Effect Of Nitric Oxide On Biofilm Formation Of Staphylococcus. Aureus

Nitric Oxide (NO) is an important active molecule in biology. It is described as a multifunctional second messenger since it plays various functions including cell differentiation and apoptosis, the relaxation of blood vessels, neurotransmission, defense during immune response, seed germination, hypocotyls elongation, leaf expansion and root growth. NO has attracted a great deal of attention from researchers in animals and high plants, however, the research about NO in bacteria especially its role in the biofilm is rarely reported. Biofilms are multicellular, matrix-enclosed microbial communities that are attached to an environmental surface throughout the biological world. Cells within a biofilm have a number of advantages over their planktonic counterparts, including protection against the immune system and enhanced resistance to antimicrobial agents and other stresses. Staphylococcus. aureus is one of the most important causes of nosocomial infections and the pathogenic mechanism of S. aureus infections is closely related with the formation of biofilm. In this paper we investigated the effect of NO on biofilm formation of S. aureus and laid the foundation for the research of the function and mechanism of NO in biofilm.First, the mRNA expression level of nitric oxide synthase (NOS) gene nos in biofilm and planktobacteria were analyzed by quantitative real-time PCR assay. The results showed that the nos gene was obviously up-regulated in biofilm, indicating that NOS may paly a important role in the biofilm of S. aureus. In order to detect the effect of NO on the biofilm of S. aureus, the NO donor SNP and NO scavenger PTIO were used in the biofilm forming assay. The results showed that SNP could enhance the growth of biofilm and PTIO could counteract the enhancement of biofilm by SNP.In order to further investigate the effect of NO on biofilm, we constructed the S. aureus nos deletion mutant via homologous recombination. In the process of mutant construction, we found it difficult to transfer exogenous DNA into S. aureus cells during transformation because of the thick compact cell wall of S. aureus and the large shuttle vector we used. So we developed a simple and efficient method for electroporation of S. aureus by pretreating cells with lysozyme, lysostaphin or Triton X-100 before electroporation and the transformation efficiency was obviously improved and reached 1.7×10~3 cfu/μg DNA to the most extent. The homologous recombination vector pMADΔnos was constructed by the ligating the upstream and downstream sequences of nos acquired by PCR, and the spectinomycin resistance gene into shuttle vector pMAD. S. aureus strain RN4220, a restriction deficient strain, was used as the initial recipient for transformation and the modified vector was then electroporated into S. aureus RN6390. The nos deletion mutants were selected by temperature sensitivity and antibiotic resistance. The biofilm formation assay showed that biofilm formation ability of the mutant is obviously weakened by 30% campared with the wild type whereas the minimum biofilm elimination concentration (MBEC) test showed that the antibiotic sensitivity of the mutant is enhanced to twice as against the wild type.In conclusion, a simple and efficiency electroporation method has been established to transfer S. aureus strains and the nos deletion mutant was constructed via homologous recombination. The results showed that NO promoted the growth of biofilm and the deletion of nos inhibited the growth of biofilm and improved the antibiotic sensitivity of biofilm. This study revealed the effect of NO on biofilm of S. aureus and laid the foundation for investigating the function and mechanism of NO in biofilm. Furthermore, this study aslo has important value in the investigation of the regulation mechanism of biofilm and the control of biofilm related infection.