The Study Of Mechanisms And Effects Of Sub-MIC Macrolides On Biofilms Formation Of Clinical Isolate Of Staphylococcus Epidermidis

Bacterial biofilm is a special structure of bacterial community enclosed in a self-produced hydrous and polymeric matrix and adherent to material surface. Biofilms may form on a wide variety of surfaces, including living tissues and indwelling medical devices. Bacterial biofilm has property of dissemination and bacteria inside biofilms usually show high resistance to antibacterial agents, so bacterial biofilm-associated infctions have chronic process invariably. Staphyloccocus epidermidis is the most common coagulase-negative staphylococci (CNS) which often form bacterial biofilms on implanted medical devices, and S epidermidis biofilm is the primary cause of medical device-related infections. The prevention and treatment of S epidermidis biofilm is a difficult but significant topic in researches on clinical infections nowadays.Some researches indicated that antibacterial agents may interfere biofilms formation at subinhibitory concentrations (sub-MICs), although it s difficult to clear bacterial biofilms. That gives the hope to prevent biofilms from forming and disseminating in clinic. Sub-MIC antibacterial agents have drawn attention to its potential clinical use. A series of researches could be found about sub-MIC macrolides for its inhibition effect on biofilms formation of Pseudomonas aeruginosa, Mycobacterium aviu, and so on. Antimicrobial activity of macrolides is broad in spectrum and widely used in infections caused by beta-hemolytic streptococci, pneumococci, staphylococci and enterococci, but little is known about what effects of sub-MIC macrolides on these pathogens biofilms formation. In order to obtain a further insight on mechanisms of S epidermidis biofilms formation and to find new methods to prevent biofilm-associated infections, we studied the mechanisms and effects of sub-MIC macrolides on clinical isolate of S epidermidis biofilms formation.In this study we first investigated 105 clinical isolate erythromycin-resistant S epidermidis strains with the 1/4 MIC EM by microtiter-plate assay, and found that 21 strains biofilms were induced. Subsequent research was performed to investigate the effect of EM and other two macrolides, AZM and CAM. IcaA genetype was surveyed by PCR to reveal the relationship between biofilm-induced effect and icaA presence. A icaA negative S epidermidis SW036 and a icaA positive S epidermidis SW029 were employed as representative strains and the time-effect and dose-effect of sub-MIC EM were studied. Then growth rate was investigated upon antibiotic exposure, and initrial adherence was estimated by count bacteria under CLSM. Biofilm formation process was observed by CLSM and SEM till the time of 48h, and biofilm associated genes expression of icaA, sigB, atlE and sarA were investigated by realtime RT-PCR. DNA array for global genes expression was used to reveal the passible mechanisms and look for the targets of sub-MIC macrolides. At the last part of this paper, we attempted to establish a new model of biofilm infection in vivo.The main results and conclusions we obtained in the study as follows:1. EM at concentrations of 1/4MIC induced 20% clinical isolate of S epidermidis biofilm formations. It is remarkable that the similar effects were found on these 20% strains by other two macrolide, AZM and CAM. The enhanced extent of AZM and CAM showed dependability with EM (R2 is 0.91 of both). It is presumed that macrolides have a biofilm-induced propertise on some certain S epidermidis strains.2. Investigation on icaA locus showed stronger biofilms formation for icaA-presenced strains, but it is indicated that the inducing effect is not correlated to icaA presence. And investigation of sub-MIC EM on Staphylococcus aureus showed that the biofilm-induced effect may be specified for S epidermidis.3. 1/4, 1/16 and 1/64 MIC AZM, CAM, EM all showed significant enhance on S epidermidis SW029 biofilm formation with no dose-dependence effect observed. Sub-MICs showed obvious dose-dependence effect on S epidermidis SW036; the significant inducing effect existed till the time 48h.4. EM(8mg/L) showed inhibition of growth of S epidermidis SW029 in 24h. There was no significant difference of initial adherence between EM-treated and control. It indicates that biofilm-inducing effect is not associated with the change of initial adherence.5. Processes of biofilm formation with and without EM (8mg/L, 1/16MIC) of S epidermidis SW029 were respectively observed by CLSM and SEM. Polysaccharide (stained green) secretion was observed apparently enhanced at the time point of 12h for EM-treated, while a little of polysaccharide were observed in the control group. At the time 48 hr, colony cultured with EM showed typical mass of biofilm in which bacteria were enclosed, and control didn t show similar structure.6. EM (8mg/L, 1/16MIC) treated for 6h, icaA, sigB, atlE and sarA expression was 1.22.0 6.2 1.7-fold respectively compared with that of the control for S epidermidis SW029, and it reached to 2.8, 0.6, 2.4, 6.9-fold in 24h. It is presumed that the regulation of atlE may contribute to biofim-formed regulation and genes of icaA and sarA showed secondary effects according to the time points and inducing extent.7. Results of DNA array for genes expression showed EM (8mg/L, 1/16MIC) impose a wide range of gene expression changes. There are 332 genes down-regulated and 395 genes up-regulated for at least 2-fold, and they account to about one third in expressed genes. Biofilm-associated genes which changed for at least 2-fold covered bacterial growth, responding to oxidative stress, adherence and bacterial envelop biosynthesis, and atlE had a high up-regulation effect among them. It is presumed that regulation of atlE may be one of primary mechanisms of biofilm-induced regulation. Similar to the antimicrobial mechanism of macrolides, we presume that the direct effect of sub-MIC EM was to inhibit proteins synthesis because most of ribosomal protein genes were up-regulated. Considering that macrolides showed significant inducing effect at a very low concentration of 1/64 MIC, there may exist some mechanisms for sub-MIC macrolides exhibit their regulated effect through interacting with ribosome, and some special mechanisms may exist such as binding site and genes mutation of ribosome. Further researches on ribosomal protein and genes may be useful.8. The novel model of biofilm infection established by Babl/c mice has advantages of minimal invasion, direct-viewing, easy to be handled, and so on. Technological verification research indicated it could reflect differences of biofilm formation for strains with different abilities on biofilms formation to some degree. But the deficiency of specificity in the detected method is the main problem, and improvements are needed.In summary, there is a tendency of inducing (enhancing) biofilms formation by macrolides as observed in our study. Because sub-MIC is a related concept, nowadays the emerging of high resistant staphylococci strains make it a great chance that patients who have (or potential have) S epidermidis caused by biofilm infections can receive macrolides at sub-MICs. However, according to our research, it should be noted that there might be some risks for the use of macrolides on them. Sub-MIC marolides may inhibit bacterial protein synthesis and through some unknown pathway to regulate biofilm-associated genes and atlE may play a role in it.Antibiotics have been searched on purpose as inhibitors (either bactericidal or bacteriostatic) of bacterial growth, but antibiotics come from molecules which are synthesized and released by bacteria, and it could be sensed by bacteria and regulate its life behaviors, including biofilm formation. Little is known about what effects and mechanisms of sub-MICs on pathogens biofilms formation. Much more relative researches in laboratories may contribute to the reasonable use of antimicrobials in clinics, and the researches of biofilm-regulated mechanisms of sub-MICs may contribute to prevention and control biofilm-associated infections.