裂解基因E和核酸酶基因串联表达载体的构建及大肠杆菌菌影的制备

目的构建噬菌体裂解基因E和核酸酶基因串联表达载体,制备高质量的大肠杆菌菌影。方法自行设计一对引物,PCR扩增PhiX174噬菌体裂解基因E,将该基因亚克隆至原核表达载体pGEX-6P-1,构建大肠杆菌菌影的表达载体pGEX-E。在E基因基础上与辅助基因葡萄球菌核酸酶A(SN)基因串联,并插入pGEX-6P-1载体,构建双基因串联高效表达载体pGEX-E-5aaLinker-SN和pGEX-E-15aaLinker-SN,采用CaCl2法将其转入大肠杆菌BL21(DE3),经IPTG诱导,制备大肠杆菌菌影。结果裂解基因E单基因及串联基因已成功插入融合表达载体pGEX-6P-1,所构建的大肠杆菌菌影表达载体pGEX-E、pGEX-E-5aaLinker-SN和pGEX-E-15aaLinker-SN,诱导后经透射电镜及活菌计数显示大肠杆菌已发生不同程度裂解,制成了大肠杆菌菌影。电镜下菌影形态完整,内容物已释放到胞外。结论已成功构建了噬菌体裂解基因E单基因及裂解基因和核酸酶基因串联基因表达载体,并制成了大肠杆菌菌影,为进一步研究菌影这一新型的疫苗及佐剂形式奠定了基础。     

一起由金黄色葡萄球菌所致学校食物中毒调查分析

目的 查明一起学校食物中毒发生的原因和可疑危险因素,提出有效控制措施防止事态蔓延,为类似食源性疾病的调查和预防提供参考.方法 利用现场流行病学、食品卫生学和实验室检测等方法,开展病例主动搜索、个案调查和现场卫生学调查,采集食品、外环境和病例的标本进行实验室检测,以判断本次事件的可疑餐次和食物.结果 通过开展主动搜索,共...     

Physicochemical and Theoretical Characterization of a New Small Non-Metal Schiff Base with a Differential Antimicrobial Effect against Gram-Positive Bacteria

Searching for adequate and effective compounds displaying antimicrobial activities, especially against Gram-positive bacteria, is an important research area due to the high hospitalization and mortality rates of these bacterial infections in both the human and veterinary fields. In this work, we explored (E)-4-amino-3-((3,5-di-tert-butyl-2-hydroxybenzylidene)amino) benzoic acid (SB-1, harboring an intramolecular hydrogen bond) and (E)-2-((4-nitrobenzilidene)amino)aniline (SB-2), two Schiff bases derivatives. Results demonstrated that SB-1 showed an antibacterial activity determined by the minimal inhibitory concentration (MIC) against Staphylococcus aureus, Enterococcus faecalis, and Bacillus cereus (Gram-positive bacteria involved in human and animal diseases such as skin infections, pneumonia, diarrheal syndrome, and urinary tract infections, among others), which was similar to that shown by the classical antibiotic chloramphenicol. By contrast, this compound showed no effect against Gram-negative bacteria (Klebsiella pneumoniae, Escherichia coli, and Salmonella enterica). Furthermore, we provide a comprehensive physicochemical and theoretical characterization of SB-1 (as well as several analyses for SB-2), including elemental analysis, ESMS, ¹H and ¹³C NMR (assigned by 1D and 2D techniques), DEPT, UV-Vis, FTIR, and cyclic voltammetry. We also performed a computational study through the DFT theory level, including geometry optimization, TD-DFT, NBO, and global and local reactivity analyses.     

乳酸乳球菌乳亚种NCU036018细菌素的分离纯化及其抗菌机制

从自然发酵泡菜中筛选到1株对金黄色葡萄球菌具有抑制作用的乳酸菌,经16S rDNA和生理生化实验鉴定其为乳酸乳球菌乳亚种（Lactococcus lactis subsp. lactis）并命名为NCU036018。NCU036018发酵上清液中的抑菌物质对胰蛋白酶、木瓜蛋白酶、蛋白酶K、α-糜蛋白酶敏感,而对胃蛋白酶、脂肪酶、淀粉酶以及过氧化氢酶不敏感,在pH 2.0～10.0、温度50～100℃的范围内分别保留了70%和90%以上的抑菌活性。随后通过硫酸铵盐析、超滤、阳离子交换层析对发酵上清液抑菌成分进行分离纯化,将得到的细菌素命名为乳球菌素036018,其分子质量在14.4～20 kDa之间,最小抑菌浓度为0.50 mg/mL。乳球菌素036018能有效降低金黄色葡萄球菌生物膜的形成率,损坏细胞表面形态、破坏细胞膜的通透性,从而抑制甚至杀死细胞。将乳球菌素036018添加至牛奶中,发现其具有显著抑制金黄色葡萄球菌生长的效果,具有良好的食品防腐保鲜的应用前景。     

SARS-CoV-2 Spike Protein and Mouse Coronavirus Inhibit Biofilm Formation by Streptococcus pneumoniae and Staphylococcus aureus

The presence of co-infections or superinfections with bacterial pathogens in COVID-19 patients is associated with poor outcomes, including increased morbidity and mortality. We hypothesized that SARS-CoV-2 and its components interact with the biofilms generated by commensal bacteria, which may contribute to co-infections. This study employed crystal violet staining and particle-tracking microrheology to characterize the formation of biofilms by Streptococcus pneumoniae and Staphylococcus aureus that commonly cause secondary bacterial pneumonia. Microrheology analyses suggested that these biofilms were inhomogeneous soft solids, consistent with their dynamic characteristics. Biofilm formation by both bacteria was significantly inhibited by co-incubation with recombinant SARS-CoV-2 spike S1 subunit and both S1 + S2 subunits, but not with S2 extracellular domain nor nucleocapsid protein. Addition of spike S1 and S2 antibodies to spike protein could partially restore bacterial biofilm production. Furthermore, biofilm formation in vitro was also compromised by live murine hepatitis virus, a related beta-coronavirus. Supporting data from LC-MS-based proteomics of spike–biofilm interactions revealed differential expression of proteins involved in quorum sensing and biofilm maturation, such as the AI-2E family transporter and LuxS, a key enzyme for AI-2 biosynthesis. Our findings suggest that these opportunistic pathogens may egress from biofilms to resume a more virulent planktonic lifestyle during coronavirus infections. The dispersion of pathogens from biofilms may culminate in potentially severe secondary infections with poor prognosis. Further detailed investigations are warranted to establish bacterial biofilms as risk factors for secondary pneumonia in COVID-19 patients.     

Atomic force microscopy study of the antimicrobial activity of aqueous garlic versus ampicillin against Escherichia coli and Staphylococcus aureus

BACKGROUND: In this study the effects of ampicillin and aqueous garlic extract on Escherichia coli (ATCC 9637) and Staphylococcus aureus (ATCC 25923) were compared. Atomic force microscopy (AFM) was used to study the possible mechanisms of membrane disruption. RESULTS: Ampicillin disrupted the cell membrane of E. coli, inducing pores and cell leakage. Aqueous garlic extract also induced leakage from the cell membrane in E. coli, but no pores were observed. The trend in Young's modulus for E. coli was E_native ≈ E_age > E_amp. In contrast, S. aureus incubated with low ampicillin (≤50µg mL^?1) and garlic (≤50 mg mL^?1) concentrations showed no significant changes in surface morphology compared with the untreated bacterium. The trend in Young's modulus for S. aureus was E_native ≈ E_age ≈ E_amp. CONCLUSION: The trend E_native ≈ E_age for E. coli and S. aureus supports the hypothesis that the compounds in garlic show intracellular activity. This proof‐of‐concept study of the aqueous crude isolate of garlic points to the feasibility of further AFM investigations to compare the antimicrobial properties of various pure thiosulfinate isolates found in garlic. Copyright © 2009 Society of Chemical Industry     

High‐dose cefazolin on consecutive hemodialysis in anuric patients with Staphylococcal bacteremia

Methicillin‐sensitive Staphylococcus aureus (MSSA) bacteremia is a leading cause of infection in hemodialysis (HD) patients. Cloxacillin, cefazolin, and vancomycin are the mainstay antimicrobials. Cloxacillin administration leads to frequent drug dosing, longer length of stay (LOS), and higher cost, while resistance and poorer outcomes are associated with vancomycin use. Dosing cefazolin during HD allows for prolonged blood therapeutic levels. We assessed the outcomes and safety of a strategy of treating MSSA bacteremia with 2–3 g cefazolin on HD only. All HD patients with MSSA bacteremia admitted in June–December 2009 at our center and receiving this regime were compared with historical controls who received cloxacillin. Demographic characteristics and outcome measures like mortality, LOS, cost, recrudescence, and adverse drug reactions were assessed. Of 27 consecutive episodes reviewed, 14 and 13 patients received cefazolin and cloxacillin, respectively. Baseline demographics were comparable between the 2 treatment groups. More than one‐third of the bacteremia was related to tunneled catheter infection. The 30‐day mortality of cloxacillin‐ and cefazolin‐treated patients was 15% and 7%, respectively (P=0.14). Two of the 11 survivors treated with cloxacillin (18%) had recrudescent bacteremia while none was observed in cefazolin‐treated survivors. Cefazolin was associated with shorter LOS (10 vs. 20 days, P<0.05) and lower cost (US$8262.00 vs. US$15,367.00, P<0.05). Cefazolin use resulted in 3 idiosyncratic adverse drug reactions. Cefazolin dosed on each HD in MSSA bacteremia leads to earlier discharge and less cost. Larger prospective studies are, however, warranted to fully assess its safety and efficacy.     

Inhibition of Staphylococcus aureus by Phenolic Phytochemicals of Selected Clonal Herbs Species of Lamiaceae Family and Likely Mode of Action through Proline Oxidation

Staphylococcus aureus, a Gram-positive organism, is responsible for numerous infections ranging in severity from skin and soft tissue infections to endocarditis and septic arthritis. Further, Staphylococcal food poisoning (SFP) caused by enterotoxigenic staphylococci is one of the main food-borne diseases. In this study, several single-seed origin clonal lines of herbs from Lamiaceae family were screened for antimicrobial applications against S. aureus. These clonal lines have consistent total phenolic content, phenolic profile and antioxidant activity as opposed to heterogenous phenolic profiles of typical herbal extracts. The clonal extracts of specific herb species were evaluated for anti- S. aureus activity. Water extracts of clonal lemon balm had the highest anti- S. aureus activity followed by clonal extracts of rosemary RoLA, sage, oregano, rosemary Ro-6, rosemary RoK-2 and chocolate mint. The anti- S. aureus activity of the water extracts correlated with gallic acid and caffeic acid content, which in pure form showed high anti- S. aureus activity. Based on the rationale that gallic acid and caffeic acid were likely proline analogs affecting proline oxidation, the countering effect of proline to overcome their anti- S. aureus activity was evaluated. Results indicated proline can overcome the inhibitory effects of gallic acid and caffeic acid. These results point to the likely role of phenolic oxidation via proline dehydrogenase, which could be an important target for inhibition by phenolic phytochemicals behaving as proline analog mimics. This mode of regulation by phenolics at the bacterial proline dehydrogenase in the plasma membrane may be an important antimicrobial defense in plants and has consequences for disruption of critical energy metabolism of invading bacterial pathogen. This rationale could be used to design new antimicrobial strategies against S. aureus and other relevant bacterial pathogens.     

Antimicrobial drug susceptibility of Staphylococcus aureus strains isolated from bovine and ovine mammary glands

The activity of selected antimicrobial agents against Staphylococcus aureus was determined with the agar disk diffusion test to determine the diameter of the zone of inhibition and the E-test for determination of the minimal inhibitory concentration (MIC). The 92 S. aureus strains used in this study were isolated from bovine (n = 76) and ovine (n = 16) intramammary infections. Four antibiotics, which are frequently used in mastitis therapy were chosen: penicillin-G, ampicillin, kanamycin, and cephalexine. The fifth compound (oxacillin) was used to detect methicillin-resistant S. aureus, but no such strains could be found. According to the evaluation criteria, 65.2 (penicillin) to 93.5% (kanamycin, cephalexine) S. aureus strains were susceptible to the antibiotics tested. Ovine S. aureus strains reveal a lower resistance rate than bovine isolates. Comparison of the results of the two methods of susceptibility testing shows, with exception of penicillin and ampicillin, satisfactory agreement. Analyzing the results of the MIC endpoints and the zone diameter values, very major errors, according to the error rate bounded method of Metzler and DeHaan, occurred at an error rate of 3.3% for penicillin and 3.8% for ampicillin.