Scanning CO2 laser bacterial inactivation systems

AIMS: The performance of three scanning CO(2) laser inactivation systems was assessed and included: a gantry system, a rapidly rotating mirror and a low-power hybrid system combining an oscillating mirror and rotary motion of the sample. METHODS AND RESULTS: Escherichia coli and Staphylococcus aureus were the target organisms on stainless steel, nutrient agar or moist collagen film and the laser power was varied from 2 to 1060 W (two laser sources). In general, a threshold energy density was identified, above which no inactivation was observed because the scanning velocity was too high (10 cm s(-1) for stainless steel, 660 W). Reducing the velocity increased the inactivation process until complete inactivation was observed at 1.3 cm s(-1) (E. coli, approximately 10(6) CFU per sample) and 0.82 cm s(-1) (S. aureus, approximately 10(8) CFU per sample); consequently, S. aureus organisms showed a greater resistance to laser irradiation. For the nutrient agar and collagen samples, the averages of the width of clearing were measured as a function of the translation velocity and the rates of inactivation (I(R), cm(2) s(-1)) were found; an optimum velocity was observed that produced the maximum rate of inactivation. At a laser power of 1060 W, the maximum value of I(R) was 140 cm(2) s(-1) ( approximately 10(7) CFU cm(-2)) for S. aureus on collagen and slightly less on nutrient agar (114 cm(2) s(-1), estimated from a best-fit polynomial, r(2) = 0.98). CONCLUSIONS: A comparison of the low- and high-power lasers produced values of 0.09 cm(2) s(-1) W(-1) (i.e. I(R) per Watt delivered) for S. aureus on nutrient agar with the low-power laser at 13 W and on collagen 0.13 cm(2) s(-1) W(-1) for 1060 W. The rate of inactivation was found to be a function of the laser power, translation velocity and properties of the substrate media. The three laser inactivation systems successfully demonstrated the potential speed, efficiency and application of such systems. SIGNIFICANCE AND IMPACT OF THE STUDY: Laser scanning systems offer the potential for rapid and efficient inactivation of surfaces, eliminating the need for chemical treatment.     

Persister cells and tolerance to antimicrobials

Bacterial populations produce persister cells that neither grow nor die in the presence of microbicidal antibiotics. Persisters are largely responsible for high levels of biofilm tolerance to antimicrobials, but virtually nothing was known about their biology. Tolerance of Escherichia coli to ampicillin and ofloxacin was tested at different growth stages to gain insight into the nature of persisters. The number of persisters did not change in lag or early exponential phase, and increased dramatically in mid-exponential phase. Similar dynamics were observed with Pseudomonas aeruginosa (ofloxacin) and Staphylococcus aureus (ciprofloxacin and penicillin). This shows that production of persisters depends on growth stage. Maintaining a culture of E. coli at early exponential phase by reinoculation eliminated persisters. This suggests that persisters are not at a particular stage in the cell cycle, neither are they defective cells nor cells created in response to antibiotics. Our data indicate that persisters are specialized survivor cells.     

A novel fluorescent probe based on naphthalimide for imaging nitroreductase (NTR) in bacteria and cells

A nitroreductase (NTR) responsive fluorescent probe, Na-NO₂, comprising p-nitrobenzyl as the unique recognition group and 1,8-naphthalimide as fluorophore, was synthesized. Na-NO₂ showed remarkable fluorescence “turn-on” signal in the presence of NTR under DMSO/H₂O (1:19, v/v) buffered with PBS (pH = 7) solution in the presence of NADH (300 µM). Furthermore, the probe has a low detection limit down to 3.4 ng/mL and it is very sensitive towards the NTR in Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), normal and tumor cells such as HL-7702, HepG-2 and MCF-7.     

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。     

The phototoxicity of phenothiazinium derivatives against Escherichia coli and Staphylococcus aureus

Phenothiazinium dyes, and derivatives, were tested for toxicity to Escherichia coli and Staphylococcus aureus. The dyes were generally lipophilic (log P>1) and showed inherent dark toxicity (minimum lethal concentrations: 3.1-1000 microM). Dye illumination (total light dose of 3.15 J cm(-1) over 30 min) led to up to eight-fold reductions in minimum lethal concentrations. Most of the illuminated dyes showed significant relative singlet oxygen yields (phi'delta: 0.18-1.35) suggesting a type II mechanism of generating a phototoxic response. Although generally up to six-fold more effective against S. aureus, the dyes tested efficiently killed E. coli and may be of particular use in combating Gram-negative pathogens.     

聚乙二醇小檗碱液对大肠埃希菌和金黄色葡萄球菌生长的抑制作用

目的 明确聚乙二醇小檗碱液在琼脂培养基表面的抑菌特点,及其对大肠埃希菌和金黄色葡萄球菌的标准菌株、抗生素敏感菌株与多重耐药菌株生长的抑制作用,研究评价药物在皮肤黏膜表面的抑菌作用的合理实验方法.方法 以大肠埃希菌和金黄色葡萄球菌(标准菌株、抗生素敏感菌株和多重耐药菌株)为研究对象,用常量肉汤稀释法测定聚乙二醇小檗碱液的最低抑菌浓度(Minimum Inhibitory Concentration,MIC);用平皿琼脂培养法和试管肉汤培养法测定不同浓度聚乙二醇小檗碱液的抑菌作用.结果 在不同浓度的聚乙二醇小檗碱液的作用下,在琼脂培养基表面上或肉汤培养基中细菌的生长受到明显抑制,抑制作用与小檗碱浓度正相关,且对抗生素敏感菌株和多重耐药菌株的抑制作用差异无统计学意义;聚乙二醇小檗碱液在平皿琼脂表面和试管肉汤中对大肠埃希菌、金黄色葡萄球菌抑制100％菌株的浓度分别为1 500和375 mg/L、1 500和375 mg/L.聚乙二醇小檗碱液在琼脂培养基表面的抑菌作用明显低于在肉汤培养基中的抑制作用,在琼脂培养基表面的抑菌浓度是肉汤培养基中的抑菌浓度的4倍.并且金黄色葡萄球菌与大肠埃希菌之间差异无统计学意义.聚乙二醇小檗碱液必须达到肉汤培养基中4倍以上浓度时,才能获得抑制100％细菌在琼脂培养基表面生长的效果.结论 高浓度的聚乙二醇小檗碱液可以抑制皮肤黏膜表面的细菌,包括抗生素耐药菌株的生长;皮肤黏膜表面应用聚乙二醇小檗碱液的适宜浓度为1 500 mg/L.琼脂培养基法适用于评价药物在皮肤黏膜表面的抑菌作用.     

Isolation and characterization of Leuconostoc oenos bacteriophages from wine and sugarcane

Twenty bacteriophages specific for Leuconostoc oenos were isolated from South African red wines and sugarcane. Leuconostoc oenos ML34 and PSU-1, used commercially by the wine industry, were sensitive to some of the phages. Ten of the 39 L. oenos strains tested were resistant or insensitive to all phages. The bacteriophages were morphologically similar to Bradley's type B phages, possessing hexagonal heads and long flexible, non-contractile tails. Restriction endonuclease analysis of phage DNA revealed the existence of five genetic groups.     

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.     

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.     

不同浓度酒精对两种常见细菌生物膜的影响

目的 探讨不同浓度的酒精对金黄色葡萄球菌和大肠杆菌生物膜形成的抑制作用.方法 配制不同浓度的酒精(1.25％、2.5％、5％和10％),作用于培养24 h形成成熟生物膜的金黄色葡萄球菌和大肠埃希菌,利用FDA/PI荧光染料染色,在激光共聚焦显微镜扫描生物膜并分析活菌与死菌比例.结果 不同浓度的酒精对两种细菌生物膜的形成均有一定破坏作用,5％、10％浓度酒精对金黄色葡萄球菌生物膜破坏最大,活菌与死菌比例为0.142 ±0.007、0.006±0.001;10％浓度酒精对大肠埃希菌生物膜破坏最大,活菌与死菌比例为5.751±1.779.结论 较低浓度的酒精可抑制金黄色葡萄球菌和大肠埃希菌生物膜的形成,且10％浓度的酒精效果最好.     

EFFICIENT PRODUCTION OF Staphylococcus simulans LYSOSTAPHIN IN A BENCHTOP BIOREACTOR BY RECOMBINANT Escherichia coli

Lysostaphin is an enzyme with bactericidal activity against Staphylococcus aureus and other staphylococcal species. In spite of many advantages and promising results of preliminary research, the enzyme is still not widely used in medicine, veterinary medicine, or as a food preservative. One of the most important factors limiting application of the enzyme in clinical or technological practice is the high cost of its production. In this study we have determined the optimal conditions for lysostaphin production in a 5-L batch bioreactor. The enzyme production was based on a heterologous, Escherichia coli expression system designated as pBAD2Lys and constructed earlier in our laboratory. An evident influence of physicochemical conditions of the process (areation, pH and temperature) and composition of the growing media on the amount and activity of produced enzyme was noticed. Efficiency of production of about 13,000 U/L has been achieved in the optimal conditions of the production process: low aeration (400 rpm of mechanical stirrer), pH 6, and temperature 37°C in classical LB medium. Further, about twofold improvement in the production efficiency of the enzyme was achieved as a result of modification of composition of growing media. Finally, more than 80,000 units of lysostaphin were obtained from one (batch) bioreactor with 3 L of culture of E. coli TOP10F’ transformed with pBAD2Lys plasmid. To the best of our knowledge, this is the most efficient method of production of recombinant lysostaphin in E. coli expression systems described to date.     

DOPA Production with Enterobacter cloacae NB 320 by Transamination Reaction

Taxonomical investigation was performed on the bacterium, strain NB 320 isolated from soil, and it was identified as Enterobacter cloacae. This bacterium produced the enzyme which catalyzed the transamination reaction between 3,4-dihydroxyphenyl pyruvate and an amino acid to form l-Dopa.

The optimum culture conditions for the enzyme production were studied along with the characteristics of the enzyme. The enzyme of the strain was different in some properties from that of Alcaligenes faecalis IAM 1015 which had been already studied. The former utilized glutamate as an amino donor best among the amino acids tested for transamination and was induced by the addition of glutamine and asparagine. Intact cells of the strain did not catalyze the reaction unless they were treated with sonication or with a detergent.     

不同类型铜合金板的杀菌特性

【目的】微生物对可接触表面的污染给公共卫生带来了极大的威胁。利用具有杀菌特性的铜及铜合金代替不锈钢等制品,可以降低消毒剂的使用和细菌的传播。【方法】通过分析3株金黄色葡萄球菌和2株大肠杆菌在铜及铜合金平板上的存活时间,对不同类型铜合金的杀菌特性进行了探索。【结果】铜合金平板的杀菌能力与其铜含量成正比;铜合金对同属细菌的杀菌能力相近,对不同属细菌则有一定差异;铜合金的杀菌效率与细菌对Cu2+抗性没有直接联系;铜合金杀菌的效率与细菌的细胞壁结构可能有很大关联。【结论】铜及铜合金是较好的杀菌材料。     

沙门菌、大肠杆菌和金黄色葡萄球菌的多重PCR检测

根据沙门菌invA基因、大肠杆菌phoA基因和金黄色葡萄球菌nuc基因序列,设计3对特异性引物进行多重PCR并对反应条件进行优化。结果表明3对引物能特异地扩增出284bp、622bp、484bp的目的条带;最佳反应条件为沙门菌、大肠杆菌、金黄色葡萄球菌的引物浓度分别为40nmol／L、40nmol／L、80nmol／L,Mg^2＋浓度2．4mmol／L,dNTP浓度2001μmol／L,Taq DNA聚合酶1.5u,退火温度55．0℃-57．4℃之间;在此条件下多重PCR同时检测DNA的敏感性分别是10．2pg、10．2pg、102．0pg,检测时间4h。建立的多重PCR是一种敏感、特异、准确、快速的方法,为同时检测食品中沙门菌、大肠杆菌和金黄色葡萄球菌奠定了基础。     

Purification of pluripotent embryonic stem cells using dielectrophoresis and a flow control system

Pluripotent stem cells (PSCs) such as embryonic stem cells and induced PSCs can differentiate into all somatic cell types such as cardiomyocytes, nerve cells, and chondrocytes. However, PSCs can easily lose their pluripotency if the culture process is disturbed. Therefore, cell sorting methods for purifying PSCs with pluripotency are important for the establishment and expansion of PSCs. In this study, we focused on dielectrophoresis (DEP) to separate cells without fluorescent dyes or magnetic antibodies. The goal of this study was to establish a cell sorting method for the purification of PSCs based on their pluripotency using DEP and a flow control system. The dielectrophoretic properties of mouse embryonic stem cells (mESCs) with and without pluripotency were evaluated in detail, and mESCs exhibited varying frequency dependencies in the DEP response. Based on the variance in DEP properties, mixed cell suspensions of mESCs can be separated according to their pluripotency with an efficacy of approximately 90%.     

High pressure homogenization inactivation of Escherichia coli and Staphylococcus aureus in phosphate buffered saline,milk and apple juice

High pressure homogenization (HPH) offers new opportunities for food pasteurization/sterilization. Escherichia coli and Staphylococcus aureus suspended in phosphate buffered saline (PBS) buffer, milk and apple juice at initial concentration of ~10⁶ log₁₀ CFU per ml were subjected to HPH treatments up to 200 MPa with inlet temperatures at 4–40°C. After HPH at 200 MPa with the inlet temperature at 40°C, the count of E. coli suspended in PBS, milk and apple juice reduced by 3·42, 3·67 and 3·19 log₁₀ CFU per ml respectively while the count of S. aureus decreased by 2·21, 1·02 and 2·33 log₁₀ CFU per ml respectively suggesting that S. aureus was more resistant. The inactivation data were well fitted by the polynomial equation. Milk could provide a protective effect for S. aureus against HPH. After HPH at 200 MPa with the inlet temperature at 20°C, the cell structure of E. coli was destroyed, while no obvious damages were found for S. aureus.     

Tissue engineering with electric fields: immobilization of mammalian cells in multilayer aggregates using dielectrophoresis

Positive dielectrophoresis can be used to create aggregates of animal cells with 3D architectures. It is shown that the cells, when pulled together into an aggregate by positive dielectrophoresis in a low-conductivity iso-osmotic solution, adhere to each other. The adherence of the cells to each other is non-specific and increases in time, and after 10-15 min becomes strong enough to immobilize the cells in the aggregate, enabling the ac electric field to be released, and the iso-osmotic buffer to be replaced by growth or other media. Cell viability is maintained. The new method of immobilization significantly simplifies the construction of aggregates of animal cells by dielectrophoresis, and increases the utility of dielectrophoresis in tissue engineering and related areas.     

乳酸杆菌细胞裂解物对家兔实验性阴道炎的治疗作用

目的观察乳杆菌DM8909裂解物及发酵物在体内对大肠埃希菌、金黄色葡萄球菌的抑制效果,探索一种替代活菌制剂的生态制剂。方法用阿莫西林和甲硝唑接种家兔建立脱污染模型,再用大肠埃希菌和金黄色葡萄球菌的混合液接种,建立实验性感染模型后,随机分为5组,分别用乳杆菌裂解物原液、裂解物稀释液、发酵上清液、乳杆菌活菌制剂及生理盐水(对照组)进行治疗,分析和考察阴道菌群变化。结果德氏乳酸杆菌裂解物具有治疗实验性家兔细菌性阴道炎的效果,其疗效与活菌制剂相近。结论德氏乳酸杆菌裂解物可作为乳酸杆菌活菌制剂的替代物。     

Nucleotide sequence of the ethidium efflux gene from Escherichia coli

The nucleotide sequence of the gene specifying the ethidium efflux system of Escherichia coli has been determined. The translated open reading frame has identified a membrane-bound polypeptide of 110 amino acids (11,960 Da) which shares 42% identity with a staphylococcal protein specifying resistance to ethidium.     

Involvement of endotoxin in the mortality of mice with gut‐derived sepsis due to methicillin‐resistant Staphylococcus aureus

MRSA causes a wide diversity of diseases, ranging from benign skin infections to life‐threatening diseases, such as sepsis. However, there have been few reports of the pathophysiology and mechanisms of sepsis resulting from the gut‐derived origin of MRSA. Therefore, we established a murine model of gut‐derived sepsis with MRSA and factors of MRSA sepsis that cause deterioration. We separated mice into four groups according to antibiotic treatment as follows: (i) ABPC 40 mg/kg; (ii) CAZ 80 mg/kg; (iii) CAZ 80 mg/kg + endotoxin 10 μg/mouse; and (iv) saline‐treated control groups. Gut‐derived sepsis was induced by i.p. injection of cyclophosphamide after colonization of MRSA strain 334 in the intestine. After the induction of sepsis, significantly more CAZ‐treated mice survived compared with ABPC‐treated and control groups. MRSA were detected in the blood and liver among all groups. Endotoxin levels were significantly lower in the CAZ‐treated group compared to other groups. Inflammatory cytokine levels in the serum were lower in the CAZ‐treated group compared to other groups. Fecal culture showed a lower level of colonization of E. coli in the CAZ‐treated group compared to other groups. In conclusion, we found that CAZ‐treatment ameliorates infection and suppresses endotoxin level by the elimination of E. coli from the intestinal tract of mice. However, giving endotoxin in the CAZ‐treated group increased mortality to almost the same level as in the ABPC‐treated group. These results suggest endotoxin released from resident E. coli in the intestine is involved in clinical deterioration resulting from gut‐derived MRSA sepsis.