乳酸杆菌细胞裂解物对金黄色葡萄球菌、大肠埃希菌的抑制作用

目的探讨乳杆菌DM8909裂解物在体内外对金黄色葡萄球菌、大肠埃希菌的抑制作用。方法通过对乳杆菌超声波破碎制成裂解物,分别用乳杆菌裂解物原液、裂解物稀释液、发酵上清液、乳杆菌活菌制剂进行体内、体外实验,观察乳杆菌各成分对金黄色葡萄球菌、大肠埃希菌的抑制作用。结果德氏乳酸杆菌裂解物对金黄色葡萄球菌、大肠埃希菌的抑制作用与乳杆菌活菌制剂的抑制作用相近。结论德氏乳酸杆菌裂解物在体内外对金黄色葡萄球菌、大肠埃希菌均有较强的抑制作用。     

A new gel tube method for the direct detection, identification and susceptibility testing of bacteria in clinical samples

We recently developed a simple new method which is designed to separate and concentrate bacteria from a sample by centrifugation in a gel system. Bacterial enzyme activity is then detected inside the gel without further manipulation using a colorimetric or fluorogenic substrate. The method provides a rapid, direct means of detecting bacteria in clinical samples, dispensing with the 24-h period normally required to isolate colonies on agar. Various applications of the method are described below, e.g. screening of negative urine samples, identification of Escherichia coli in urine samples, identification of Staphylococcus aureus in blood culture broths and detection of oxacillin-resistant S. aureus in blood culture broths. The advantages of the gel system and other applications are discussed.     

新抗菌靶点分选酶基因（srtA）在两种原核载体中的克隆表达

【目的】革兰氏阳性菌的表面蛋白在病原菌致病性方面具有重要作用,表面蛋白锚定到细胞壁过程的关键酶—分选酶成为抗感染的新靶点。【方法】本文利用GenBank中的分选酶A基因（srtA）序列设计特异性引物,以金黄色葡萄球菌基因组DNA为模板进行PCR扩增,获得618 bp的DNA片段。按照常规分子克隆操作成功构建两种原核表达载体pet22-srtA和pTRX-srtA,转入大肠杆菌感受态BL21（DE3）中,在1 mmol/LIPTG诱导下进行表达。利用SDS-PAGE和western blot进行鉴定和分析,【结果】结果显示：(1)重组载体pet22-srtA和pTRX-srtA分别表达出相对分子量为约45 kDa和39 kDa的外源蛋白;【结论】（2）分子伴侣硫氧还蛋白（Trx）有利于分选酶A基因的可溶性表达。该实验为后续的分选酶酶学性质研究特别是抑制剂筛选研究奠定良好基础。     

一种能同时富集沙门菌、大肠杆菌和金黄色葡萄球菌的增菌培养基

研究设计出一种能同时富集培养沙门菌、大肠杆菌和金黄色葡萄球菌的缓冲盐水肉汤(buffed saline broth,BSB),主要组分和含量为:蛋白胨10g、牛肉膏3g、磷酸氢二钠9g、磷酸二氢钾1.5g、添加剂50g、蒸馏水定容至1000mL,pH7.2。将1cfu/mL的上述3种菌同时分别接种到97mLBSB、蛋白缓冲水、乳糖肉汤、营养肉汤、大肠杆菌肉汤、氯化镁孔雀绿增菌液、7.5%氯化钠肉汤增菌培养基中,37℃增菌18h。结果表明BSB能使这3种菌以相对一致的速度增殖,分别达到106、106、107cfu/mL,并且多重PCR能同时分别扩增沙门菌invA基因284bp、大肠杆菌phoA基因622bp和金黄色葡萄球菌nuc基因484bp的特异性条带。相反,其他几种培养基则不能同时协调增殖上述3种菌。这些结果表明,BSB有较好的应用前景。     

Functional expression,purification, and antimicrobial activity of a novel antimicrobial peptide MLH in Escherichia coli

In this study, a novel heterozygous antimicrobial peptide MLH was synthesized, expressed, purified, and characterized. The peptide Md-cec-LL-37_Hp (MLH) was selected through bioinformatic analysis using musca domestica antimicrobial peptide (Cec-Med), human antimicrobial peptide LL-37, and helicobacter pylori antimicrobial peptide (Hp) as parent peptides. The target gene was synthesized by overlap extension PCR (SOE-PCR) and connected to the expression vector pET-32a (+), and the recombinant plasmid pET-32a-MLH was transformed to Escherichia coli for constructing pET-32a-MLH/BL21 (DE3). Isopropyl β-D-thiogalactoside (IPTG) was used to induce protein expression, and SDS-PAGE and western blot were adopted to test the target protein. And fermentation condition was optimized to get the mass expression of the fusion protein. The Ni²⁺ affinity chromatographic column was used to purify. Active heterozygous peptide was obtained after renaturation. Finally, the activity of the heterozygous antimicrobial peptide was identified. The fusion peptide showed significant antimicrobial effect on both E. coli and Staphylococcus aureus.     

A high prevalence of antimicrobial resistant Escherichia coli isolated from pigs and a low prevalence of antimicrobial resistant E. coli from cattle and sheep in Great Britain at slaughter

The incidence of antimicrobial resistance and expressed and unexpressed resistance genes among commensal Escherichia coli isolated from healthy farm animals at slaughter in Great Britain was investigated. The prevalence of antimicrobial resistance among the isolates varied according to the animal species; of 836 isolates from cattle tested only 5.7% were resistant to one or more antimicrobials, while only 3.0% of 836 isolates from sheep were resistant to one or more agents. However, 92.1% of 2480 isolates from pigs were resistant to at least one antimicrobial. Among isolates from pigs, resistance to some antimicrobials such as tetracycline (78.7%), sulphonamide (66.9%) and streptomycin (37.5%) was found to be common, but relatively rare to other agents such as amikacin (0.1%), ceftazidime (0.1%) and coamoxiclav (0.2%). The isolates had a diverse range of resistance gene profiles, with tet(B), sul2 and strAB identified most frequently. Seven out of 615 isolates investigated carried unexpressed resistance genes. One trimethoprim-susceptible isolate carried a complete dfrA17 gene but lacked a promoter for it. However, in the remaining six streptomycin-susceptible isolates, one of which carried strAB while the others carried aadA, no mutations or deletions in gene or promoter sequences were identified to account for susceptibility. The data indicate that antimicrobial resistance in E. coli of animal origin is due to a broad range of acquired genes.     

Susceptibility of food-borne bacteria to binary combinations of antimicrobials at selected a(w) and pH

AIM: To evaluate the antibacterial susceptibilities of food-borne bacteria to individual and binary mixtures of a synthetic antimicrobial agent with a natural phenolic compound. METHODS AND RESULTS: Antibacterial susceptibilities of Escherichia coli, Listeria innocua, Salmonella Typhimurium and Staphylococcus aureus to individual and binary mixtures of potassium sorbate with a phenolic compound (thymol, carvacrol, or eugenol) were evaluated, at selected water activity (a(w); 0.99 or 0.97) and pH (5.5 or 4.5). The bacteria studied were susceptible to the action of the antimicrobials individually with minimal inhibitory concentrations that varied from 800-ppm potassium sorbate for Staph. aureus at a(w) 0.99, and pH 5.5 to 100-ppm thymol or carvacrol for the four studied bacteria at a(w) 0.97 and pH 4.5. Several binary mixtures of potassium sorbate with thymol, carvacrol or eugenol inhibited bacterial growth. Antimicrobial agent inhibitory concentrations in the mixture varied among bacteria, additionally depending on the a(w) and the pH tested. CONCLUSIONS: Synergistic binary mixtures with fractional inhibitory concentration index <0.6 include 100- or 200-ppm potassium sorbate with 50- or 100-ppm thymol, carvacrol or eugenol. SIGNIFICANCE AND IMPACT OF THE STUDY: The synergistic combinations could be useful in reducing the amounts of antimicrobials needed to inhibit growth, thus diminishing consumer concerns regarding chemical preservatives.     

Antibacterial properties of nine pure metals: a laboratory study using Staphylococcus aureus and Escherichia coli

Bacterial attachment and growth on material surfaces are considered to be the primary steps leading to the formation of biofilm. Biofilms in hospital and food processing settings can result in bacterial infection and food contamination, respectively. Prevention of bacterial attachment, therefore, is considered to be the best strategy for abating these menaces and therefore the development of antibacterial metals becomes important. In this study, nine pure metals, viz. titanium, cobalt, nickel, copper, zinc, zirconium, molybdenum, tin, and lead have been tested for their antibacterial properties against two bacterial strains, Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. This was accomplished using two assay methods, the film contact method and the shaking flask method. The results show that the antibacterial properties varied significantly with different metals and the effectiveness of metals to resist bacterial attachment varied with the bacterial strain. Among the metals tested, titanium and tin did not exhibit antibacterial properties. TEM images showed that metal accumulation resulted in the disruption of the bacterial cell wall and other cellular components.     

Effect of laser and environmental parameters on reducing microbial contamination of stainless steel surfaces with Nd:YAG laser irradiation

AIMS: The effect of laser (pulse repetition frequency, pulse energy and exposure time) and environmental parameters (pH, NaCl concentration and wet or dry samples) of Nd:YAG laser decontamination of stainless steel inoculated with Escherichia coli, Staphylococcus aureus and Listeria monocytogenes was investigated. METHODS AND RESULTS: Stainless steel discs were inoculated with the bacterial samples and exposed to laser energy densities to about 900 J cm(-2). These inactivation curves allowed selection of laser parameters for two-level multifactorial designed experiments, the results of which allowed comparisons to be made between effects of individual and combined parameters on the laser inactivation efficiency. Escherichia coli was inactivated most effectively as a wet film with L. monocytogenes and S. aureus showing similar response. For the multifactorial experiments all laser parameters were significant and were smallest for S. aureus as a wet film. CONCLUSIONS: pH and NaCl concentration had little effect on the efficacy of laser inactivation but dry or wet states and all laser parameters were significant. SIGNIFICANCE AND IMPACT OF THE STUDY: Such systems may prove to be applicable in industrial processes where stainless steel may be contaminated with acidic solutions or salt, e.g. in the food industry with laser inactivation seeming to be independent of these parameters. Parameters have been identified that allow optimization of the treatment process.     

Antimicrobial action and efficiency of silver-loaded zeolite X

Aims: To synthesize silver-loaded zeolite X and establish the extent to which it persist in its antimicrobial action against strains of Escherichia coli K12W-T, Pseudomonas aeruginosa NCIMB8295 and Staphylococcus aureus NCIMB6571. Methods and Results: The antimicrobial action and efficacy of silver-loaded zeolite X on Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa were investigated. Zeolite X was synthesized and loaded with Ag⁺ by ion exchange. This resulted in 2·0% (w/w) loading of Ag⁺ in the zeolite framework and 5·8% (w/w) on the zeolite. Escherichia coli and Pseudomonas aeruginosa and Staphylococcus aureus suspended in tryptone soya broth were exposed to 0·15, 0·25, 0·5 or 1·0 g l⁻¹ of silver-loaded zeolite X for a period up to 24 h. No viable cells were detected for any of the three micro-organisms within 1 h. Silver-loaded zeolite X, retrieved three times from the first exposure cultures, was washed with de-ionized water and added to fresh bacterial suspensions. The results showed that the silver-loaded zeolite X retained its antimicrobial action. Conclusions: Silver-loaded zeolite X persisted in its antimicrobial action against all three micro-organisms. Significance and Impact of the study: The results are significant for the longevity of antimicrobial action of silver-loaded zeolite X.     

藏绵羊子宫内膜炎主要致病菌多重PCR检测方法的建立与评价

本研究旨在建立一种多重PCR方法检测青海藏绵羊子宫内膜炎主要的病原菌。首先,提取5种标准菌株基因组,筛选出特异性引物;然后以标准菌株的基因组为模板,建立多重PCR方法。用无菌棉拭子涂抹藏绵羊子宫,置于LB培养液中培养并编号,48 h后提取样品基因组。运用单一PCR法对600份样品基因组进行检测,记录阳性样品;再挑取单一PCR法检测的阳性样品进行多重PCR检测,再次记录阳性样品,通过计算两种检测方法的符合率验证多重PCR方法;随机挑出30份阳性样品,进行病原菌分离鉴定菌种种类。单一PCR检测的样品中,无乳链球菌感染比例占47.33%,大肠杆菌占34.83%,金黄色葡萄球菌占6.5%,未检出沙门氏菌和化脓隐秘杆菌;多重PCR检测的阳性样品中,无乳链球菌感染比例占45.50%,大肠杆菌占33.50%,金黄色葡萄球菌占6.5%;两种检测结果相比较,多重PCR检测出的符合率均高于95%;分离鉴定的病原菌与两种PCR方法检测出的菌种结果基本一致。成功建立了多重PCR方法并检测出引起青海藏绵羊子宫内膜炎的主要病原菌为无乳链球菌、大肠杆菌和金黄色葡萄球菌。     

中国静灰球体外抑菌活性的研究

采用纸片扩散法(Kirby-Bauer法)和菌饼法(活菌活性测定法),系统研究了中国静灰球的菌丝体、发酵液、活菌菌饼及其子实体的水、乙醇、乙酸乙酯、乙醚、三氯甲烷和石油醚6种溶剂提取液对金黄色葡萄球菌、大肠埃希氏菌、鼠伤寒沙门氏菌、黑曲霉和绿色木霉5种测试菌的体外抑菌活性。结果表明:中国静灰球的菌丝体、发酵液及活菌菌饼对金黄色葡萄球菌、鼠伤寒沙门氏菌和黑曲霉均有不同程度的抑菌作用,即具有广谱的抑菌活性;其水和乙酸乙酯的浸提液对金黄色葡萄球菌均具有较显著的抑菌作用。推测中国静灰球菌丝体中含有对金黄色葡萄球菌、大肠埃希氏菌、鼠伤寒沙门氏菌、黑曲霉、绿色木霉5种测试菌的广谱抑菌活性物质。     

In vitro activity of the antimicrobial peptide AP7121 against the human methicillin-resistant biofilm producers Staphylococcus aureus and Staphylococcus epidermidis

Abstract

In vitro activity against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis biofilm producers from blood cultures of patients with prosthetic hip infections was evaluated. The Minimum Inhibitory Concentration (MIC) for AP7121 was determined and the bactericidal activity of AP7121 (MICx1, MICx4) against planktonic cells was studied at 4, 8 and 24?h. The biofilms formed were incubated with AP7121 (MICx1, MICx4) for 1 and 24?h. The anti-adhesion effect of an AP7121-treated inert surface over the highest MIC isolate was studied with scanning electron microscopy (SEM). The bactericidal activity of AP7121 against all the planktonic staphylococcal cells was observed at 4?h at both peptide concentrations. Dose-dependent anti-biofilm activity was detected. AP7121 (MICx4) showed bactericidal activity at 24?h in all isolates. SEM confirmed prevention of biofilm formation. This research showed the in vitro anti-biofilm activity of AP7121 against MRSA and S. epidermidis and the prevention of biofilm formation by them on an abiotic surface.     

Effect of temperature on the release of carvacrol and cinnamaldehyde incorporated into polymeric systems to control growth and biofilms of Escherichia coli and Staphylococcus aureus

This study assessed the effect of temperature on the release of essential oil components incorporated by melt compounding into polymeric films. Specifically, polyethylene-co-vinylacetate (EVA) films containing carvacrol (CAR) and cinnamaldehyde (ALD), alone and in combination, were prepared and their surface and mechanical properties and antibacterial and anti-biofilm activity against Escherichia coli and Staphylococcus aureus were evaluated. The addition of ALD and CAR did not provoke variation in the surface morphology of EVA and allowed their delivery. At 37°C, films containing CAR, ALD or their combination (25+75%) were found to have the strongest bactericidal effect, whereas at lower temperatures a lower killing rate was observed. There was no clear evidence of the influence of temperature on the anti-biofilm activity of the essential oil component-based polymeric films. The biomass formed on EVA containing ALD, CAR or their combination (25+75) was significantly lower (60–80% reduction) than that formed on the EVA control at both 37° and 22°C.     

Impact of prescribed cleaning and disinfectant use on microbial contamination in the home

Aims: To identify and quantify the presence of Escherichia coli, Staphylococcus aureus, Salmonella, hepatitis A and norovirus in households and to assess the effect of chlorine and quaternary ammonium–based disinfectants following a prescribed use. Methods and Results: Eleven sites distributed in kitchen, bathroom, pet and children′s areas of two groups of 30 homes each: (i) a nonprescribed disinfectant user group and (ii) a disinfectant protocol user group. During the 6‐week study, samples were collected once a week except for week one when sample collection occurred immediately before and after disinfectant application to evaluate the disinfectant protocol. The concentration and occurrence of bacteria were less in the households with prescribed use of disinfectants. The greatest reductions were for E. coli (99%) and Staph. aureus (99·9999%), respectively. Only two samples were positive for HAV, while norovirus was absent. Disinfection protocols resulted in a significant (P < 0·05) microbial reduction in all areas of the homes tested compared to homes not using a prescribed protocol. Conclusions: The study suggests that disinfectant product application under specific protocol is necessary to achieve greater microbial reductions. Significance and Impact of the Study: Prescribed protocols constitute an important tool to reduce the occurrence of potential disease‐causing micro‐organisms in households.     

Room temperature sterilization of surfaces and fabrics with a One Atmosphere Uniform Glow Discharge Plasma

We report the results of an interdisciplinary collaboration formed to assess the sterilizing capabilities of the One Atmosphere Uniform Glow Discharge Plasma (OAUGDP). This newly-invented source of glow discharge plasma (the fourth state of matter) is capable of operating at atmospheric pressure in air and other gases, and of providing antimicrobial active species to surfaces and workpieces at room temperature as judged by viable plate counts. OAUGDP exposures have reduced log numbers of bacteria, Staphylococcus aureus and Escherichia coli, and endospores from Bacillus stearothermophilus and Bacillus subtilis on seeded solid surfaces, fabrics, filter paper, and powdered culture media at room temperature. Initial experimental data showed a two-log₁₀ CFU reduction of bacteria when 2 × 10² cells were seeded on filter paper. Results showed ≥3 log₁₀ CFU reduction when polypropylene samples seeded with E. coli (5 × 10⁴) were exposed, while a 30 s exposure time was required for similar killing with S. aureus-seeded polypropylene samples. The exposure times required to effect ≥6 log₁₀ CFU reduction of E. coli and S. aureus on polypropylene samples were no longer than 30 s. Experiments with seeded samples in sealed commercial sterilization bags showed little or no differences in exposure times compared to unwrapped samples. Plasma exposure times of less than 5 min generated ≥5 log₁₀ CFU reduction of commercially prepared Bacillus subtilis spores (1 × 10⁶); 7 min OAUGDP exposures were required to generate a ≥3 log₁₀ CFU reduction for Bacillus stearothermophilus spores. For all microorganisms tested, a biphasic curve was generated when the number of survivors vs time was plotted in dose-response cures. Several proposed mechanisms of killing at room temperature by the OAUGDP are discussed. Received 06 June 1997/ Accepted in revised form 01 November 1997     

Addition of a surfactant to tryptic soy broth allows growth of a lactic acid bacteria food antimicrobial, Escherichia coli O157:H7, and Salmonella enterica

Aims: This study aimed to determine the survival and growth of Escherichia coli O157:H7 and Salmonella enterica subsp. enterica in a medium supporting the growth of a Lactic Acid Bacteria (LAB) food antimicrobial culture. Methods and Results: Foodborne pathogens and LAB were cultured individually in tryptic soy broth (TSB), tryptic soy broth supplemented with one g l^?1 Tween 80^® (TSB‐T80), and de Man, Rogosa and Sharpe (MRS) broth. Growth of E. coli O157:H7 and Salmonella was similar in TSB and TSB‐T80 but was significantly less in MRS. Conversely, LAB growth was similar in MRS and TSB‐T80 but was significantly less in TSB. Conclusions: Supplementation of TSB with Tween 80^® allows growth of LAB to levels similar to that observed with MRS but does not inhibit the growth of E. coli O157:H7 and Salmonella. We present the formulation of a medium useful in studies useful for evaluating competitive inhibition of foodborne pathogens by LAB in vitro. Significance and Impact of the Study: This study reports the utility of TSB‐T80 for the completion of in vitro competitive inhibition assays incorporating a Lactic Acid Bacteria food safety culture.     

Xenorhabdus antibiotics: a comparative analysis and potential utility for controlling mastitis caused by bacteria

Aims: The role of antibiotics produced by bacterial symbionts of entomopathogenic nematodes is to suppress growth of microbes in the soil environment. These antibiotics are active against Gram‐positive and Gram‐negative bacteria, and were tested against mastitis isolates from dairy cows. Methods and Results: Two bioassays were adapted for Xenorhabdus antibiotics; an overlay method on agar plates, and serially diluted, cell‐free, Xenorhabdus cultures. The antimicrobial activities of the liquid cultures of 13 strains from five Xenorhabdus species were further evaluated. Antimicrobial activities of the type strains of X. nematophila, X. budapestensis and X. szentirmaii were tested on mastitis isolates of Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae with both bioassays. A previously reported antibiotic from X. nematophila, nematophin, was synthesized in three steps from tryptamine and 4‐methyl‐2‐oxovaleric acid sodium salt. Conclusions: The antibiotics of all three Xenorhabdus strains were powerful in either bioassay, but the sensitivity of the isolates differed from each other. While Kl. pneumoniae was the least susceptible, Staph. aureus had the highest sensitivity to each Xenorhabdus strain. Xenorhabdus szentirmaii and X. budapestensis were more potent antibiotic producers than X. nematophila, and raceme nematophin was ineffective against all mastitis isolates. Significance and Impact of the Study: These results indicate that Xenorhabdus antibiotics are effective against mastitis isolates and should be further evaluated for their potential in mastitis control or prevention.