关键词: 金黄色葡萄球菌, 生物被膜, 耐药性, 分子分型, 毒力基因

Abstract: 【Objective】 To investigate the epidemiological characteristics of staphylococcus aureus in the biofilm producing strains and to explore the correlation between biofilm forming ability and molecular typing, so as to provide theoretical basis for the treatment of Staphylococcus aureus infection. 【Method】 The biofilm producing ability of all strains of Staphylococcus aureus was determined by crystal violet semi-quantitative method.The minimum inhibitory concentrations of 22 common antibiotics were determined by the membrane producing strains. Molecular typing was conducted by three common typing methods of Staphylococcus aureus (spa typing, MLST typing and PFGE typing), and the correlation between membrane production capacity and molecular typing was analyzed. Finally, whole genome sequencing technology was used to analyze the antibiotics resistance gene and virulence genes in the biofilm producing strains. 【Result】 The semi-quantitative results of crystalline violet showed that a total of 23 strains (23.47%) of 98 S. aureus strains were able to produce biofilm, including 22 strains (25.29%, 22/87) from cow milk source, 14 strains (60.87%, 14/22) from Zhejiang dairy farms, 8 strains (39.13%, 8/22) from Fujian dairy farms, and 1 strain from pig source ( 9.10%, 1/11) from Guangdong slaughterhouse, indicating that the film-producing potential of Staphylococcus aureus from cow's milk source was higher than that of pig source of which 22 strains (95.65%) were from cow's milk source and 1 strain (4.35%) was from pig source; the film-producing ability was classified into strong, medium and weak, and among the 23 film-producing strains, 2 strains (8.70%, 2/23) were strong film-producing strains and 9 strains (39.13%, 9/23) were medium film-producing strains. (39.13%, 9/23), 12 (52.17%, 12/23) of weak film-producing strains were strong (2/23=2.70%), medium (9/23=39.13%), and weak (12/23=52.17%), respectively; the results of drug sensitivity test showed that the film-producing strains of cow milk origin were sensitive to all antibacterial drugs tested, while the film-producing strains of pig origin were sensitive to all antibacterial drugs including penicillin, clindamycin, and tilmicin. The results of the drug sensitivity test showed that the bovine milk-derived membrane-producing strains were sensitive to all the tested antibacterial drugs, while the pig-derived membrane-producing strains showed resistance to 13 antibacterial drugs, including penicillin, amoxicillin, ceftiofur, cefoxitin, enrofloxacin, ciprofloxacin, clindamycin, doxycycline, erythromycin, flupenthixol, cotrimoxazole, tiamulin, and tilmicosin. The spa typing results showed that 98 strains of S. aureus obtained 8 spa types, and 23 strains of film-producing S. aureus accounted for 3 of them: 1 strain t2922 from porcine origin in Guangdong, 14 strains t2119 from Zhejiang cow milk source, and 8 strains t189 from Fujian cow milk source; MLST typing results showed that spa typing and MLST typing results showed that all strains were classified into 8 spa types and 9 ST types, and 98 strains were classified into 9 ST types, of which 6 ST types did not have the ability to produce biofilm, namely ST398, ST522, ST705, ST ST1651, ST479 and ST151, and only 3 strains of ST type had biofilm production ability, namely ST9, ST7 and ST188; we found that the molecular types of strong film-producing strains were mainly ST7-t2119, medium film-producing strains were mainly ST7-t2119 and ST188-t189, and weak film-producing strains were ST9-t2922, ST7-t2119 and ST188-t189. The strains with membrane producing ability were mainly ST7-t2119, medium membrane producing strains were mainly ST7-t2119 and ST188-t189, weak membrane producing strains were ST9-t2922, ST7-t2119 and ST188-t189. ST7-t2119 and ST188-t189, the ST type of weak film-producing strains can be well distinguished from the medium and strong film-producing strains, and only the specific ST type of S. aureus has the ability to produce biofilm. 23 film-producing strains PFGE typing all successful PFGE typing results show that the results show that each strain of film-producing bacteria in Guangdong, Fujian and Zhejiang provinces are divided into a total of three The results show that there are geographical distribution characteristics of PFGE types, and isolates from the same region may have clonal transmission, and strains in the province are clonal to each other, but there are significant differences in biofilm production ability between clones; whole genome sequencing results show that the drug resistance genes and virulence genes in the film-producing strains are diverse according to the molecular type. 【Conclusion】 Staphylococcus aureus from different sources have different potential to produce biofilm and all carry different film-producing genes; the film-producing potential of S. aureus from bovine milk source is much higher than that of porcine source and all carry different film-producing genes; whether strains can produce film or not may be strongly correlated with ST type, and specific ST types such as ST9, ST7 and ST188 are more likely to produce biofilm; however, at the same time, strains with the same molecular type have different abilities to produce biofilm.

Key words: Staphylococcus aureus, Biofilm, Antibiotics resistance gene, Molecular typing, Virulence genes