| Background and Objectives Staphylococcus epidermidis(S. epidermidis), a bacterium with low virulence and weak pathogenicity, is the leading pathogen causing biofilm-associated infections of surgical implants, central venous catheters and artificial pacemaker and so on. This has attracted the close attention of researchers. The serious harms of bacterial biofilms were that the bacteria resistance to antibiotics would be enhanced very greatly after biofilm formation, and it is very difficult to kill these bacteria in the biofilm with antibiotics commonly used and this usually leads to the failures of biological materials treatment. Antibiotic therapy is a very important means of treating bacterial infections. After going into the body, the plasma drug concentration in vivo increases gradually to reach the peak concentration, then gradually reduces. Before using the next dose, the state of plasma drug concentration will continue to meet the minimal plasma drug concentration(MIC), until less than MIC, namely sub-minimal inhibitory concentration(sub-MIC) and it is an inevitable process during antibiotic treatment. Therefore, it is very important to investigate the effects of sub-MIC antimicrobial agents on bacteria biofilm formation for clarifying the biological behavior, features and mechanisms of bacteria in response to sub-MIC antibiotics. It can provide a theoretical reference for the development of new drugs treating biofilm infections. Macrolides is one of the antimicrobial drugs used commonly for treating infections caused by Staphylococcus susceptible to it. Recently we reported that sub-MIC levels of erythromycin(ERY), azithromycin(AZM) and clarithromycin(CLR) markedly enhanced the biofilm formation of 20% macrolide-resistant clinical isolates of S. epidermidis in vitro, however, didn’t find biofilm-inhibition phenomenon and didn’t investigate whether macrolides can affect clinical isolates of ERY-susceptible S. epidermidis on biofilmformation. Recent studies have shown that there is a relationship between the biofilm formation and the antibiotic resistance phenotype, but there aren’t only reports about whether there is a relationship between the biofilm formation affected by sub-MIC antibiotics and the antibiotic resistance features. Polysaccharide intercellular adhesin(PIA) plays an important role in the process of biofilm formation in S. epidermidis and its synthesis is regulated by the production of ica gene cluster. Some investigations suggested that the mechanism of sub-MIC antibiotics enhancing the biofilm formation of S. epidermidis was to increase PIA secretion, but how the upstream of ica gene cluster was affected by sub-MIC antibiotics was still not clear. Some studies have shown that Lux S/AI-2 quorum sensing systems(QSS), protein Sar A and Ica R may regulate the transcription expression of ica gene or PIA generation. So the aim of this study was to use the clinical isolates of S. epidermidis with different ERY-resistant phenotype as research subjects and investigate the ability to biofilm formation in S. epidermidis clinical isolates, the effects of sub-MIC ERY on biofilm formation of clinical isolates of S. epidermidis and the relationships between the effects of sub-MIC ERY on biofilm formation of clinical isolates of S. epidermidis and ERY resistance features of the strains and the upstream regulators of PIA in S. epidermidis clinical isolates.Methods 1. Susceptibility testing The determination of the MICs was performed by standard agar dilution method according to the suggestions of Clinical and Laboratory Standards Institute(CLSI). The standard strain of S. aureus ATCC 29213 was as a quality control strain. 2. Biofilm assay Biofilm formation was tested by growth of isolates in 96-well corning flat bottom plates, essentially as described previously. The criteria of biofilm formation were the OD value of biofilm formation was equal to or greater than twice that of the measured OD values of biofilm formation of Staphylococcus epidermidis ATCC12228. The definition for the biofilm formation to be categorized as enhanced, inhibited, or unaffected with the addition of 1/4 MIC ERY was the following: firstly, to compare whether the biofilm formation of per strain with or without 1/4 MIC ERY were similar(p>0.05) orsignificantly different(p<0.05) with t-test. If the arithmetical mean of OD value with 1/4 MIC ERY was greater than that of OD value without 1/4 MIC ERY and p value <0.05, then the biofilm formation of the strain can be considered as enhancement. If the arithmetical mean of OD value with 1/4 MIC ERY was less than that of OD value without 1/4 MIC ERY and p value <0.05, then the biofilm formation of the strain can be considered as inhibition. If p value >0.05, the biofilm formation of the strain would be considered as no affection, in spite of the arithmetical mean of OD value with 1/4 MIC ERY was less or greater than that of OD value without 1/4 MIC ERY. The screening criterias for subsequent experiments strains include that the ability to biofilm formation is strong, the biofilm formation is enhanced or inhibited obviously by 1/4 MIC ERY and the standard deviation of OD value of the strain is small. 3. Serial passage of induction resistance in vitro This was performed as previously described with modifications. A 24 h culture of each ERY-susceptible S. epidermidis isolate was first transferred with a swab onto Mueller-Hinton agar containing 1/4 MIC of ERY. Surface growth after 24-72 h of incubation was then transferred with a swab onto ERY-free agar for isolation and onto Mueller-Hinton agar containing doubled amount of ERY. These plates were then incubated for 24-72 h. This process was repeated serially until no growth occurred. All experiments for induction of resistance were performed on the same media, incubation temperature and atmosphere. The MIC of ERY was determined for isolates that were grown with the highest concentration of ERY. Isolates were also transferred three times on ERY-free agar, followed by a re-determination of the MIC of ERY to assess the stability of the selected resistance. 4. Dose-effects experiments and time-effects experiments of biofilm formation The ranges of ERY concentration are from one-Two hundred and fifty sixth MIC to three-quarters MIC in dose effects experiments. The time points of biofilm formation tested are 2, 4, 6, 12 and 24 h in time effects experiments. 5. Determination of growth Bacteria culture solution was incubated for 0, 1, 2, 3, 4, 6, 8, 10, 12 and 24 h, and then was added to the 96-well plate and the absorbance measurements were taken with Sunrise Tecan ELIASA at 530 nm. If the absorbance value of the bacteria culture solution exceeded 3.0, we would properly dilute it to assay.6. PCR analysis for macrolide resistant genes The genomic DNA was isolated from the planktonic cells of S. epidermidis isolates using a Wizard® Genomic DNA Purification Kit. Then the genomic DNA was used as a template for PCR using Go Taq Green Master Mix. The primers used were synthesized by Sangon Biotech(Shanghai) Co, Ltd.. The gyr B gene was the positive control. All amplification reactions were prepared in a 25 μl volume. PCR products were analyzed by agarose gel electrophoresis and further sequenced in Sangon Biotech(Shanghai) Co.,Ltd.. 7. Real time RT-PCR for erm C, ica R, sar A, lux S, pfs and ica A genes Total RNA was isolated from intact S. epidermidis cells grown in liquid TSB medium with or without 1/4 MIC ERY for 2 hs using ultrasonication and a rapid bacterial RNA extraction kit. The incubated time was determined according to the results of the time-effect tests of transcriptional expression of erm C gene. Then the c DNA was used as a template for real-time PCR using SYBR® Premix Ex Taq? Ⅱ in an i Cycler Real Time PCR instrument TY0678. All reactions were normalized to the gyr B gene, which encodes the protein DNA gyrase β-subunit. 8. Biofilm formation process and the secretion of PIA were observed by Confocal Laser Scanning Microscope(CLSM). The processes of biofilm formation and PIA secretion were assessed using microscopic observations under a confocal laser scanning microscope(CLSM), as described by previous researches. Biofilm cells that grew on a 1 cm × 1 cm coverslip for 6, 12 and 24 h were fixed with 2.5% glutaraldehyde for 1.5 h and washed with phosphate-buffered saline. Concanavalin A conjugated to fluorescein isothiocyanate green was added at a concentration of 50 μg/ml and incubated at 4°C for 30 min, followed by staining in the dark with propidium iodide at a concentration of 5 μg/ml at 4°C for 15 min. Polysaccharide stained green, and the nucleoplasm of cells stained red. 9. Determination of PIA with sulfuric acid phenol method Bacterial polysaccharide extraction was performed as previously described with modifications. An overnight culture of cells grown in TSB was incubated for 6, 12 and 24 h with shaking. The ultrasonication was performed for 10 min(action 8 sec and stop 8 sec). A 37% formaldehyde solution(300 μl) was added, and the cells were preserved at 4°C for 3 h. Then, a 1 mol/L sodium hydroxide solution was added, and the cells were preserved at 4°Cfor 3 h. Each test was repeated 3 times. The polysaccharide assay was performed using sulphuric acid-phenol method as described previously. 10. The synthesis of AI-2 was measured using bioluminescence. An overnight culture of a S. epidermidis strain grown in TSB was incubated for 0, 2, 4, 5, 6, 8, 10, 12, 16, 20 and 24 h. Cell-free conditioned culture medium(CFM) was prepared by centrifuging cultures at 8,000 g for 15 min, passing the medium through a 0.2 μm pore-size sterile syringe filter and storing at-20°C until tested. CFM from V. harveyi BB170 was prepared in the same manner, except the cultures were grown in DMB at 30°C for 24 h. Bioluminescence assays were performed as described [14] with modifications. V. harveyi BB170 was grown at 30°C for 16 h, and cultures were diluted 1:10,000 in fresh DMB. A 180 μl dilution and 20 μl 10% CFM from bacterial cells were added to white 96-well Thermo Scientific Microtiter plates with 4 replicates. After samples were added for 3 h, bioluminescence was measured using a multifunction ELIASA.Results 1. ERY resistance phenotype and biofilm formation in response to sub-MIC ERY Among the 96 clinical isolates of S. epidermidis, we found 85 resistant to ERY and the resistance rate was 88.5%. The ERY MICs of these resistant isolates ranged from 8 to 256 μg/ml, most of which were highly resistant to ERY, with MIC≥128 μg/ml. We examined the biofilm phenotypes in response to 1/4 MIC of ERY for all of the 96 S. epidermidis clinical strains. Twenty-three of the 85(27%) ERY-resistant isolates exhibited biofilm induction. There were 18 out of these 23 strains in which biofilm induction intensity ranged from 1.11-fold to 2.0-fold, and 5 strains in which biofilm induction intensity were more than 2-fold. At the same time, we found that there were 4 out of the 85 ERY-resistant isolates of S. epidermidis whose biofilm formation was inhibited by sub-MIC ERY. However, the biofilm phenotype modification in response to sub-MIC ERY was observed in none of the 11 ERY-susceptible isolates. There was a statistically significant difference of the incidence of biofilm phenotype modification in response to sub-MIC of ERY between ERY-resistant isolates and ERY-susceptible isolates(27/85(31.8%) vs. 0/11(0.0%), P=0.031). 2. Biofilm formation in response to 1/4 MIC ERY after induction resistance All of the 11 ERY-susceptible isolates became resistant to ERY after serial passage technique of induction resistance and the new MIC of the 11 S. epidermidis isolatesdistributed from 16 to 256 μg/ml. Parallel experiments were performed in which biofilm formation of induced and un-induced cells in the presence and absence of their respective sub-MIC ERY was determined. There were three isolates, in which biofilm formation of induced cells in response to 1/4 MIC of ERY was significantly enhanced comparing with that of induced cells without ERY and that of un-induced cells in the presence and absence of the original sub-MIC ERY. There were four isolates, in which biofilm formation of induced cells in response to 1/4 MIC of ERY was significantly inhibited comparing with that of induced cells without ERY and that of un-induced cells in the presence and absence of the original sub-MIC ERY. 3. The dose-effect tests of biofilm formation in S. epidermidis strains in response to sub-MIC ERY incutated for 24 h The biofilm formation of S. epidermidis ATCC35984 TM wasn’t affected by 1/256-3/4 MIC ERY(p>0.05). The biofilm formation of S. epidermidis SH04 was inhibited by 1/4, 1/2 and 3/4 MIC ERY(p<0.05) and the biofilm formation in response to 1/4, 1/2 and 3/4 MIC ERY was similar and the inhibition level was 13.6%, 17.1% and 18.5%, respectively(p>0.05). The biofilm formation of S. epidermidis SH10 was enhanced by 1/128-3/4 MIC ERY(p<0.05) and the modification was very obvious in response to 1/4 MIC ERY. 4. The time-effect tests of biofilm formation in S. epidermidis strains in response to 1/4 MIC ERY We examined biofilm formation of S. epidermidis strains at 6, 12 and 24 h with or without 1/4 MIC ERY. Biofilms of S. epidermidis ATCC35984 at 6, 12 and 24 h were not affected by 1/4 MIC ERY compared with the control group at the same time points(p>0.05). Biofilms of the clinical isolate S. epidermidis SH04 at 6, 12 and 24 h were significantly inhibited 19.5%, 10.7% and 13.6%, respectively, compared with the control group at the same time points(p<0.01). The biofilm of the clinical strain S. epidermidis SH10 was obviously inhibited compared with the control group at 6 h(p<0.01). However, biofilms of S. epidermidis SH10 at 12 and 24 h were markedly enhanced 49.4% and 99.7% compared with the control group, respectively(p<0.01). These results suggested that the effects of 1/4 MIC ERY on biofilm formation were different for different isolates of S. epidermidis. 5. The effects of 1/4 MIC ERY on the growth of S. epidermidis strainsThe growth of S. epidermidis ATCC35984 TM and S. epidermidis clinical isolate SH04 in response to 1/4 MIC ERY was similar to that of their respective control group(p>0.05). The growth of S. epidermidis SH10 at the time point of 2, 3, 6, 8 and 10 h was obviously inhibited by 1/4 MIC ERY(p<0.05), however, the growth of it at the time point of 0, 12 and 24 h in response to 1/4 MIC ERY was similar to its control group(p>0.05). 6. The prevalence of erm A, erm B and erm C genes in 96 S. epidermidis isolates tested The positive PCR amplification of erm C gene was obtained for 91 out of the 96(94.8%) S. epidermidis isolates tested, and the positive amplification for both erm A and erm B gene was 6 out of 96(6.2%). There was no correlationship between the distribution of erm A, erm B and erm C genes and the biofilm formation of 96 S. epidermidis isolates in response to 1/4 MIC ERY(p>0.05). The distribution of erm C gene in biofilm-inhibited,-enhanced and unaffected strains was 100%, 95.7% and 94.2%, respectively. We determined the base sequence of the amplification product of erm C gene, and compared it with the homologous sequence in Pubmed NCBI gene bank, and found that the homology was 99%. 7. Correlationship between the transcription of erm C gene and the rate of OD value of biofilm formation in response to 1/4 MIC ERY We investigated the transcription expression of erm C gene incubated with sub-MIC ERY for 2 hours in 25 ERY-resistant isolates(including 7 induced resistant isolates) with different biofilm formation phenotypes(9 enhanced isolates, 8 inhibited ones and 8 un-affected ones) and found that there was no correlation between the transcription expression of erm C gene and the ratio of biofilm OD value of ERY-treated and-untreated group(n=25,r=-0.07,R2=0.0044). However, with statistic stratification analysis, a linear and positive correlation was identified between the transcription of erm C gene and the rate of OD value of biofilm formation in response to 1/4 MIC ERY in the biofilm-enhanced strains(n=9, r=0.70, R2=0.4992); a linear and negative correlation in biofilm-inhibited strains(n=8, r=-0.61, R2=0.3686) and a weakly positive and linear correlation in biofilm-unaffected strains(n=8, r=0.21, R2=0.0512). 8. The affects of 1/4 MIC ERY on biofilm formation and PIA tested by doubleFluorescence staining Biofilm formation and the distribution of polysaccharide matrix of ERY group in S. epidermidis ATCC35984 TM were always similar to those in the control group at 6, 12 and 24h. Biofilm formation and the distribution of polysaccharide matrix of ERY group in S. epidermids SH04 were always thinner than those of control group at 6, 12 and 24 h. Biofilm formation and the distribution of polysaccharide matrix of ERY group in S. epidermids SH10 at 12 and 24 h were always thicker than thoseof control group, however, those of ERY group in it were thinner than those of control group at 6 h. 9. The affects of 1/4 MIC ERY on PIA tested by sulphuric acid-phenol method The PIA production of S. epidermidis strains was tested, but we found that the observed differences in the PIA production of S. epidermidis ATCC35984 at 6, 12 and 24 h between the ERY group and the control group were not statistically significant(p>0.05). The PIA production of S. epidermidis SH04 at 6, 12 and 24 h was significantly inhibited by 1/4 MIC ERY(p<0.01). However, the PIA production of S. epidermidis SH10 at 12 and 24 h was significantly enhanced by 1/4 MIC ERY(p<0.01), but at 6 h, it was obviously inhibited by 1/4 MIC ERY(p<0.01). These changes in PIA production were consistent with biofilm formation in response to ERY. 10. Affects of 1/4 MIC ERY on the transcription expression of ica A、pfs、lux S、ica R and sar A genes The transcription expression of ica A gene in S. epidermidis ATCC35984 TM at the time of 2, 4, 6, 12 and 24 h wasn’t affected by 1/4 MIC ERY(p>0.05). The transcription expression of ica A gene in S. epidermidis SH04 at the time of 4, 6, 12, 24 h was inhibited by 1/4 MIC ERY(p<0.05). The transcription expression of ica A gene in S. epidermidis SH10 at the time of 6, 12, 24 h was enhanced by 1/4 MIC ERY(p<0.05); however that of ica A gene in it at 2 h was inhibited by 1/4 MIC ERY(p<0.05), and that of ica A gene in it at 4 h wasn’t affect by 1/4 MIC ERY(p>0.05). Incubated with 1/4 MIC ERY for 2, 4, 6, 12 and 24 h, the transcription expression of pfs gene in S. epidermidis SH04 was significantly enhanced(p<0.05); that in S. epidermidis SH10 was obviously inhibited(p<0.05); but the transcription expression of pfs gene in S. epidermidis ATCC35984 TM wasn’t affected(p>0.05). 1/4 MIC ERY didn’t affect the transcription expression of lux S and ica R genes in S. epidermidis ATCC35984 TM, SH04 and SH10 at the time of 2, 4, 6, 12 and 24 h, and it didn’t affect the transcription expression of sar A gene in S. epidermidis ATCC35984 TM, SH04 and SH10 at the time of 4, 6, and 12 h. However, it inhibited the transcription expression ofsar A gene in S. epidermidis SH10 at the time of 2 h and the transcription expression of sar A gene in all of the three strains at the time of 24 h. 11. Affects of 1/4 MIC ERY on signal molecule AI-2 We examined the bioluminescence activity of S. epidermidis strains incubated with 1/4 MIC ERY within 24 h using the V. harveyi AI-2 reporter assay. The V. harveyi strain BB170 only exhibits bioluminescence in response to AI-2 or AI-2-like molecules. The growing of a culture of strain BB170 overnight and diluting it 1:10,000 yielded low cell density and reduced the level of endogenous AI-2 below the threshold required for luminescence. In this experimental system, the addition of exogenous AI-2 molecules from other bacteria restored the bioluminescence phenotype of the reporter strain BB 170. Sterile TSB added to the strain BB170 dilution served as the luminous background(Luminous intensity = Measured value – background value). The luminous intensity of AI-2 in S. epidermidis SH04 at the time of 2, 4, 6, 8 and 10 h was markedly enhanced by 1/4 MIC ERY(p<0.05). The luminous intensity of AI-2 in S. epidermidis SH10 at the time of 2, 4, 6 and 8 h was markedly inhibited by 1/4 MIC ERY(p<0.05); but the luminous intensity of AI-2 in S. epidermidis ATCC35984 TM at the time of 2, 4, 6, 8, 10, 12 and 24 h wasn’t affected by 1/4 MIC ERY(p>0.05).Conclusions 1. The biofilm formation of clinical isolates of ERY-resistant S. epidermidis can be enhanced or inhibited by 1/4 MIC ERY. 2. There was a correlation between the phenotype of ERY resistance and the affection of 1/4 MIC ERY on biofilm formation in S. epidermidis clinical isolates. 3. The effect of 1/4 MIC ERY on biofilm formation in S. epidermidis clinical isolates was probably related with the transcription expression of erm C gene. 4. The effect on the secretion of PIA and the upstream regulator of lux S/AI-2 QSS is proberbly one of the important mechanisms, by whic 1/4 MIC ERY affectted the biofilm formation in clinical isolates of S. epidermidis. |
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