硫酸铜溶液诱导对耐甲氧西林金黄色葡萄球菌抗菌药物抗性效应的初步研究

旨在探究硫酸铜溶液连续诱导对耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus，MRSA）抗菌药物抗性及相关基因表达的影响。采用微量肉汤稀释法测定经硫酸铜诱导前后MRSA菌株（MR-YB1224、MR-YS3和MR-P318）对硫酸铜溶液及β-内酰胺类（氨苄西林、苯唑西林和头孢西丁）、氟喹诺酮类（氧氟沙星）、大环内酯类（罗红霉素）和氨基糖苷类（链霉素）等抗菌药物的最小抑菌浓度（minimum inhibitory concentration，MIC）；采用荧光定量PCR方法测定硫酸铜诱导对菌株抗重金属基因（copA和arsB）和耐药基因（mecA，norA，ermB和aac6'/aph2″）表达的影响。连续诱导7 d后，MRSA菌株对硫酸铜溶液的MIC值均增加；特别是对β-内酰胺类抗菌药物的MIC值增加显著（P<0.05）；MRSA菌株对6种抗菌药物MIC值变化大小依次为苯唑西林 > 氨苄西林 > 头孢西丁 > 氧氟沙星 > 链霉素 > 罗红霉素。另外，连续诱导后，MRSA菌株的抗重金属基因和相关耐药基因的表达均显著上调（P<0.05）。本试验结果表明，硫酸铜溶液诱导对MRSA菌株抗菌药物抗性效应具有较强的协同效应。     

宁夏地区牛源金黄色葡萄球菌β-内酰胺酶的分析及作用方式的研究

旨在深入了解宁夏地区牛源金黄色葡萄球菌(Staphylococcus aureus, SA)所产生β-内酰胺酶的进化及结构特征并揭示其作用方式。分别以药敏试验和PCR方法检测5种β-内酰胺类药物耐药情况和耐药基因;通过生物信息学手段对检出的β-内酰胺酶BlaZ作进化分析及结构功能预测;利用分子对接和动力学模拟,分析BlaZ的作用方式。结果表明,青霉素类药物的耐药率明显高于头孢菌素;blaZ基因检出率为82.37%,并检出SA中较少报道的blaZTEM-1基因,检出率为26.05%;进化分析可知,BlaZ属于A型β-内酰胺酶,包含63个重要且承受进化压力的踪迹残基;分子对接发现,BlaZ易与青霉素类药物及阿维巴坦等抑制剂结合并形成稳定复合物;动力学模拟显示,BlaZ的结构柔性小于同型酶TEM-1和TEM-52,其中Ω-loop区域柔性存在差异(P<0.05)且能影响与药物的作用,即该区域柔性与结合活性呈正相关;BlaZ分别与氨苄西林和头孢噻呋结合时结构稳定性差异显著(P<0.05)。宁夏地区牛源SA产生的β-内酰胺酶BlaZ属A型,并从菌株中检出blaZTEM-1基因;BlaZ对青霉素类药物的结合活性高于头孢菌素且易于结合阿维巴坦等抑制剂,其中Ω-loop区域的结构柔性是BlaZ与药物结合活性的重要影响因素。     

Study on Infection of Staphylococcus aureus Small Colony Variants on Mammary Epithelial Cells in Vitro

In order to study the situation of S. aureus small colony variants (SASCVs) and the normal strains hitting the mammary epithelial cells of primary culture, we observed and compared the degree of damage to the cellular structure and the effects on mammary epithelial cells. This test was carried out with a strain of SASCV which was separated by Yunnan Agricultural University Animal Medical Experimental Teaching Center Laboratory via infecting mice mammary epithelial cells in vitro. We put the SASCVs and quality control strains ATCC 25923 respectively into the mammary epithelial cells cultivated in vitro in the laboratory, and observed the morphological change of mammary epithelial cell apoptosis induced by quality control strains ATCC 25923 and SASCVs using ordinary light microscope and scanning electron microscope; At the same time, we also observed the mammary gland epithelial cells apoptosis induced by quality control strains ATCC 25923 and SASCVs using flow cytometry (Annexin V-FITC/PI double staining). The results showed that 3 h after quality control strains and small colony mutant strains infecting the mammary epithelial cells, the mammary epithelial cells could be induced to apoptosis. It showed some typical characteristics of apoptosis such as the nucleus shrivelled, chromatin marginalized, mammary gland epithelial cells could be induced apoptosis, expression was shrinking nucleus, chromatin marginalized, the increase of cell intracytoplasmic vacuoles and so on. After testing using the Annexin V-FITC/PI double staining, we found that compared with the control group, the apoptosis rate of infection group was obviously higher, and the apoptosis rate of the positive control group was obviously higher than that of the experimental group. The results showed that in the process of infecting cells, the SASCVs reached a certain number and then stopped; The apoptosis rate of SASCVs was obviously lower than the normal of S.aureus. Therefore, SASCVs could also induce mammary epithelial cells apoptosis. It had the typical morphological characteristics of apoptosis. It provided experimental basis for the prevention and control of SASCVs chronic mastitis cows.     

小花棘豆生物碱对表皮葡萄球菌的抗菌作用及生物膜形成的影响

探讨小花棘豆生物碱对表皮葡萄球菌的抗菌作用及其对细菌生物膜的抑制作用。以表皮葡萄球菌为研究对象,通过微量肉汤稀释法测定最小抑菌浓度(MIC)和最小杀菌浓度(MBC),微量板半定量法测定其对表皮葡萄球菌生物膜形成的影响,以去离子水和氯化钠检测小花棘豆生物碱对细菌细胞壁的渗透能力影响。结果表明,小花棘豆生物碱对表皮葡萄球菌的抑菌作用较强,其MIC和MBC分别为1.6mg/mL和3.2mg/mL,1~1/4MIC的小花棘豆生物碱可明显抑制表皮葡萄球菌生物膜的形成,而且1~1/8MIC的小花棘豆生物碱对表皮葡萄球菌细胞壁的渗透能力有一定的影响。说明小花棘豆生物碱对表皮葡萄球菌具有抗菌作用,对其生物膜形成具有明显的抑制作用。     

恩诺沙星在健康及实验性感染乳房炎奶山羊间的比较药动学

 每毫升 10 6菌落形成单位的金黄色葡萄球菌悬液 1ml单侧乳房内接种 ,人工诱发奶山羊乳房炎。 15只泌乳期健康奶山羊 ,按给药途径随机分为 3组 ,采用交叉试验设计 ,分别进行静注、肌注和乳房灌注恩诺沙星 (2 .5mg·kg-1)对健康和人工感染乳房炎的奶山羊进行比较药动学研究。采用HPLC法测定血中恩诺沙星及其代谢产物环丙沙星的浓度 ,用统计矩原理处理药物浓度 时间数据。结果表明 ,实验性乳房炎可以使静注给药后恩诺沙星在奶山羊的消除缓慢 (P <0 .0 5 ) ,消除半衰期 (t1/ 2 β)由 1.32± 0 .2 6h增至 1.77± 0 .12h ;使肌注给药恩诺沙星的吸收、生物转化和代谢均发生显著变化 (P <0 .0 1) ,药 时曲线下面积 (AUC)由 3.0 4mg·h-1·L-1增至 4 .36mg·h-1·L-1,代谢率 (f)由 4 4 .0 6 %降至 2 4 .0 2 % ,环丙沙星的AUC由 1.2 9降至 1.0 1mg·h-1·L-1;使乳房灌注给药恩诺沙星的平均滞留时间 (MRT)由 1.86h增至 2 .2 9h(P <0 .0 5 ) ,f由 4 5 .73%降至 30 .0 6 % (P <0 .0 1)。     

Strains of Staphylococcus aureus and pathology associated with chronic suppurative mastitis in rabbits

Staphylococcal mastitis is one of the main reasons for culling adult does from commercial rabbitries. The aim of this study was describe the spectrum of gross and microscopic lesions in 178 cases of chronic staphylococcal mastitis in adult does and to determine whether there is a correlation between Staphylococcus aureus genotypes and pathology. On the basis of histopathology, chronic mastitis was differentiated into abscesses (66.3%), suppurative mastitis with a lobular pattern (7.9%), cellulitis (19.6%) and mixed lesions (6.2%). Pathological presentations were not related to S. aureus genotype.     

Genotypic characterization of Staphylococcus aureus isolated from bovines, humans, and food in Indonesia

The present study determined the genetic relationships between 41 Staphyloccocus (S.) aureus isolates from bovines, humans, and food using a single enzyme amplified fragment length polymorphism (AFLP) technique. We evaluated the prevalence of staphylococcal enterotoxin (SE) genes and other virulence gene determinants by PCR. The identification of S. aureus was based on culturing and biochemical tests, and by amplifying a specific section of the 23S rRNA gene. PCR amplification of the SE genes (sea, seb, sec, see, seg, seh, and sei) singly or in combination was observed. Most isolates of bovine origin harbored hla (84%) and cap5 (74%), while most isolates from humans harbored hla (73%), cap8 (91%), and fnbA (100%). Strains from food sources were positive for hla (100%), cap5 (100%), and cap8 (64%) unlike isolates from humans or bovines. A single enzyme AFLP analysis revealed a correlation between AFLP clusters of some strains and the source of the isolates The genotypic results of the present study might help to better understand the distribution of prevalent S. aureus clones among humans, bovines, and food and will help control S. aureus infections in Indonesia.     

Carriage of methicillin-resistant Staphylococcus aureus by veterinarians in Australia

Objective To estimate the prevalence of carriage of methicillin‐resistant Staphylococcus aureus (MRSA) among Australian veterinarians. Methods Individuals attending veterinary conferences in Australia in 2009 were recruited to provide nasal swabs and complete a questionnaire about their professional activities. Swabs were processed by standard methods for detecting MRSA and questionnaire responses were used to group veterinarians according to their areas of major work emphasis (species and practice type). Prevalence was estimated for each of these grouping and contingency tables and regression tree analysis used to explain the variation in MRSA carriage. Results Among the 771 respondents ‘industry and government veterinarians’ (controls) had the lowest prevalence of MRSA carriage at 0.9%. Veterinarians with horses as a major area of work emphasis had a prevalence of 11.8% (13‐fold that of controls) and those whose only major emphasis was horses had a prevalence of 21.4% (23‐fold that of controls). Veterinarians with dogs and cats as a major activity had a 4.9% prevalence (5‐fold that of controls). Prevalence rates for other major activities (pigs, dairy and beef cattle, avian and wildlife) were also increased, but were estimated from smaller numbers of respondents. Regression tree analysis clearly isolated equine veterinarians and dog and cat practitioners as groups at increased risk of carriage of MRSA. Conclusion Carriage of MRSA is a notable occupational health issue for veterinarians in clinical practice in Australia, particularly those who work with horses.     

Dynamic of nasal colonization by methicillin-resistant Staphylococcus aureus ST398 and ST1 after mupirocin treatment in a family in close contact with pigs

Nasal colonization by methicillin-resistant Staphylococcus aureus (MRSA) was evaluated after a mupirocin treatment in a family previously colonized by MRSA sequence type ST398 and ST1, who lived close to a pig farm. Eight nasal samples were swabbed from each of the four family members on different moments after mupirocin treatment. The efficacy of treatment was low in those family members who worked in the farm, and higher in the remaining two family members with sporadic contact with pigs. In addition, nasal and skin swabs from randomly selected pigs of the farm were taken. MRSA were detected in 33% of pigs tested. All MRSA isolates obtained were characterized by Staphylococcal-Cassette-Chromosome mec (SCCmec) determination, Multilocus-Sequence-Typing (MLST), spa- and agr-typing, Pulsed-field-gel-electrophoresis (PFGE), antimicrobial susceptibility, detection of antimicrobial resistance genes, and toxin gene profiling. Spa-types t011, t1255 and t1197 were detected in humans and animals, with indistinguishable PFGE patterns, suggesting animal to human MRSA transmission. Each spa-type was ascribed to a specific pulsotype. Spa-types t127 and t108 were only detected in MRSA isolates obtained from humans, and t012 only in those from animals. MRSA ST1-t127 isolates and some ST398-t011 and ST398-t1197 isolates presented a multiantimicrobial-resistance phenotype. None of them harbored lukF/lukS, tst, eta and etb virulence genes. This study showed that the efficacy of nasal MRSA decolonization in healthy people with very close contact with pigs is especially low.