MIC of norfloxacin. The optical density at 600 nm (OD₆₀₀) was used to monitor cell growth at designated time points.
 
Colony forming unit assay
 
The number of viable cells of S. suis in the presence of norfloxacin was tested using the protocol described (Chan et al., 2017). In brief, the 96-well microtiter plate were filled with S. suis (1/100 dilution) and added the presence of norfloxacin. After formation of biofilm for 24 hours at 37oC, the each well was washed twice using 200 µL PBS to remove non-adherent cells and sonicated in a sonicator bath (Scientz, SB-5200D, China) for 10 min with 100 µL PBS. The number of viable cells of S. suis was investigated by inoculating into TSB. The experiments were repeated three times.
 
RT-PCR RNA extraction and quantitative RT-PCR
 
The expression of virulence genes of S. suis were determined by quantitative real-time PCR. In brief, an overnight culture was inoculated into TSB medium at anOD₆₀₀ = 0.1 and aliquots were subsequently exposed for 6 h at 1/4 MIC norfloxacin.Total RNA of S. suis was extracted using the TRIzon (CoWin Biosciences Co., Ltd, Biejing, china) method from the bacteria after 6 hours of incubation. Total RNA was reverse-transcribed to cDNA using the PrimeScript™ reagent kit (CoWin Biosciences Co., Ltd, Biejing, china). The primers used to amplify cps, mrp, ef, sly,fbps, gdh, gapdh and 16S rRNA( endogenous control gene) are listed in Table 1. Real-time PCR was performed according to previous reports (Wang et al., 2019).The 2^-ΔΔCT method using the 16S rRNAgene as an internal control was used to calculate and analyze the threshold cycle values (CT) obtained according to the melting curve.
 

 
Statistical analysis
All experiments were performed in triplicate. The statistical analysis was carried out by Graphpad Software package (GraphPad Software, La Jolla, CA). One-way analysis of variance (ANOVA) and Student’s t test (p < 0.05) were used for biofilm formation and CFU counts. Results are expressed as average value ± SE of determinations made in each sample. Differences were considered statistically significant when the P value was < 0.05.

Biofilm Formation of Sub-MIC Norfloxacin
 
The MIC and MBC listed in Table 2 for norfloxacin on the selected field isolates of S. suis strain ranged from 2.5 to 10 μg mL^-1 and 10 to 20 μg mL^-1, respectively. We tested the effects of sub-MIC norfloxacin on biofilm formation isolates in S. suis are shown in Fig 1. Analysis of the biofilm formation of S.suis [Fig,1(a) to (e)] indicates that sub-MIC norfloxacin promote biofilm formation. Norfloxacin demonstrated a significant increase in the growth of biofilm at 1/4 MIC and resulted in did not increase biofilm formation isolated SS_9801, SS_1886 and SS_595 at 1/8 and 1/16 MIC compared to the untreated norfloxacin.
 

 

       
Some studies have strongly shown that the sub-MIC antibiotic can promote biofilm formation (Yuksel et al., 2018; Sato et al., 2018; Rachid et al., 2000). Our research brings clear evidence that sub-MICs of norfloxacins enhance the ability of S. suis to form biofilms. Recent studies have shown that the formation of bacterial biofilms under the influence of sub-MICs of antibiotics relies on signalling pathways such as RND (Resistance-nodulation-cell division) systems of AdeABC, AdeFGH and AdeIJK(Sato et al., 2018), the CpxRA system (Hathroubi et al., 2015), type VI secretion system (Jones et al., 2013) and the AI-2/LuxS system (Ahmed et al., 2009).
 
Bacterial growth and viable cells at different concentrations
 
The effects of sub-MIC on norfloxacin of S. suis were evaluated and the results are listed in Fig 2. Compared with the control groups, bacterial growth in the norfloxacin groups was significantly inhibited at 1/2 MIC. However, When S. suis strains were grown in the presence of 1/4, 1/8, or 1/16 MIC norfloxacin, their growth was similar to that of the control sample. Thus, growth was reduced at 1/2 MIC, but was restored at lower concentrations of S. suis. The number of viable cells at sub-MIC norfloxacin exhibited significant increases in 1/4 and 1/8 MIC of norfloxacin when compared with drug-free culture, as shown in Fig 3. The CFU counts of S. suis treated with 1/2 MIC of norfioxacin exhibited significant increase relative to the control but showed no significant difference at other concentrations exception of isolates SS_9801 and SS_1886 [Fig 3(a) and (b)] differences in CFU counts were observed for the 1/16 MIC about all isolated S.suis.
 

 

 
       
To assess the influence of sub-MICs norfloxacin on bacterial biofilm and virulence gene, we initially investigated their effects on bacterial growth and the number of viable cells in biofilm of S. suis. As shown in Fig 2, the bacterial growth rates of the studied S. suis strains were significantly reduced by the addition of ½ MIC of norfloxacin in the media, but recovered at lower concentrations of antibiotics, which induced the formation of biofilm (Hathroubi et al., 2015; Andersson and Hughes, 2014). Our CFU results shows that the viable cell count at a concentration of ¼ MIC of norfloxacin increased significantly. Bruchmann have already observed that sub-MICs of antibiotics influence bacterial behavior in biofilms, increase biomass and biofilm thickness and induce phenotypical changes in biofilm architecture (Bruchmann et al., 2013). We speculate that increasing the number of viable cells in biofilm exposed to sub-MICs of antibiotics is a bacterial self-defense mechanism, which increase S. suis survival and persistence within the swine.
 
PCR Real-time quantitative PCR
 
We measured the expression of gdh, mrp, gapdh, sly, fbps, ef, cpsin untreated S. suis and in S. suis treated with ¼ MIC of norfloxacin using calculated Ct values (Fig 4). Relative expression levels of treated cultures were compared with untreated cultures and the data were analysed using the 2^-ΔΔCT method. The mRNA expression levels of cps, ef, sly, fbps, gdh and gapdh were significantly decreased compared with the mRNA levels in the absence of antibiotics in S. suis, whereas the mRNA levels of mrp was increased (Fig 4).
 

 
The gene expression of cps, ef, sly, fbps, gdh andgapdh were suppressed exposed to ¼ MIC norfloxacin inthis study. In S. suis, fbps and gapdh gene were previously shown to mediate cell adhesion and play important roles in bacterial infection and invasion (Wang et al., 2014a). The decreased expression of these two genes may result in the increased biofilm formation and adherence ability. Expression levels of the gene cps, ef,sly and gdh were down-regulated, which reduced virulence and pathogenicity of S.suis and more conducive to the formation of persistent bacteria and persistent infection. In our study, ¼ MIC of norfloxacin increased mrp mRNA levels. Muramidase-released protein, or Mrp, induces the bacteria’s ability to form biofilms (Wei et al., 2009). Because the mrp gene contributes to the virulence and biofilm formation (Wang et al., 2014a; Wang et al., 2011a), the upregulation of mrp genes leads to the development of infection of S. suis (Wang et al., 2011b). Taken together, these results indirectly suggest that norfloxacin sub-MICs may increase biofilm formation and persistent infection by increasing the expression of the mrp gene. These results show that exception of mrp virulence gene, gene expression of all other virulence genes decreased at ¼ MIC of norfloxacin, which contribute to the biofilm formation and bacterial persistent infection.

Norfloxacin sub-MICs affects biofilm formation and virulence gene expression in S. suis. These findings suggest that investigating the effect of the administration of antibiotics sub-MICs on bacterial biofilms may lead to develop antibiotic treatments modalities. It may also support emergent views on how to correctly use antibiotics to fight infections.

This work was supported by the National Natural Science Foundation of China (31902309, 31772761).