A Study Of Drug Resistance About Staphylococcus Epidermidis And Its Molecular Mechanism Of Polysaccharide Intercellular Adhesion (PIA) Synthesis

Staphylococcus epidermidis (SE) is part of the normal mucosa and skin microflora, usually showing low pathogenicity. In recent years, however, S. epidermidis, being as an opportunistic organism, become an important etiologic agent of infections associated with venous catheters, other indwelling medical devices and abuse of broad-spectrum antibiotics.After infection in the host, S. epidermidis is liable to form biofilm in the involved sites, especially on the surface of high-molecular biomaterials, such as indwelling catheters, implanted materials and etc. The bacteria in the biofilm can not only be resistant to antibiotics, but also resistant to immunologic phagocytosis of the host. Therefore, they can produce chronic infection and even septicemia, causing critical results. Just because of that, in nowadays, the mechanism of biofilm formation becomes an actively ongoing study. It is of great significance for preventing such infections as well as looking for sensitive antibiotics against such infections. At the same time, the study about mechanism of biofilm formation may actually help to inhibit biofilm fonnation and then ultimately eliminate slime-producing bacteria.Biofilm fonnation is a very complex and multistep process. Usually it is divided into two phages: adherence phage and collection phage. In adherence phage, S. epidermidis specifically combine to the surface of biomaterials owing to some physical factors and various adherence factors. After the adherence, it is collection phage. Firstly there is only one cellular layer, and then, mediated by polysaccharide intercellular adhesion (PIA) and other factors, bacteria adhere to each other, followed by differentiation and proliferation, and hence produce multilayer cellular mass as well as abundant slime. At last, on the surface of biomaterials, a thick slime layer comes into being, in which S. epidermidis studded, forming biofilm that can be observed by electron microscopy.PIA is an extremely important substance during the process of biofilm formation. It is investigated that S. epidermidis lack of PIA shows normal ability of adherence to the biomaterials in early stage, and remarked decrease of adherence ability among bacteria in late stage, causing inability to form biofilm. It suggests that PIA is indispensable in mediation of intercellular adherence.ica gene is the basis of PIA synthesis. Studies indicate that ica gene comprises operon icaA, icaD, icaB, icaC, which are necessary structural genes for PIA production. Among those, co-expression of icaA and icaD is the most important link to promote PIA production. icaR gene locates in the upper stream of ica operon, being able to inhibit icaA/icaD. Therefore it can decrease PIA production, and consequently inhibit biofilm formation. Since it is poorly studied about the molecular mechanism of PIA synthesis in S. epidermidis, the aim of this study was to primarily investigate the molecular mechanism of PIA synthesis on the basis of analyzing the status of drug resistance of S. epidermidis, and to elaborate the relationship between PIA synthesis and expression of icaA, icaD, and icaR.Objective: to study the situation of drug resistance of 61 S. epidermidis stains collected from various specimens of different patients in all clinical departments of our hospital in the recent two years, and to analyze the tendency of its drug resistance. To detect the status of PIA syntheses and Amplifying icaA, icaD, icaR genes in various strains and search the relationship between PIA production and icaA, icaD and icaR genes.Methods: 1. After resuscitation and culture of 61 SE strains, drug sensitivity test for common antibiotics was performed.2. In order to analyze the tendency of drug resistance, statistics of drug resistance was made on the basis of the results of drug sensitivity test.3. The situation of PIA synthesis of various strains was detected in Congo red agar medium.4. Amplifying icaA, icaD, icaR genes, and then investigating the relationship between them and PIA synthesis. 5. Detecting mRNA expression of icaA and icaR genes, and investigating the relationship between icaR and PIA synthesis.Results: 1. All the 61 SE strains were completely resuscitated and cultured. Among them, 41 strains were resistant to Oxacillin (67.21%), 20 strains sensitive to Oxacillin. The strains presenting drug resistance to over 3 kinds of antibiotics accounted for 90.16% (including Oxacillin) in all the specimens (55/61 )₅ with multi-drug-resistance rate up to 100% (41/41) in MRSE and 70.00% (14/20) in MSSE. There was significant difference between the two groups in multi-drug-resistance rate (x² —10.47, P<0.005). The 61 SE strains showed total drug resistance to all the 13 antibiotics at 40.86% (324/793), with total drug resistance at 47.28 (252/533) for MRSE and 27.69% (72/260) for MSSE. It showed extremely significant difference in total drug resistance between the two groups (x² —27.75, P<0.005).2. The drug sensitive test in vitro showed that SE preserved a relatively lower drug resistance to cefazolin, cefuroxime, gentamicin and ampicillin-sulbactam (<30%), and a higher drug resistance to SMZ-TMP, penicillin, erythromycin, clindamycin and levofloxacin (>50%). In this study, we did not find any SE strain resistant to vancomycin.3. The results of detecting PIA in the 61 SE strains showed that 22 strains were PIA positive, accounting for 36.07% (22/61). Among them, there were 19 strains of MRSE, accounting for 46.34% (19/41), while only 3 strains of MSSE, accounting for 15.00% (3/20). There was significant difference between the two groups (X² =4.448, P<0.05).4. After extraction of DNA and amplification of ica genes, 25 SE strains turned out icaA positive, among which PIA-producing strains accounted for 76.00% (19/25), while there were only 8.33% (3/36) of icaA-negative strains being able to produce PIA. The two groups had extremely significant difference (x² =26.44, P<0.005). The results of icaD detection was completely consistent with that of icaA.5. According to the investigation of mRNA expression of icaA and icaR genes in 61 SE strains, we found that icaR gene no longer expressed mRNA in 20 PIA-positive strains, but icaA remained normal expression of mRNA; meanwhile, in PIA-negative strains, icaA gene did not express at the level of mRNA, while icaR gene remained normal.Conclusions: 1. S. epidermidis showed a serious situation about drug resistance, with a higher multi-drug-resistance rate and total drug resistance rate in MRSE,2. Co-expression of icaA/icaD genes is the key link of PIA synthesis.3. The expression of icaR gene may inhibit the synthesis of PIA.