تولید پروتئین دگرسان در زیست لایه های اشرشیا کولی: یک فرم متداول کشت چگالی سلول بالا

Heterologous protein production in Escherichia coli biofilms: A non-conventional form of high cell density cultivation

چکیده
1-مقدمه
2-منابع و روشها
2-1 صفت موروثی باکتریایی، پلاسمیدها و شرایط کشت
2-2 سیستم تشکیل زیست لایه و نمونه برداری
2-3 روشهای تحلیلی
2-4 بیان پروتئین دگرسان
2-6 تحلیل آماری
3- نتایج
4- بحث
5- نتیجه گیری

چکیده

اشرشیا کولی، یکی از میزبانان مساعد برای تولید پروتئین دارای صفات ارثی جدید است و به صورت یک مدل عالی برای مطالعات زیست لایه شناخته میشود. کشتهای چگالی سلول بالای (HCDC) این باکتری، بازده های تولید حجمی جذابی را میسر میسازند و سلولهایی که در زیست لایه رشد می یابند در برخی چالشهای کشتهای پلانکتونی چگالی بالای سلول دخیل هستند.
این اثر، پتانسیل تولید سلولهای زیست لایه E.coli JM109 (DE3) را ارزیابی میکنند که یک پروتئین الگو را بیان میکنند، پروتئین فلوئورسان سبز ارتقا یافته (eGFP )، از یک پلاسمید دارای صفات ارثی متشکل جدید. یک صفت موروثی پرورش دهنده ی تیره ی پلاسمیدی یکسان اما دچار کمبود ژن eGFP برای ارزیابی اثر تولید پروتئین دگرسان بر تشکیل زیست لایه بکار رفت. نتایج نشان میدهد تولید eGFP از سلولهای زیست لایه در حدود 30 برابر بالاتر از حالت پلانکتونی بود. به علاوه، سلولهای بیان کننده ی eGFP ، تشکیل زیست لایه را در مقایسه با سلولهای کنترل ارتقا داده بودند. مقادیر تولید حجمی، دو برابر مقادیر قبلا گزارش شده با همان پروتئین بود و در طیف چیزی هستند که میتواند با HCDC در تولید پروتئینهای حلال بدست آید. گرچه چگالی بالای سلولی که بدست آمد از HCDC متداول پایین تر بود (0.5 برابر)، این سیستم نوین به مقادیر تولید خوبی دست یافت که احتمالا پس از بهینه سازی شرایط کشت، بهبود می یابند.
1- مقدمه
باکتری گیاهی گرم-منفی اشرشیا کولی، یک میزبان ارجح برای تولید پروتئینهای دارای صفات ارثی متشکل جدید محسوب میگردد که به خاطر رشد سریع آن در چگالی های بالای سلولی، شرایط مورد نیاز غذایی ساده، ژنتیک کاملا شناخته شده و دسترسی به شمار زیادی از حاملهای تولید مثل غیر جنسی و صفات موروثی میزبان جهش یافته است. این باکتری گیاهی دارای توانایی انباشته کردن بسیاری از پروتئینهای دارای صفات ارثی متشکل به حداقل 20% از پروتئین سلولی کل میباشد و در پاره ای از موارد، آنها را از سیتوپلاسم به پریپلاسم منتقل میکند. کشت ای.کولی در پرورشهای چگالی بالای سلول (HCDC)، مزایای زیادی را ارائه میدهد همانند حجم کشت کاهش یافته، فراوری پایین دست ارتقا یافته و هزینه های تولید پایین تر. با وجود این مزایا، هنوز هم چالشهای زیادی وجود دارد که باید در HCDC مورد بررسی قرار گیرند و آنها عبارتند از انتقال اکسیژن ناکافی، شرایط لازم محیط کشت ویژه، بازده ترکیب کاهش یافته در راکتور، تجمع کربن دی اکسید که مقادیر رشد تولید استات بیشتر را کاهش میدهند. تولید پروتئین دارای صفات ارثی متشکل جدید توسط سلولهای زیست لایه در برخی از چالشهای HCDC متداول بویژه پخش مواد مغذی و اکسیژن از طریق زیست لایه و هم چنین در انباشتن تولیدات ضایعات سمی دخیل است.
بیان پروتئین دارای صفات ارثی متشکل جدید در زیست لایه های ای.کولی توسط هوآنگ و همکارانش پیشتاز شد که تولید بتا-گالاکتوسیداز را در E.coli DH5a حامل یک پلاسمید حاوی ترویج دهنده ی tac بررسی کرده اند. بعدها، اُکونل و همکارانش، نخستین سیستم برای تولید پروتئین دگرسان سطح بالا را در سلولهای زیست لایه ای.کولی با استفاده از یک حامل مبتنی بر pUC برای بیان پروتئین فلوئورسان سبز ارتقا یافته (e.GFP) توصیف نموده اند. با وجود پتانسیل عظیم این سیستم بیان کننده، تولید پروتئین دگرسان از سلولهای زیست لایه ای.کولی به طور وسیعی بررسی نشده باقی می ماند.
5- نتیجه گیری
استفاده از سلولهای ای.کولی در HCDC یک شیوه ی بسیار جذاب تولید پروتئینهای دگرسان از طریق بهبود بهره وری های حجمی و کاهش سرمایه گذاری پولی و هزینه های عملیات در تاسیسات تولید است. تولید پروتئینهای دگرسان در سلولهای زیست لایه در بخشی از چالشهای HCDC دخیل است اما برخی مطالعات نشان داده اند سلولهای زیست لایه از یک ظرفیت تولید ویژه عالی برخوردارند زمانیکه با همتاهای پلانکتونی خود مقایسه میشوند. این اثر نشان میدهد سلولهای زیست لایه ای.کولی میتوانند یک پروتئین دگرسان الگو را در مقادیر بسیار بالاتر از سلولهای پلانکتونی تولید کنند و چگالی های سلولی و بهره وری های حجمی از قبل در طیف چیزی هستند که میتوانند توسط HCDC بدست آیند حتی بدون بهینه سازی شرایط کشت. اثر آینده، بهینه سازی این شرایط را برای مهار قدرت این زیست لایه های تولید کننده بررسی خواهد کرد.

Abstract

Escherichia coli is one of the favourite hosts for recombinant protein production and is recognized as an excellent model for biofilm studies. High cell density cultures (HCDC) of this bacterium enable attractive volumetric production yields and cells growing in biofilms share some of the challenges of conventional high cell density planktonic cultures. This work assesses the production potential of E. coli JM109(DE3) biofilm cells expressing a model protein, the enhanced green fluorescent protein (eGFP), from a recombinant plasmid. A control strain harbouring the same plasmid backbone but lacking the eGFP gene was used to assess the impact of heterologous protein production on biofilm formation. Results show that specific eGFP production from biofilm cells was about 30 fold higher than in planktonic state. Moreover, eGFP-expressing cells had enhanced biofilm formation compared to control cells. Volumetric production values were 2 fold higher than those previously reported with the same protein and are within the range of what can be obtained by conventional HCDC in the production of soluble proteins. Although the cellular density that was obtained was lower than in conventional HCDC (0.5 fold), this novel system reached good production values which are likely to be improved after optimization of culture conditions.

1- Introduction

The Gram-negative bacterium Escherichia coli is a preferred host for the production of recombinant proteins [1,2] due to its fast growth at high cell densities, simple nutrient requirements, well-known genetics and the availability of a large number of cloning vectors and mutant host strains [3]. This bacterium has the ability to accumulate many recombinant proteins to at least 20% of the total cell protein [4] and, in some cases, to translocate them from the cytoplasm to the periplasm [5]. E. coli cultivation in high cell density cultures (HCDC) presents many advantages such as reduced culture volume, enhanced downstream processing and lower production costs [6]. Despite these advantages, there are still many challenges that have to be addressed in HCDC and these include insufficient oxygen transfer, specific culture medium requirements, reduced mixing efficiency in the reactor, accumulation of carbon dioxide which decreases growth rates and increased acetate production [5,6]. Recombinant protein production by biofilm cells shares some of the challenges of conventional HCDC, namely in diffusion of nutrients and oxygen through the biofilm and also in the accumulation of toxic waste products [7,8]. Recombinant protein expression in E. coli biofilms was pioneered by Huang et al. [9- 11] who have studied the production of -galactosidase in E. coli DH5α carrying a plasmid containing the tac promoter. Later, O’Connell et al. [12] have described the first system for high level heterologous protein production in E. coli biofilm cells using a pUC-based vector for the expression of enhanced green fluorescent protein (eGFP). Despite the enormous potential of this expressing system, heterologous protein production from E. coli biofilm cells remains largely unexplored.

5 -Conclusions

Using E. coli cells in HCDC is a very attractive way of producing heterologous proteins by improving volumetric productivities and reducing capital investment and operation costs in production facilities. Producing heterologous proteins in biofilm cells shares some of the challenges of HCDC, but some studies have shown that biofilm cells have a superior specific producing capacity when compared to their planktonic counterparts. This work shows that E. coli biofilm cells can produce a model heterologous protein at much higher levels than planktonic cells and that the cellular densities and volumetric productivities are already within the range of what can be obtained by HCDC, even without optimization of culture conditions. Future work will address the optimization of these conditions in order to harness the power of these productive biofilms..

فیتاز از باکتری Weissella halotolerans: تخلیص، مشخصات جزئی و اثر برخی فلزات

Phytase from Weissella halotolerans: purification, partial characterisation and the effect of some metals

Abstract

In the study, phytase was purified in three simple steps comprising ammonium sulphate precipitation, anion exchange and gel filtration chromatography from Weissella halotolerans. The enzyme was obtained with a specific activity of 227.73 EU/mg and 6.52% recovery. The molecular mass of the enzyme was determined to be 41.52 kDa. The optimum pH and temperature for the enzyme were 6.0 and 50°C, respectively. Furthermore, the effects of metal ions on the enzyme were investigated. Ag+ , Zn2+, Cr2+ and Fe2+ ions inhibited phytase by 21.86, 25.63, 32.82 and 90.43%, respectively, whereas Co2+, Cu2+, Pb2+, Cd2+ ve Mn2+ ions increased the enzyme activity.

1 Introduction

Phytic acid (myo-inositol hexakisphosphate) is a common compound of plant-derived foods and constitute 1–5% of edible cornflakes, pollen, and hazelnut oil and seeds in weight. It is an organic form of phosphorus that constitute 60–90% of the total phosphorus content and plays an anti-nutritive role.[1–3] Although they are important in many physiological functions, especially in seed germination, phytates are solely considered as anti-nutritive compounds because they bind starch and proteins. Phytases affect protein digestion and interact with proteins, and even inhibit the activity of digestive enzymes. They also have a strong chelating effect on divalent minerals including Ca2+, Mg2+, Zn2+ and Fe2+.[4] Although it is a strong chelator of iron and zinc in herbal foods, as a result of being a mediator for these metals, phytate actually acts as an antioxidant that reduces their free radical forms.[4] The effective degradation of phytic acid is obtained through both enzymatic and non-enzymatic degradation.[5] Enzymatic degradation is obtained by using phytase that was derived from a variety of fungus and bacteria. Non-enzymatic hydrolysis is used during food processing or the physical removal of phytate from phytate-rich plant seeds to reduce the phytate levels in end food products.[6]

Phytase (myo-inositol hexakisphosphate phosphohydrolase E.C. 3.1.3.8) disintegrates the phosphoric monoester bonds in phytic acid and phytate. This results in a series of phosphate ester release from myoinositol; therefore, it gained a quick worldwide acceptance as a food additive and animal feed.[7] In the last two decades, it drew the attention of scientists and entrepreneurs for its nutrition, environmental protection and other biotechnological applications. Phytases have many applications in myo-inositol phosphate preparation. Moreover, they are used as precursors and soil improvers in the semi-synthesis of peroxidases in the paper industry and in plant growth.[2] In addition, along with their applications in aquaculture, [8–10] they have important applications in human nutrition; [11] hence, phytases are widely used as food and feed additives and are used to reduce phosphorus pollution in animal feed.[12]

Bacterial phytases have a significant advantage over fungal phytases thanks to their high temperature stability, phytate substrate specifity, resistance to proteolysis and wide pH profile; thus, the purification and characterisation of phytases, which have thermostability and are active at neutral pH, are important in industrial applications. In the study, phytase was purified from lactic acid bacteria, Weissella halotolerans, in three steps comprising ammonium sulphate precipitation, Q-Sepharose anion exchange chromatography and Sephadex G-100 gel filtration column. The characteristic properties of the enzyme were determined and the effects of metal ions on the enzyme activity were investigated.

تعیین مشخصات و شناسایی LAB احتمالی پروبیوتیکی با فعالیت فیتاز

Screening, Characterization and Identification of Probiotic Potential LAB with Phytase Activity

Abstract

Probiotics have been extensively studied and explored commercially in many different products in the world. Looking to the demand and health prospects extensive screening for Lactic Acid Bacteria (LAB) with probiotic potentials was investigated. Total 57 bacterial isolates were collected from the 32 samples. Out of which, 33 LAB isolates were confirmed on the basis of morphological and preliminary biochemical characterization. On the basis of probiotic traits such as low pH and high bile salt tolerance, 8 LAB isolates with probiotic potentials were selected. These were further evaluated for phytase activity which is very important for availing various minerals and proteins in the gut. Two LAB isolates found to show significant phytase activity. Considering the probiotic potential along with phytase activity these isolates show health prospects and therefore they were characterized for identification. Morphological, biochemical and molecular characterization studies revealed Isolate 21 as strain of Pediococcus acidilactici (KU173544) and Isolate 24 (KU173545) as a strain of Pediococcus lolii.

1 Introduction

According to world health organization probiotics defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. There is growing evidence for their efficacy in protecting against acute diarrhoeal disease in children, gastroenteritis and antibioticassociated diarrhoea, inflammatory bowel diseases and pouchitis (Reid et al., 2003; Geier et al., 2007; Nomoto K., 2005; Weng and Walker, 2006). There is also evidence to support further investigation of the use of probiotics and prebiotics in the treatment of illnesses affecting sites other than the intestinal tract, e.g. urinary tract infections, vaginal infections, arthritis, atopic eczema, pharyngitis and otitis media (Reid et al., 2003; Geier et al., 2007; Tagg and Dierksen, 2003; Lenoir et al., 2007).The numerous perceived health benefits and the growing awareness about probiotics have caught the attention of the food industry (Saarela et al., 2002; Salminen and Gueimonde, 2004). Food companies are increasingly manufacturing foods with incorporated probiotic bacteria, which fall under the new category of foods so called functional foods. Probiotic dairy products such as yogurts containing L. acidophilus and Bifidobacterium spp. constitute a significant amount among the commercially available probiotic foods (Reid et. al., 2003). The goal of functional foods is to improve the quality of life, enhance health status and increase lifespan while maintain health preventing diseases.

Besides imparting all these beneficial attributes probiotics are also known to produce various enzymes that help in the digestion of monogastric animals, phytases and lipases being the important ones. Phytic acid (myo-inositol hexakis phosphate, phytate) is the major storage form of phosphorus in cereals, oil and legumes (Khattak et al., 2006). Phytase, a specific group of phosphatase, hydrolyses phytic acid to myo-inositol and phosphoric acid. In the context of human and animal nutrition, two aspects of phytate are critically important (Reddy et al. 1982; Wodzinski and Ullah, 1996): (i)monogastric animals have only low levels of phytate-degrading enzymes in their digestive tracts, and since phytic acid itself is not absorbed. (ii) phytic acid is considered to be an anti-nutrient factor as it forms complexes with proteins and a variety of metal ions and therefore decreases the dietary availability of these nutrients. Because of these problems, there is considerable interest in phytate-degrading enzymes. Inorganic phosphate is therefore added to their diet to meet the phosphorus requirement, while undigested phytate phosphorus is excreted in manure and poses a serious phosphorus pollution problem, contributing to the eutrophication of surface water in areas of intensive livestock production (Bajaj and Wani, 2011). The formation of insoluble mineral-phytate complexes at physiological pH values is regarded as the major reason for the poor mineral bioavailability, because these complexes are essentially non absorbable from the human gastrointestinal tract (Greiner and Konietzny, 2006). Minerals are involved in activation of intracellular and extracellular enzymes, in regulation of critical pH levels in body fluids necessary for the control of metabolic reactions and in osmotic balance between the cell and its environment. A deficiency of any one of the essential minerals can result in severe metabolic disorders and compromise the health of the organism. Some minerals deficiencies are common in developing countries, but mineral sub-deficiencies may also occur in developed countries (Lopez et al., 2002).

The objectives of present studies were to isolate and identify phytase producing probiotic potential LABs for its exploitation for better health prospects.

تقویت تولید فیتاز از یک سوش جدید پروبیوتیک Bacillus Subtilis P6

Enhancement of Phytase Production from a New Probiotic Strain Bacillus subtilis P6

Abstract

An integrated classical and statistical optimization approach involving the combination of Placket–Burman design (PBD) and Central Composite Design (CCD) was employed for increasing phytase yield. PBD was used to evaluate the effect of 9 variables related to phytase production from probiotic strain B. subtilis, and three statistically significant variables, namely, glucose, beef extract and potassium phosphate were selected for further optimization studies. The levels of five variables for maximum phytase production were determined by a CCD. The optimum values for the factors were determined via response surface methodology (RSM) as: 6.59 gl-1 of glucose, 6 gl-1 of beef extract and 2.75 gl-1 of potassium phosphate respectively. Phytase production improved from 2.74 EUml-1 to 46.76 EUml-1 indicating 17-fold increase in activity after optimization.

1 Introduction

The growing awareness of the diet and health has prompted an increasing demand of food products that can support health apart from providing basic nutrition (Haros et al., 2009). Since last six decades, a lot of antibiotics have been incorporated in animal feed to improve their growth, efficiency and to protect them from pathogenic microorganisms.

But, antibiotic resistance has become a major public health concern today (Sharma et al., 2014). The gastrointestinal microflora plays beneficial role in the health and nutrition of animals, and probiotics, live microorganisms help in the maintenance of gut microflora. Probiotics have been strongly recommended as alternatives to antibiotics for food animals (Reid and Friendship, 2002). Recently, a great deal of attention has been devoted to the genuine value of bacterial species as multifunctional probiotics, which secrete various extracellular enzymes for enhancing feed digestibility as well as many antimicrobial compounds for improving animal performance (Lee et al., 2012); among them phytase holds the key position. Phytate is a natural phosphate reservoir in plant based animal feed and acts as an anti-nutritional factor in gut of animals and humans by restricting absorption of proteins, carbohydrates, amino acids and metals viz. Zn2+, Fe3+, Ca2+ and Mg2+ . However, in order to meet the phosphorus requirement, diet of monogastric animals are supplemented with inorganic phosphorus which causes the excretion of large amount of phosphorus in the excreta resulting in environmental pollution and several human health problems (Jorquera et al., 2011). Phytase (myo-inositol hexakisphosphate phosphohydrolase, E.C. 3.1.3.8 or E.C. 3.1.3.26) are group of enzymes that hydrolyze phytate to inositol phosphate, myo-inositol and inorganic phosphate. Today, phytase extracted from fungi (Nautophos® , Allzyme® SSF, Finase® P/L) is commercialized as feed supplement; on the other hand several bacterial species have been used as probiotics.

But till date, phytase producing probiotic strains have not been commercialized. However, a probiotic strain with phytase activity can perform double function and enhance productivity of animals manifolds. Simultaneously, they can also reduce the serious environmental problems caused by undigested phytate, which is main byproduct of human and animal excreta. Hence, from last few years, this area has become a major public health concern and is drawing the interest of health and research professionals all around the world.

One of the major cornerstones in biotechnology today is the optimization of the cultivation conditions for enhancing the productivity. Screening and evaluation of nutritional requirements of microorganisms is an important step in any bioprocess development.

Optimization studies involving one factor-ata-time approach is tedious, tends to overlook the interaction among the factors and might lead to misinterpretation of results. In contrast, statistical strategies are preferred and more advantageous and mitigate the error in determining the effects of parameters in an economical manner (Sharma and Satyanarayana, 2006). The empirical technique is a traditional optimization method employing one-factor-at-a-time strategies which is simple, easy and explains the individual effect of different components. Unfortunately, it is tedious and fails to explain the interactions among the factors (Awad et al., 2011). Statistical optimization is a proven tool for overcoming the limitations of the “one-factor at a time‟ method. It is more efficient technique since it can provide statistical data with a relatively small number of experiments.

In our previous endeavor, we isolated phytase producing potential probiotic strain Bacillus subtilis P6 from exotic environment (Sharma and Trivedi, 2015). Considering the high potential of phytase for use as feed supplement and its future prospective in animal feed and human nutrition, the present appraisal therefore aims at achieving the multifold improvement in the phytase production through sequential classical and statistical optimization strategy from probiotic strain Bacillus subtilis P6 so as to verify whether it can become a new kind of feed additive for food and animal feeding in the future.

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فهرست مقاله:

چکیده 
1. مقدمه 
2. مواد و روش ها 
2.1. مواد شیمیایی 
2.2. میکروارگانیسم ها 
2.3. مدل معدی روده ای in vitro 
2.4. ارزیابی سلول های باکتریایی آزاد برای مقاومت آن ها در شرایط معده و روده کوچک 
2.5. ارزیابی سلول های باکتریایی انکپسوله شده برای مقاومت در شرایط معده و روده کوچک 
2.6. ارزیابی مقاومت دمایی سلول های باکتریایی آزاد 
2.7. ارزیابی مقاومت دمایی سلول های باکتریایی انکپسوله
2.8. آماده سازی عصاره ی پلوروتوس استراتوس
2.9. تخمین بتا گلوکان در قارچ خشک شده
2.9.1. اندازه گیری گلوکان کلی (آلفا گلوکان + بتا گلوکان) به اضافه ی D گلوکز در الیگوساکاریدها، سوکروز، و D – گلوکز آزاد
2.9.2. اندازه گیری آلفا گلوکان (فیتوگلیکان و نشاسته) به اضافه ی D گلوکز در سوکروز، و D گلوکز آزاد
2.9.3. تخمین بتا گلوکان در عصاره های قارچ 
2.10. آماده سازی میکروکپسول سین بیوتیک و کاربرد آن برای فرمولاسیون محصولات غذایی عملکردی خشک
2.10.1. ارزیابی قابلیت بقا باکتری ها در طی ذخیره سازی در میکروکپسول های سین بیوتیک الحاق شده در ماتریکس غذای خشک (مخلوط سلامتی (سالم) غلات) 
2.10.2. ارزیابی قابلیت بقا باکتری ها در طی ذخیره سازی در میکروکپسول سین بیوتیک در یک اسنک غذایی خشک هندی 
2.10.3. ارزیابی قابلیت بقا باکتری ها در طی ذخیره سازی در میکروکپسول های سین بیوتیک الحاق شده در مخلوط سلامت خشک (نوشیدنی سلامت مالت) 
2.11. آنالیز آماری 
3. نتایج و بحث 
3.1. ارزیابی سلول های باکتریایی آزاد برای مقاومت آن ها در شرایط معده و روده کوچک 
3.2. ارزیابی سلول های باکتریایی انکپسوله برای مقاومت آن ها در شرایط معده و روده کوچک
3.3. ارزیابی مقاومت دمایی سلول های باکتریایی آزاد 
3.4. ارزیابی مقاومت دمایی سلول های باکتریایی انکپسوله 
3.5. محتوای بتا گلوکان در قارچ P. اسراتوس
3.6. آماده سازی میکروکپسول سین بیوتیک و کاربرد آن برای فرمولاسیون محصولات غذایی عملکردی خشک
3.6.1. مطالعه قابلیت بقا در طی ذخیره سازی باکتری ها در میکروکپسول های باکتریایی و میکروکپسول های سین بیوتیک الحاق شده به درون یک ماتریکس غذایی خشک محلی (مخلوط سلامت غلات)
3.6.2. ارزیابی قابلیت بقا در طی ذخیره سازی میکروکپسول های سین بیوتیک در یک اسنک غذایی خشک سنتی هندی 
3.6.3. ارزیابی قابلیت بقا در طی ذخیره سازی میکروکپسول سین بیوتیک در نوشیدنی سلامت مالت خشک
4. نتیجه گیری

بخشی از ترجمه فارسی مقاله:

1. مقدمه
یک هیاهویی از محصولات پروبیوتیک سلامت محور در طی یک دهه اخیر به وجود آمده است. فعالیت بیولوژیک باکتری های پروبیوتیک، مدیون توانایی اتصال آن ها به انتروسیت ها می باشد که از این رو، با روش مستثنی کردن رقابتی، مانع از اتصال پاتوژن های روده ای، می شود. باکتری های پروبیوتیک، به دلیل خصوصیات سودمند مختلف خودشان، از جمله کاهش علایم سندرم روده تحریک پذیر، اثرات تنظیم کنندگی ایمنی و کاهش کلسترول، در صنعت غذایی مورد استفاده قرار گرفته اند (FAO/WHO, 2006). شمول باکتری های پروبیوتیک در محصولات تخمیری، ارزش آن ها را به عنوان غذاهای با عملکرد درمانی بهتر افزایش می دهد. برای همه ی محصولاتی که ادعای سلامت پروبیوتیک دارند لازم است که حداقل CFU/ml106 باکتری پروبیوتیک، تا تاریخ انقضا داشته باشند، چون مینیمم سطح درمانی در هر روز، 109-108 سلول در نظر گرفته می شود (Kailasapathy & Chin, 2000). کریمی، مرتضویان و دا کروز (2011) بیان کردند که محصولات پروبیوتیک زمانی که 100 گرم در روز مصرف می شوند حدود 109 سلول زنده را درون روده تحویل می دهند. گزارش ها نشان دهنده ی بقا ضعیف پروبیوتیک ها در محصولات غذایی و همچنین در سیستم معدی روده ای انسان می-باشد. بقا پروبیوتیک ها در یک محصول در طی مصرف، برای اثر بخشی آن ها دارای اهمیت است، چون بقا آن ها در طی فراوری و ذخیره سازی محصولات غذایی ضروری است (Mortazavian, Mohammadi, & Sohrabvandi, 2012). انتخاب سویه های پروبیوتیک بهتر و ارائه ی لایه بندی فیزیکی به آن ها، برای افزایش بقا آن ها، از جمله، استفاده از پروبیوتیک های مناسب و ترکیب نرمالی از پروبیوتیک ها و پری بیوتیک ها (سین بیوتیک ها) می تواند تحویل پروبیوتیک های زنده ی کافی در محصولات غذایی عملکردی به مصرف کنندگان را افزایش دهد. بقا پروبیوتیک-ها در ماتریکس غذایی توسط عواملی مانند PH، اسیدی شدن در طی ذخیره سازی محصولات تخمیری، تولید هیدروژن پروکسید، سمیت اکسیژن، دماهای فراوری و ذخیره سازی، نرخ و نسبت تلقیح، میکروانکپسولاسیون و پایداری در طی ذخیره سازی تحت تاثیر قرار می گیرد (Mortazavian et al., 2012). به منظور فعالیت به عنوان پروبیوتیک در دستگاه گوارش (GIT) و به منظور اعمال اثرات سودمند خودشان روی میزبان، برای باکتری ها ضروری است که مکانیسم های محافظتی برای مقاومت در برابر PH پایین در معده، آنزیم های هاضمه و صفرای موجود در روده کوچک داشته باشند (Argyri et al., 2012). مدل معدی روده ای in vitro (سیستم تک ظرفی) ترجیحا برای تقلید این میکرو محیط GIT مورد استفاده قرار می گیرد.
میکروانکپسولاسیون به عنوان یک فرایندی تعریف می شود که در آن، سلول ها درون یک غشا حفظ می شوند تا آسیب سلولی و مرگ را کاهش دهند که ذراتی در حد نانومتر (نانوانکپسولاسیون)، میکرومتر (میکروانکپسولاسیون) یا مقیاس میلی متری ایجاد می کنند (Burgain, Gaiani, Linder, & Scher, 2011). انکپسولاسیون سلول های پروبیوتیک را پایدار می کند، که به طور معنی داری بقا و پایداری آن ها در تولید و مدیریت غذاهای عملکردی و همچنین در طی هیدراتاسیون مجدد و لیپوفیلیز شدن آن ها را افزایش می دهد. آن همچنین از فعالیت متابولیک پروبیوتیک ها در دستگاه معدی روده ای محافظت می کند (Picot & Lacroix, 2004) و پایداری در طی ذخیره سازی درازمدت را تضمین می کند (Zuidam & Nedovic, 2010). به علاوه، انکپسولاسیون، خصوصیات چشایی غذا را بهبود می دهد و پایدار می کند و همچنین به توزیع همگن پروبیوتیک ها در کل محصول کمک می کند (Krasaekoopt, Bhandari, & Deeth, 2003). به نظر می رسد که قارچ ها یک کاندیدای بالقوه برای پری بیوتیک ها باشند چون آن ها حاوی کربوهیدرات هایی مانند کیتین، همی سلولز، بتا و آلفا گلوکان، مانان، زایلان و گالاکتان-ها هستند. مطالعات پیشین پیشنهاد می کنند که پلی ساکاریدهای گرفته شده از قارچ، خصوصیات تعدیل کننده ی ایمنی مانند افزایش تکثیر لنفوسیت و تولید آنتی بادی (Bao, Liu, Fang, & Li, 2001) و همچنین خصوصیات ضد سرطانی (Wasser, 2002) و کمک به حذف کلسترول و جلوگیری از چاقی دارند. آخرین یافته توسط هرست و همکارانش (2009) و تسایی و همکارانش (2009)، به ترتیب، خصوصیات ضد میکروبی و آنتی اکسیدانی قارچ ها را نشان داد. به جز خصوصیات پزشکی آن ها، قارچ های خوراکی، بهبود سلامتی معنی داری را نشان دادند چون آن ها محتوای پایینی از کالری، سدیم، چربی و کلسترول دارند و در عین حال در پروتئین، کربوهیدرات، فیبر، ویتامین ها و مواد معدنی غنی هستند. این خصوصیات تغذیه ای به قارچ ها، این پتانسیل را اعطا می کند که یک مکمل غذایی و همچنین یک عامل دارویی باشند. آن ها قادر به دستکاری ترکیب میکروبیوتای کولون در روده انسان توسط مهار پاتوژن های اگزوژن هستند (Rycroft, Jones, Gibson, & Rastall, 2001)، بنابراین، سلامت میزبان را بهبود می دهند (Roberfroid, 2002). سینیتسیا و همکارانش (2009) نشان دادند که عصاره های قارچ قادر به تحریک رشد پروبیوتیک ها هستند. نوشیدنی های پروبیوتیک عموما حاوی باکتری-های زنده هستند. آن ها همچنین باید حاوی یک منبعی از مواد مغذی برای باکتری ها برای تغذیه باشند (Savini et al., 2010). در نتیجه، مسایل کوتاه شدن ذخیره سازی و دوام در قفسه برای برخی محصولات زنده وجود دارد. به علاوه، رقابت برای مواد مغذی بین سویه های باکتریایی درون یک نوشیدنی یک عارضه ی مهم دیگر می باشد. به علاوه، پروبیوتیک های مایع نیاز به نگه داری در یخچال دارند. از طرف دیگر، پودرهای پروبیوتیک شامل پروبیوتیک هایی هستند که تحت دما و فشار پایین با سرما خشک شده اند بدون اینکه سلول ها آسیب ببینند. این یک رشد مناسب را در حالت معلق برای ذخیره سازی درازمدت باکتری های پروبیوتیک فراهم می کند. بعداز هضم، به محض اینکه مجددا رطوبت در دسترس قرار می گیرد، آن ها مجددا هیدراته می شوند و سپس مجددا یک نسبتی از سلول ها، همانند چیزی که قبل از خشک شدن با سرما بوده اند، آغاز به تقسیم شدن می-کنند. محققین، فهمیده اند که پروبیوتیک هایی که مجددا هیدراته شده اند، قادر به فراهم کردن موثر مزایای مربوطه ی خودشان هستند (Bohbot & Cardot 2012). اینکه آیا نگه داری در یخچال برای پروبیوتیک ها مورد نیاز هست یا نه بستگی به سویه های معین دارد، برخی از آن ها مقاوم به گرما و قفسه هستند و برخی از آن ها اینطور نیستند. بنابراین، کار اخیر برای مطالعه ی اثربخشی میکروکپسولاسیون در حفاظت از LAB از اسید، صفرا، و آنزیم های گوارشی برنامه ریزی شده است. این کار همچنین به ارزیابی اثربخشی انکپسولاسیون LAB با مولکول های پری بیوتیک به منظور افزایش قابلیت بقا آن ها در طی ذخیره سازی در ماتریکس غذایی خشک کمک کرده است.

بخشی از مقاله انگلیسی:

1. Introduction

There has been an explosion of health-based probiotic products since over a decade. The biological activity of probiotic bacteria owes to their ability of attachment to enterocytes thereby inhibiting the enteric pathogens from binding as a result of competitive exclusion. Probiotic bacteria are used in the food industry due to various beneficial properties including reduction of irritable bowel syndrome symptoms, immunomodulatory effects, and cholesterol reduction (FAO/WHO, 2006). Inclusion of probiotic bacteria in fermented products enhances their value as better therapeutic functional foods. It is necessary for all products having probiotic health claims to have minimum 106 CFU/mL probiotic bacteria till expiry date, since minimum therapeutic level per day is considered to be 108 –109 cells (Kailasapathy and Chin, 2000). Karimi et al. (2011) stated that probiotic products when consumed 100 g per day deliver about 109 viable cells into the intestine. Reports indicate poor survival of probiotics in food products as well as in the human gastro-intestinal system. Viability of probiotics in a product during consumption is important for their efficacy, as their survival is essential during processing and storage of food products (Mortazavian et al., 2012). Selecting better probiotic strains and providing them physical layering to enhance their survival, including the use of appropriate prebiotics and the optimal combination of probiotics and prebiotics (synbiotics), can increase the delivery of sufficient viable probiotics in functional food products to the consumers. Viability of probiotics in food matrix is affected by factors like pH, acidification during storage of fermented products, production of hydrogen peroxide, oxygen toxicity, processing and storage temperatures, rate and proportion of inoculation, micro-encapsulation, and stability during storage (Mortazavian et al., 2012). In order to act as probiotic in the gastrointestinal tract (GIT) and to exert their beneficial effect on the host it is essential for the bacteria to have protective mechanisms to withstand the low pH in the stomach, digestive enzymes, and bile in the small intestine (Argyri et al., 2012). In-vitro gastro intestinal model (one pot system) is preferably used to mimic this GIT micro-environment. Microencapsulation is defined as a process in which the cells are retained within a membrane to reduce cell injury and death, producing particles in the nanometer (nanoencapsulation), micrometer (microencapsulation) or millimeter scale (Burgain et al., 2011). Encapsulation stabilises the probiotic cells, significantly enhancing their viability and stability in the production and handling of functional food as well as during their rehydration and lyophilisation. It also preserves the metabolic activity of probiotics in the gastrointestinal tract (Picot and Lacroix, 2004), and ensures viability during long-term storage (Zuidam and Nedovic, 2010). In addition, encapsulation improves and stabilises the sensory properties of the food as well as aids in the homogeneous distribution of probiotics throughout the product (Krasaekoopt et al., 2003). Mushrooms seem to be a potential candidate for prebiotics as it contains carbohydrates like chitin, hemicellulose, β and α-glucans, mannans, xylans, and galactans. Previous studies suggested that the polysaccharides from mushroom have immunomodulating properties like enhancement of lymphocyte proliferation and antibody production (Bao et al., 2001) as well as antitumor properties (Wasser, 2002) and help in cholesterol removal and prevention of obesity. Latest finding by Hearst et al. (2009) and Tsai et al. (2009) revealed antimicrobial and antioxidant properties of mushrooms, respectively. Other than its medicinal properties, edible mushrooms also show significant health improvement as they have low content of calories, sodium, fat, and cholesterol, while they are rich in protein, carbohydrate, fibre, vitamins, and minerals. These nutritional properties give mushrooms the potential to become a food supplement as well as a pharmaceutical agent. They are able to manipulate the composition of colonic microbiota in human gut by inhibition of exogenous pathogens (Rycroft et al., 2001), thus improving the host health (Roberfroid, 2002). Synytsya et al. (2009) showed that mushroom extracts were able to stimulate the growth of probiotics. The probiotic drinks generally contain live bacteria. They must also contain a source of nutrition for the bacteria to feed upon (Savini et al., 2010). Consequently, there is storage and shelf-life issue shortened for some live products. Furthermore, competition for nutrition between bacterial strains within a drink is another important complication. In addition, liquid probiotics all require refrigeration. On the other hand probiotic powders consist of probiotics that have been freeze-dried under low temperature and pressure without damaging the cells. This provides a suitable growth suspended state for the long-term storage of probiotic bacteria. Once moisture becomes available again after ingestion they rehydrate and subsequently, a proportion of cell start to divide again as before being freeze-dried. Researchers have found that the rehydrated probiotics are capable of effectively providing their respective benefits (Bohbot and Cardot, 2012). Whether or not refrigeration is required for probiotics is dependent upon the actual strains, some of which are heat and shelf stable and some of which are not. Hence, the present work was planned to study the efficiency of microencapsulation in protecting the LAB from acid, bile, and digestive enzymes. This work also aimed to evaluate the efficiency of encapsulation along with prebiotic molecules to increase their survivability during storage in dry food matrix.

یک بررسی در مورد روش های تشخیصی بیماری تب مالت

A Review on Diagnostic Methods of Brucellosis

Abstract

We reviewed different diagnostic methods of brucellosis in different livestock and humans. Bacteriological diagnosis, immunohistochemistry (an alternative technique for direct diagnosis) and different molecular methods for Brucella species genotyping are among the direct methods for diagnosis of brucellosis discussed in this review. The well-established indirect methods for diagnosis of brucellosis, serological and brucellin allergic skin tests were also critically conferred. Finally, for effective control and prevention of brucellosis around the world, the direct diagnostic methods are advised in order to develop vaccine against the circulating Brucella strain in the specific country.

1 Introduction

Brucellosis is an ancient disease that can possibly be traced back to the 5th plague of Egypt around 1600 BC. Recent examination of the ancient Egyptian bones, dating to around 750 BC, showed evidence of sacroilitis and other osteoarticular lesions, common complications of brucellosis [1]. David Bruce isolated Brucella melitensis (B. melitensis) (Micrococcus melitensis at that time) in 1887 from the spleen of a British soldier who died from a febrile illness (Malta fever) common among military personnel stationed on Malta. For almost 20 years aіer isolation of M. melitensis, Malta fever remained a mystery and was thought to be a vector-borne disease until Нemistocles Zammit accidentally demonstrated the zoonotic nature of the disease in 1905 by isolating B. melitensis from goat’s milk. It was believed that goats were not the source of infection since they did not become ill when inoculated with Brucella cultures. Нe discovery that healthy goats could be carriers of the disease has been termed one of the greatest advances ever made in the study of epidemiology [2,3].

Brucellosis is caused by Gram-negative coccobacilli of the genus Brucella [4,5]. In livestock, the disease results in significant economic losses due to reproductive impairment caused by abortion, stillbirth or weak calves and neonatal mortality, infertility [6]. In humans, Brucella spp. infection causes a febrile disease that may be associated with a broad spectrum of symptoms, and it may be fatal in some cases [5,7]. Currently, there are ten spp. described in the genus Brucella. Each one may infect diوٴerent host spp., but each Brucella spp. has a preference for its host spp., B. melitensis (sheep and goats), B. abortus (cattle), B. suis (pigs), B. ovis (rams), B. canis (dogs), B. microti (rodents-Microtus arvalis), B. neotomae (rodents – Neotoma lepida), B. pinnipedialis (pinnipeds), B. ceti (cetacea), and B. inopinata (originally isolated from a human patient, but its preferential host is not known) [8,9]. Нree of this Brucella spp. can be subdivided in biotypes [10,11].

Нerefore, three biotypes (1-3) have been identified in B. melitensis; eight biotypes (1-7,9) in B. abortus; and five biotypes (1-5) in B. suis [12]. All Brucella spp. are considered potentially pathogenic for humans, with the exceptions of B. neotomae, B. microti, and B. ovis [6,9].

A precise diagnosis of Brucella spp. infection is important for the control of the disease in animals and consequently in man. Clinical diagnosis is based usually on the history of reproductive failures in livestock, but it is a presumptive diagnosis [13] that must be confirmed by laboratory methods [13,14]. Нe “gold standard” in the diagnosis of brucellosis is bacterial isolation from blood or bone marrow specimens that requires long cultivation periods (4 to 7 days up to 40 days) and oіen the blood cultures are unsuccessful [15]. Serological tests, such as serum agglutination test (SAT), rose Bengal plate test (RBPT), complement fixation test (CFT), and enzyme-linked immunosorbent assay (ELISA) are still frequently used [5,16]. Since the routine identification and diوٴerentiation of brucellosis suspected specimens, based on culture isolation and phenotypic characterization, requires biosafety level-3 (BSL-3) protocols for the high risk of laboratoryacquired infections [17], molecular methods have been explored in order to overcome these diٹculties. Furthermore, the polymerase chain reaction (PCR)-based assays have shown a higher sensitivity with respect to the standard microbiological assay for the diagnosis of brucellosis [18].

Нerefore, the objectives of this paper are to review a diagnostic methods that are used for isolation, screening, monitoring or epidemiological surveillance and complementary or confirmatory for brucellosis in livestock and humans.

شناسایی تنوع باکتریهای واژینال و رابطه آن با واژینوز باکتریال تشخیص داده شده بالینی با دناتورینگ الکتروفورز ژل گرادیانی و تحلیل تطابق

Identification of vaginal bacteria diversity and it’s association with clinically diagnosed bacterial vaginosis by denaturing gradient gel electrophoresis and correspondence analysis

Abstract

Bacterial vaginosis (BV) is a common complex associated with numerous adverse health outcomes, affecting women of different ages throughout the world. The etiology of BV remains poorly understood due to the difficulty of establishing a molecular genetic criterion to recognize the vaginal microbiota of BV-positive women from that of the normal women. We used techniques of broad-range PCR-DGGE and gel imaging analysis system cooperated with 16S rRNA gene sequencing and statistical analysis to investigate the community structure of the healthy and BV-affected vaginal microbial ecosystems. The community of vaginal bacteria detected in subjects with BV was far more luxuriant and diverse than in subjects without BV. The mean number of microbial species in 128 BV-positive women was nearly two times greater than in 68 subjects without BV(4.05 ± 1.96 versus 2.59 ± 1.14). Our sequencing efforts yielded many novel phylotypes (198 of our sequences represented 59 species), including several novel BV-associated bacteria (BVAB) and many belonging to opportunistic infections, which remain inexplicable for their roles in determining the health condition of vaginal microflora. This study identifies Algoriphagus aquatilis, Atopobium vaginae, Burkholderia fungorum, Megasphaera genomosp species as indicators to BV and subjects with BV harbor particularly taxon-rich and diverse bacterial communities. Maybe Bifidobacterium, Staphylococcus or even more alien species are commensal creatures in normal vaginal microbiota.

1 Introduction

Bacterial vaginosis (BV) is a common vaginal disorder characterized by an alteration of the normal vaginal bacterial microbiota to a heterogeneous state containing a complex population of anaerobic and microaerophilic organisms. BV is associated with sexually transmitted infections and adverse pregnancy outcomes such as low birth weight of foetus, premature birth, neonatal death and secondary infection after delivery and is also the risk factor for premature rupture of fetal membranes (PROM) (Goldenberg et al., 2008). Antibiotics treatment of the condition used to failure due to the uncertain variation of BV-associated bacteria (BVAB) (Beigi et al., 2004; Ferris et al., 2004). Many BV associated bacteria are resistant to culture and (or) difficult to detect by traditional methods, which thwart the exploration of the pathogenesis of BV. So far, there is a lack of effective prevention and treatment measures.

According to Nugent’s classification (Nugent et al., 1991), a score ranged from 7 to 10 is considered as BV, a clinical condition dominated by the morphological identification of Gram-negative and-positive bacteria, without evidence of Lactobacillus morphotypes. In contrast, the score from 0 to 3 is considered an undisturbed vaginal microflora dominated by the Lactobacillus genus, identified as the principal Gram-positive rods bacteria.

In general, race (nationality) (Ness et al., 2003; Uscher-Pines et al., 2009), behavioral habits (Bradshaw et al., 2013), personal hygiene status (Brotman et al., 2008), and so on have been considered as causes of BV. Although there is agreement in the literature that no single agent likely causes BV, there is no consensus about what constitutes a pathogenic bacterial community in this syndrome. Comprehensive cultivation-independent comparisons of the vaginal bacterial communities between subjects with and without clinically defined BV have been rare.

With the development and application of culture-independent nucleic acid amplification test (NAAT) in recent years, it is possible to explore the structure and dynamic shift of microbiota (Larin et al., 2007; Burton and Reid, 2002; Zozaya-Hinchliffe et al., 2010; Ravel et al., 2011). Molecular methods have identified in the vagina of healthy, nonpregnant women the Lactobacillus genus living with a spectrum of bacteria including Gardnerella, Atopobium, Eggerthella, Megasphaera, Leptotrichia, Prevotella, Enterococcus, Bifidobacterium, Staphylococcus, Corynebacterium, Streptococcus, Bacterioides, Mycoplasma, Escherichia, Peptostreptococcus, Ureaplasma, Veillonela, and Candida species (Zhou et al., 2004; Ling et al., 2013; Hyman et al., 2005; Vitali et al., 2007; Nam et al., 2007). However, at present the spectrum of bacterial species that are correspondent to clinical diagnosis, i.e. the Nugent’s score is not well defined.

In this study, we investigated the composition of BVAB in countryside women of reproductive age. Women with and without BV was diagnosed by Nugent’s criteria and recruited as the research subject and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and DNA sequencing techniques were used to analysis the genetic information of 16S rRNA genes (16S rDNA) of vaginal bacteria. The sequence information of 16S rDNA of every detected bacterium was identified with gene bank data. In addition, DGGE Gel Image System was used to calculate the relative abundance of each bacterial species to the whole microbial population of one sample (vaginal swab). Eventually, appropriate statistical method was used to analyze the relationship between BV and bacterial composition, as well as the relationship between BV and other risk factors.

ارزیابی تشخیصی سنجش PCR ریل تایم کمی multiplex برای واژینوز باکتریال

Diagnostic Evaluation of a Multiplex Quantitative Real-Time PCR Assay for Bacterial Vaginosis

Abstract

Background: Quantitative multiplex PCR assay for Bacterial Vaginosis (BV) based on the detection of the predominant contributory targets was evaluated against the conventional Nugent Score that is laborious and subjective due to morphological assessment bias of BV-associated bacteria.

Methods: 125 dual vaginal specimens were collected from patients aged ≥18 years at the time of presentation at the provider office to perform real time PCR and Nugent Testing. PCR assessment of BV was performed by quantitation of DNA amounts of Gardnerella vaginalis, Atopobium vaginae, Lactobacillus spp., and total amount of bacterial DNA using a multiplex RT-PCR kit (ATRiDA, Netherlands). Discordant results were resolved by the Amsel criteria or ancillary testing such as BD Affirm, when available.

Results: Nugent score classified 36.36% of the patients in BV and 15.45% in transitional BV categories. In contrast, the PCR method called 48.18% as BV and 12.72% as transitional BV or BV of unspecified origin categories. The overall concordance between the two methods was 81.81%. None of the BV positives by Nugent method were missed by the PCR. There were only 2 intermediates by Nugent that were called normal by PCR. PCR method was more sensitive than the Nugent and picked an additional 11% positives.

Conclusions: PCR based molecular BV diagnosis can standardize women health testing by removing the bias due to subjective interpretation of Nugent scoring. Our study shows that PCR method is more sensitive than conventional testing and may be a promising replacement for laborious Nugent scoring method in an era of shrinking microbiology expertise.

1 Introduction

Bacterial vaginosis (BV) is a complex polymicrobial syndrome characterized by alterations of the vaginal flora with acquisition of diverse communities of anaerobic and facultative bacteria and depletion of the usually dominant Lactobacillus flora. BV is a cause of malodorous vaginal discharge, vulvovaginal irritation, and/or dysuria. Recurrent and/or untreated infections are associated with several obstetric and gynecologic complications, including preterm rupture of membranes, chorioamnionitis, puerperal endometritis, pelvic inflammatory disease, urinary tract infection, postoperative cellulitis, cervical dysplasia, and human immunodeficiency virus acquisition. The current diagnostic gold standard Nugent Score is based on morphological assessment of BV associated bacteria. The technique is laborious and subjective as some morphologically similar non-contributory bacteria can skew the results. As a consequence, the use of molecular techniques to detect predominant contributory targets is desirable to make the BV diagnosis. Molecular methods that solely depend on the detection of a single target such as Gardnerella vaginalis have limited utility due to low sensitivity and specificity [1]. Quantitative ratios of specific anaerobic microflora such as G vaginalis and Atopobium vaginae in relation to Lactobacillus spp. are highly sensitive and specific for the diagnosis of BV and is associated with disease recurrence. Since A. vaginae have advanced resistance to metronidazole, its identification can play an important role in helping to change therapy decisions [2,3].

The aim of the study was to evaluate the diagnostic value of a multiplex quantitative real-time PCR assay (AmpliSens® Florocenosis/ Bacterial vaginosis-FRT PCR kit, ATRiDA, Netherlands) for Bacterial Vaginosis. A combination of Amsel critera and Nugent Graded gram stain (used as a gold standard) was used as the reference method.

شناسایی و مشخصات فیلوژنتیکی واژینولیزین Gardnerella vaginalis در نمونه های برگرفته از زنان بلغاری مبتلا به واژینوز باکتریال

Detection And Phylogenetic Characterization Of Gardnerella Vaginalis Vaginolysin In Samples From Bulgarian Women With Bacterial Vaginosis

Abstract

Recently, the intensive use of more advanced methods, including molecular genetic analysis, has aided the studies on the etiological role of Gardnerella vaginalis as the major pathogen of bacterial vaginosis (BV). The objective of our study was to propose a polymerase chain reaction (PCR)-based method detecting the G. vaginalis vaginolysin gene (vly) and to perform a phylogenetic analysis in an attempt to find possible correlations between clinical manifestations and gene mutations. Vaginal samples were collected from 1145 women with symptoms for vaginitis and 378 asymptomatic ones of reproductive age. After PCR detection of G. vaginalis 289 DNA samples were tested for vly using primers targeted at amplification of the whole gene. The presence of vly was detected in all 289 samples with positive PCR for G. vaginalis. Sixteen vly-positive PCR products were sequenced. The phylogenetic analysis based on the reference nucleic acid sequences deposited in the NCBI GenBank, covering the whole G. vaginalis vly gene, placed the studied isolates into three main groups. In conclusion, our experiments confirmed the major G. vaginalis virulence factor to be vaginolysin. The multiplex PCR results are promising and further studies should be carried out to determine the sensitivity range of the method and its applicability to diagnostics. Our phylogenetic analysis results, where some samples were grouped together, might imply a possible correlation between the clinical signs, the tendency to chronification, the cytotoxicity of vaginolysin and the vly gene mutations.

1 Introduction

Gardnerella vaginalis is a small, non-motile, Gram-variable coccobacillus that can grow in microaerophilic conditions. In the years following the discovery taxonomic status of this microbe remained uncertain due to some specifics in its cell wall structure: the cell wall stains Gram-positively in the exponential growth phase but, as the culture ages, the peptidoglycan layer becomes thinner and gives Gram-negative staining [1 ]. Thus, this Gram-variable microorganism was initially classified in the Corynebacterium and Haemophilus genera, but was later moved to a separate genus under the name G. vaginalis [ 1 ]. This bacterial species was proved to be a causative agent of genital tract infections in studies with volunteers [2 ] and in animal models [3 ]. It was also detected in exfoliated vaginal epithelium, in the absence of an inflammatory infiltrate. In recent years, the intensive use of more advanced methods, including molecular genetic analysis, has aided the studies on the etiological role of G. vaginalis, identifying it to be the major pathogen (in terms of frequency and quantity) in women with bacterial vaginosis (BV) [4–6]. Several members of the genus Lactobacillus inhibit pathogenic bacteria in healthy women‘s vaginal ecosystem but some substances produced by G. vaginalis could provide a synergistic effect for the growth of the other pathogens as well as an inhibitory effect against lactobacilli [3 ]. BV is the most frequent vaginal infection among women in the reproductive age group, and surveys on different continents alarm that it is especially frequent in younger women, under 30 years of age [5,7].

Several attempts have been made to classify G. vaginalis strains based on the phenotype; however, this proved to be clinically irrelevant [8 ]. The major G. vaginalis virulence factor is an exotoxin with cytotoxic activity (including hemolytic activity) against human cells. The hemolysin triggers a local immune response and the synthesis of secretary immunoglobulin A (sIgA) can therefore be used as a diagnostic marker for BV. This toxic product is a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins [9 ]. The G. vaginalis hemolysin, termed vaginolysin (VLY), shows selective toxicity and specifically targets host cells that carry complement regulatory molecule CD59. The cytotoxicity mechanism is based on VLY-mediated pore formation, with the proline residue in the undecapeptide of domain 4 of the toxin molecule playing the main role. Mutations in this region generate a VLY toxoid and could possibly aid in the development of a vaccine [9 ].

The aim of the present study was to propose a polymerase chain reaction (PCR)-based method for detection of the G. vaginalis vly gene and to perform a phylogenetic analysis in an attempt to find possible correlations between some clinical manifestations and certain gene mutations.

نانوذرات ZnO به دست آمده با استفاده از لیزر پالسی و ترکیب آنها با پارچه نخی: آماده سازی و مطالعه فعالیت های ضد باکتریایی

ZnO nanoparticles obtained by pulsed laser ablation and their composite with cotton fabric: Preparation and study of antibacterial activity

Abstract

A simple deposition method was used to prepare a ZnO/cotton fabric composite from water and ethanol dispersions of ZnO nanoparticles obtained by the pulsed laser ablation method. The structure and composition of the nanoparticles from dispersions and as-prepared composites were studied using electron microscopy, X-ray diffraction, and spectroscopy. The nanoparticles and composite obtained exhibited antibacterial activity to three different pathogenic microorganisms—Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. An attempt to understand a mechanism of bactericidal effect of ZnO nanoparticles was made. It was shown that zinc ions and hydrogen peroxide were not responsible for antibacterial activity of the particles and the composite, and surface properties of nanoparticles played an important role in antibacterial activity of zinc oxide. The proposed composite is a promising material for use as an antibacterial bandage.

1 Introduction

Semiconductor nanosized particles are widely used in different fields such as optoelectronics, catalysis, and sensorics. One of such materials is a wide-gap semiconductor (band gap of 3.37 eV), zinc oxide [1]. One more prospective application of ZnO is in biomedicine. Among antimicrobial agents, inorganic so-called “nanoantibiotics” [2] have attracted more and more attention. They exhibit great stability, high activity at low concentrations, and long shelf-life [3–5]. Zinc oxide is known as one of the most promising inorganic antibacterial agents. ZnO was found to demonstrate strong antibacterial and antifungal activity [6–10]. It was also shown [11] that after contact with ZnO slurry the sensitivity of Escherichia coli to some organic antibiotics increased. In addition, ZnO nanoparticles are active at pH values from 7 to 8 [12], which is suitable for use in water for washing and drinking. But despite the abundance of research in this area, the mechanism of antibacterial action of the zinc oxide is still not completely investigated [5,9,11]. ZnO nanoparticles can be used for disinfection purposes not only in the form of dispersions but also as a component of different materials, for example as filler for polymer film or as a coating [13]. Different materials covered with ZnO have been reported, including polymers [14], nano-fiber [15], silk [16], wool fibers [17], and cotton fabric [18–22]. It seems that antibacterial bandages based on different materials covered with ZnO nanoparticles have a great potential for hygienic and medical applications. Cotton fabric has many advantages in this area, such as natural composition, hypoallergenicity, and accessibility, and also it is an air penetrable material. Thus, this material is a very attractive as a basis for bandages containing ZnO.

The method that is the most frequently used for obtaining ZnO/cotton fabric composites is in situ synthesis of zinc oxide on the surface of the fabric. Zinc nitrate [18,20,23], zinc acetate [24], and zinc sulfate [21] are usually used as the source of zinc. Moreover,there is research in which pre-prepared ZnO was deposited on the cotton fabric from its dispersions using a pad-dry cure method [19,23] or layer-by-layer deposition [22]. In our opinion, using preprepared nanoparticle dispersions for obtaining ZnO/cotton fabric composites is more attractive from the practical point of view, since it does not require as precise control of the conditions as in situ methods require.