Monday, September 30, 2019

Economics story

The Indian Economy Since Independence India Wins Freedom On 14 August 1947, Nehru had declared: â€Å"Long years ago we made a tryst with destiny, and now the time comes when we shall redeem our pledge. The achievement we celebrate today is but a step, an opening of oopportunity, to the great triumph and achievments that await us. † He reminded the country that the tasks ahead iincluded â€Å"the ending of poverty and ignorance and disease and inequality of oopportunity†. These were the basic foundations on which India embarked upon its path of development since gaining independence in 1947.The purpose of this talk is to analyze how much has India really achieved in the last 55 years in fulfilling the aspirations on which it was founded. Indian Planning process The objective of India’s development strategy has been to establish a socialistic pattern of society through economic growth with self-reliance, social justice and alleviation of poverty. These objectives were to be achieved within a democratic political framework using the mechanism of a mixed economy where both public and private sectors co-exist. India initiated planning for national economic development with the establishment of the Planning Commission.The aim of the First Five Year Plan (1951-56) was to raise domestic savings for growth and to help the economy resurrect itself from colonial rule. The real break with the past in planning came with the Second Five Year Plan (Nehru-Mahalanobis Plan). The industrialization strategy articulated by Professor Mahalanobis placed emphasis on the development of heavy industries and envisaged a dominant role for the public sector in the economy. The entrepreneurial role of the state was evoked to develop the industrial sector. Commanding heights of the economy were entrusted to the public sector.The objectives of industrial ppolicy were: a high growth rate, national self-reliance, reduction of foreign dominance, building up of indigenous c apacity, encouraging small scale industry, bringing about balanced regional development, prevention of concentration of economic power, reduction of income inequalities and control of economy by the State. The planners 1 and ppolicy makers suggested the need for using a wide vvariety of instruments like state allocation of investment, licensing and other regulatory controls to steer Indian industrial development on a closed economy basis.The strategy underlying the first three plans assumed that once the growth process gets established, the institutional changes would ensure that benefits of growth trickle down to the poor. But doubts were raised in the early seventies about the effectiveness of the ‘trickle down’ approach and its ability to banish poverty. Further, the growth itself generated by the planned approach remained too weak to create adequate surpluses- a prerequisite for the ‘trickle down’ mechanism to work. Public sector did not live upto the e xpectations of generating surpluses to accelerate the pace of capital accumulation and help reduce inequality.Agricultural growth remained constrained by perverse institutional conditions. There was unchecked population growth in this period. Though the growth achieved in the first three Five Year Plans was not insignificant, yet it was not sufficient to meet the aims and objectives of development. These brought into view the weakness of economic strategy. We discuss the failure of the planning process in more detail in the next section. A shift in ppolicy was called for. The Fifth Plan (1974-79) corrected its course by initiating a program emphasizing growth with redistribution.To accelerate the process of production and to align it with contemporary realities, a mild version of economic liberalization was started in the mid 1980s. Three important committees were set up in the early 1980s. Narsimhan Committee on the shift from physical controls to fiscal controls, Sengupta Committe e on the public sector and the Hussain Committee on trade ppolicy. The result of such thinking was to reorient our economic policies. As a result there was some progress in the process of deregulation during the 1980s. Two kinds of delicencing aactivity took place.First, thirty two groups of industries were delicensed without any investment limit. Second, in 1988, all industries were exempted from licensing except for a specified negative list of twenty six industries. Entry into the industrial sector was made easier but exit still remained closed and sealed. Hence, the roots of the liberalization program were started in the late 80’s when Rajiv Gandhi was the Prime Minister of India, but the reach and force of the reform 2 program was rather limited. There were political reasons as to why this program could not be enhanced which we talk about later.The Failure of the Planning Process While the reasons for adopting a centrally directed strategy of development were understanda ble against the background of colonial rule, it, however soon became clear that the actual results of this strategy were far below expectations. Instead of showing high growth, high public savings and a high degree of self-reliance, India was actually showing one of the lowest rates of growth in the developing world with a rising public deficit and a periodic balance of payment crises.Between 1950 and 1990, India’s growth rate averaged less than 4 per cent per annum and this was at a time when the developing world, including Sub-Saharan Africa and other least developed countries, showed a growth rate of 5. 2 % per annum. An important assumption in the choice of post-independence development strategy was the generation of public savings, which could be used for higher and higher levels of investment. However, this did not happen, and the public sector-instead of being a generator of savings for the community’s good- became, over time, a consumer of community’s sa vings.This reversal of roles had become evident by the early seventies, and the process reached its culmination by the early eighties. By then, the government began to borrow not only to meet its own revenue expenditure but also to finance public sector deficits and investments. During 1960-1975, total public sector borrowings averaged 4. 4 % of GDP. These increased to 6 % of GDP by 1980-81, and further to 9 % by 1989-90. Thus, the public sector, which was supposed to generate resources for the growth of the rest of the economy, gradually became a net drain on the society as a whole.I will now try to give some reasons for the deterioration of the public sector in India. 1) The legal system in India is such that it provides full protection to the private interests of the so called ‘public servant’, often at the expense of the public that he or she is supposed to serve. In addition to complete job security, any group of public servants in any public sector organization ca n go on strike in search of higher wages, promotions and bonuses for themselves, irrespective of the costs and 3 nconvenience to the public. Problems have become worse over time and there is little or no accountability of the public servant to perform the public duty. 2) The ‘authority’ of governments, at both center and states, to enforce their decisions has eroded over time. Government can pass orders, for example, for relocation of unauthorized industrial units or other structures, but implementation can be delayed if they run counter to private interests of some (at the expense of the general public interest). ) The process and procedures for conducting business in government and public service organizations, over time, have become non-functional. There are multiplicity of departments involved in the simplest of decisions, and administrative rules generally concentrate on the process rather than results. There is very little decentralization of decision-making power s, particularly financial powers. Thus, while local authorities have been given significant authority in some states for implementing national programmes, their financial authority is limited.Hence during early 90’s it was imperative for India to correct its clearly faulty developmental process. There have been several reasons put forward for the failure of the developmental path which necessitated the reforms of Manmohan Singh in 1991. The way I would approach the analysis is through the approach of comparing and contrasting the viewpoints of two of the most prominent Indian economists of our times. The Bhagwati-Sen debate Jagdish Bhagwati and Amartya Sen, probably the two most influential voices amongst Indian economists, represent the two divergent ways of thinking about the development path.Though formally no such debates exists, apart from occasional jibes against Sen in the writings of Bhagwati, I believe by scrutinizing their positions a lot of introspection can be don e. As Bhagwati says â€Å"my view as to what went wrong with Indian planning is completely at odds with that of Prof Sen†. My objective in this section is bring 4 out the intellectual divergence amongst these two great minds and possibly to learn something from that. Let us start with the points on which they agree.I think the fact that India needs an egalitarian development path is quite well acknowledged by both of them. The Nehruvian dream of an egalitarian growth process was what both of them would endorse. As Bhagwati says â€Å"I have often reminded the critics of Indian strategy, who attack it from the perspective of poverty which is juxtaposed against growth, that it is incorrect to think that the Indian planners got it wrong by going for growth rather than attacking poverty: they confuse mearns with ends.In fact, the phrase â€Å"minimum income† and the aim of providing it to India’s poor were very much part of the lexicon and at the heart of our think ing and analysis when I worked at the Indian Planning Commission in the early 1960’s†. The key strategy that defined the resulting developmental effort was the decision to target efforts at accelerating the growth rate. Given the immensity of the poverty, the potential of simple redistribution was considered to be both negligible in its immediate impact and of little sustained value. Accelerated growth was thus regarded as an instrumental variable; a ppolicy outcome that would in turn reduce poverty.He goes on to argue â€Å"Those intimately associated in India’s plans fully understood, contrary to many recent assertions, the need for land reforms, for attention to the possibility of undue concentration of economic power and growth in inequality. These ‘social tasks’, which of course also can redound to economic advantage, were attended to and endlessly debated in the ensuing years, with reports commissioned (such as the Mahalanobis Committee report on income distribution in 1962) and policies continually revised and devised to achieve these social outcomes†.If we follow the writings of Sen on the other hand, in his recent book â€Å"Development As Freedom†, Sen argues that â€Å"the usefulness of wealth lies in the things that it allows us to do- the substantive freedoms it helps us to achieve†¦. an adequate conception of development must go much beyond the accumulation of wealth and the growth of gross national product and other income-related variables. Without ignoring the importance of economic growth, we must look well beyond it†. I don’t think that there is any divergence of view on this front with that of what Bhagwati says.It is worth mentioning at this juncture that this has been a common misconception amongst economists about the divergence of two different developmental paths. It is often misunderstood that Bhagwati’s 5 view stresses just on economic growth while Sen argues ag ainst economic growth and the importance of markets. The above paragraphs reveal that this is certainly not the case. Both of them is sufficiently concerned with economic growth as well as the basic issues of poverty, health and social issues. The points of divergence I believe the real disparity concerns the mearns of achieving these common goals.Bhagwati’s arguments can be summarized as follows. The development process consists of two steps. As a first step, a growth accelerated strategy would generate enhanced investments and whose objective was to jolt the economy up into a higher investment mode that would generate a much higher growth rate. The planning framework rested on two legs. First, it sought to make the escalated growth credible to private iinvestors so that they would proceed to invest on an enhanced basis in a self-fulfilling prophecy.Second, it aimed at generating the added savings to finance the investments so induced. His argument crucially rested on the fo llowing logical theory. For the higher growth rate to achieve it is very important for the economy concerned to be open. If the effective exchange rate for exports over the effective exchange rate for imports (signifying the relative profitability of the foreign over the home market), ensured that the world markets were profitable to aim for, guaranteeing in turn that the inducement to invest was no longer constrained by the growth of the domestic market.It is worthwhile to recount India’s performance as far as the public sector savings is concerned, which was considered a major hindrance towards the success of the Indian plans. Continuing with the argument, the generation of substantial export earnings enabled the growing investment to be implemented by imports of equipment embodying technical change. If the Social Marginal Product of this equipment exceeded the cost of its importation, there would be a ‘surplus’ that would accrue as an income gain to the econom y and boost the growth rate.The role of literacy and education comes at the next stage. The pproduactivity of the imported equipment would be greater with a workforce that was literate and would be further enhanced if many had even secondary education. Now his argument is based on the fact that the enhanced growth would demand and lead to a more educated workforce. Thus 6 he considers that primary education and literacy plays an enhancing, rather than initiating role in the developmental process. Sen on the other hand considers a larger view of development.He believes that questions such as whether certain political or social freedoms, such as the liberty of political participation and dissent, or opportunities to receive basic education, are or not â€Å"conducive to development† misses the important understanding that these substantive freedoms are among the constituent components of development. Their relevance for development does not have to be freshly established throug h their indirect contribution to the growth of GNP or to the promotion of industrialization.While the causal relation, that these freedoms and rights are also very effective in contributing to economic progress, the vindication of freedoms and rights provided by this causal linkage is over and above the directly constitutive role of these freedoms in development. I think that it is precisely at this point where some of Sen’s writings on economics and philosophy should be considered. According to Sen, economics as a discipline has tended to move away from focusing on the value of freedoms to that of utilities, incomes and wealth. This narrowing of focus leads to an underappreciation of the full role of the market mechanism.For example, take the example of the most important finding on the theory of the markets- the Arrow-Debreu equilibrium. That theorem shows that a competitive economic system can achieve a certain type of efficiency (Pareto efficiency to be precise) which a c entralized system cannot achieve, and this is due to reasons of incentives and information problems. But if we suppose that no such imperfections do exist and the same competitive equilibrium can be brought about by a dictator who announces the production and allocation decision, then are these two outcomes the same?In a much celebrated paper, Sen brings out the distinction between â€Å"culmination outcomes† (that is, the only final outcomes without taking any note of the process of getting there) and â€Å"comprehensive outcomes† (taking note of the process through which the culmination outcomes come about). Along these lines we can argue that Sen would disagree with Bhagwati’s point of view in that it does not consider the â€Å"comprehensive outcomes†.Though the outcomes may be the same if we bring about a simultaneous increase in investments in education, health and other social activities, with that of growth, as against a 7 framework where growth b rings about a derived demand for those activities (a la Bhagwati), these are not the same thing. So as we can see, the primary difference in the approach is that Bhagwati argues that poverty and social dimensions can be taken care of in the second step of the development process while Sen argues that social oopportunity is a constitutive element in the developmental process.In this respect it is helpful to scrutinize the East Asian case, where countries like Japan, South Korea, Taiwan (so called Asian Tigers) achieved phenomenal rates of growth in the 80’s and much of the early 90’s. The interesting fact about these countries is that they achieved this with a significant high record on the social dimensions. Both Bhagwati and Sen has commented directly on the achievement of these countries. As Bhagwati states â€Å"The East Asian investment rate began its take-off to phenomenal levels because East Asia turned to the Export promotion (EP) strategy.The elimination of th e ‘bias against exports’, and indeed a net excess of the effective exchange rate for exports over the effective exchange rate for imports (signifying the relative profitability of the foreign over the domestic market), ensured that the world markets were profitable to aim for, guaranteeing in turn that the inducement to invest was no longer constrained by the growth of the domestic market as in the IS strategy†. I personally think that there is nothing disputable in this analysis but it does not strengthen his argument that the social achievements in these countries followed their phase of growth.In fact the pioneering example of enhancing economic growth through social oopportunity, especially in basic education, is Japan. Japan had a higher rate of literacy than Europe even at the time of the Meiji restoration in the mid nineteenth century, when industrialization had not yet occurred there but had gone on for many decades in Europe. The East Asian experience was also based on ssimilar connections. The contrasts between India and China are also important in this aspect. The governments of both China and India has been making efforts for sometime now to move toward a more open, internationally active, market-oriented economy.While Indian efforts have slowly met with some access, the kind of massive results that China has seen has failed to occur in India. An important factor in this contrast lies in the fact that from a social preparedness standpoint, China is a great deal ahead of India in being able to make use of the market economy. While prereform China was deeply skeptical of markets, it was not skeptical of basic education and 8 widely shared health care. When China turned to marketization in 1979, it already had a highly literate people, especially the young, with good schooling facilities across the bulk of the country.In this respect, China was not very far from the basic educational situation in South Korea or Taiwan, where too an educated population had played a major role in seizing the economic opportunities offered by a supported market system. Indeed it is often argued that it is a mistake to worry about the discord between income achievements and survival chances-in general- the statistical connection between them is observed to be quite close. It is interesting, in this context, to refer to some statistical analyses that have recently been presented by Sudhir Anand and Martin Ravallion.On the basis of intercountry comparisons, they find that life expectancy does indeed have a significantly positive correlation with GNP per head, but that this relationship works primarily through the impact of GNP on (I) the incomes specifically of the poor and (2) public expenditure particularly in health care. In fact, once these two variables are iincluded on their own in the statistical exercise, little extra explanation can be obtained from including GNP per head as an additional causal influence. The basic point i s that the impact of economic growth depends much on how the fruits of economic growth are used.Sen argues that a focus on issues on basic education, basic health care and land reforms made widespread economic participation easier in many of the East Asian and Southeast Asian economies in a way it has not been possible in, say, Brazil or India, where the creation of social opportunities has been much slower and that slowness has acted as a barrier to economic development. I believe that one has to take note of the examples of say, Sri Lanka, the Indian State of Kerala or pre-reform China where on the contrary, impressive high life expectancy, low fertility, high literacy and so on, have failed to translate into high economic growth.I would like to see a theory which explains this. But to elucidate Sen’s view, he would rather prefer a situation of that of Kerala or Sri Lanka than that of Brazil or India. I would suggest that what one needs is such critical studies which would illuminate the failure of Brazil on one hand as against Sri Lanka on the other, to illustrate the fact that why an egalitarian growth process was not successfully implemented in these cases. We will have useful lessons to learn in that case for the future of development. The debate 9 between Bhagwati and Sen (or rather the created debate in this paper) gives rise to such an agenda.The Reforms of Manmohan Singh At the beginning of 90’s the reform process was started by the then Finance Minsiter of India, Manmohan Singh. The way I will organize this section is the following: First, I will give a short summary of the reform process , in the sense what were its general goals and ideas. Then I will mention some aspects of the reforms which I think are very encouraging. After that I will scrutinize the reforms more stringently in order to assess whether there is real cause for such jubilation that we tend to observe regarding India. (i)The Background: India’s economic reform s began in 1991 under the Narsimha Rao Government. By that time the surge in oil prices triggered by the Gulf War in 1990 imposed a severe strain on a balance of payments already made fragile by several years of large fiscal deficits and increasing external debt as was discussed before. Coming at a time of internal political instability, the balanceof-payments crises quickly ballooned into a crisis of confidence which intensified in 1991 even though oil prices quickly normalized. Foreign exchange reserves dropped to $1. billion in 1991, barely sufficient for two weeks of imports and a default on external payments appeared inevitable. The shortage of foreign exchange forced tightening of import restrictions, which in turn led to a fall in industrial output. A digression: The politics of reforms In a very engaging article on the politics of reforms Ashutosh Varshney has raised an extremely important question as to why was India’s minority government in 1991 successful in introd ucing economic reforms, whereas a much sthronger government, with a three-fourth majority in parliament, was unable to do so in 1985 (under the Prime Ministership of Rajiv Gandhi)?His argument draws a distinction between mass politics and elite politics. He believes that â€Å"this distinction has not been adequately appreciated 10 in the voluminous literature on the politics of economic reforms. Scholars of economic reforms have generally assumed that reforms are, or tend to become, central to politics. Depending on what else is making demands on the energies of the electorate and politicians- ethnic and religious strife, political order and stability, corruption and crimes of the incumbents- the assumption of reforms centrality may not be right†.In the largest ever survey of mass political attitudes in India conducted between April-July 1996, only 19 ppercent of the electorate reported any knowledge of economic reforms, even though reforms had been in existence since July 1 991. Of the rural electorate, only about 14 per cent had heard of reforms, whereas the comparable proportion in the cities was 32 per cent. Further nearly 66 ppercent of the graduates were aware of the dramatic changes in economic ppolicy, compared to only 7 per cent of the poor, who are mostly illiterate.In contrast, close to three-fourths of the electorate – both literates and illiterates, poor and rich, urban and rural- were aware of the 1992 mosque demolition in Ayodhya; 80 per cent expressed clear opinions about whether the country should have a uniform civil code or religiously prescribed and separate laws for mmarriage, divorce, and property inheritance; and 87 per cent took a stand on caste-based affirmative action. Thus according to Varshney, elite politics is typically expressed in debates and struggles within the institutionalized settings of a bureaucracy, of a parliament or a cabinet.Mass politics takes place primarily on the streets. In democracies, especially p oor democracies, mass politics can redefine elite politics, for an accumulated expression of popular sentiments and opinions inevitably exercises a great deal of pressure on elected politicians. The economic reform’s during 1991 kept progressing because the political context had made Hindu-Muslim relations and caste animosities the prime determinant of political coalitions. The reforms were crowded out of mass politics by issues that aroused greater passion, and anxiety about the nation.And hence the reforms could go as far as they did. (ii) The Reforms in a Nutshell: The reforms had two broad objectives. One was the reorientation of the economy from a statist, centrally directed and highly controlled economy to what is referred to in the current jargon as a ‘marketfriendly economy’. A reduction direct controls and physical planning was expected to improve the efficiency of the economy. It was to be made more 11 ‘open’ to trade and external flows thr ough a reduction in trade barriers and liberalization of foreign investment policies.A second objective of the reform measures was macro-economic stabilization. This was to be achieved by substantially reducing fiscal deficits and the government’s draft on society’s savings. (iii) Results: Compared with the historical trend, the impact of these policies has been positive and significant. The growth rate of the economy during 1992-93 to 1999-2000 was close to 6. 5 per cent per annum. The balance of payments position has also substantially improved.Despite several external developments, including the imposition of sanctions in 1998 and sharp rise in oil prices in 2000-01, foreign exchange reserves are at a record level. Current account deficits have been moderate, and India’s external debt (as a ppercentage of GDP) and the debt servicing burden have actually come down since the early nineties. There is also evidence of considerable restructuring in the corporate s ector with attention being given to cost-competitiveness and financial viability.The rate of inflation has also come down sharply. (iv) A Closer Scrutiny: When we talk about GDP growth we talk about the aggregate figures. Let us closely look at the sectoral composition. If we look at the growth rates with respect to different sectors we find that the growth rates of agricultural and industrial production have not increased at all in the nineties, compared with the eighties. The increase in overall growth in the 1990’s is overwhelmingly driven by accelerated growth of the ‘service’ sector.The service sector includes some very dynamic fields, such as uses of information technology and electronic servicing, in both of which India has made remarkable progress. This was largely a result of the liberalization policies initiated by Manmohan Singh. Ssimilar comments apply to the phenomenal expansion of software-related export services. Now the relevant question is, what is wrong in the fact that the services sector is driving the growth process in India?What is a bit disturbing is the fact that it is not clear as to the extent to which the rapid growth of the service sector as a whole contributes to the generation of widely-shared employment, the elimination of poverty, and the 12 enhancement of the quality of life. And also employment in the service sector is often inaccessible to those who lack the required skills or education. The current restructuring to the Indian economy towards this skill and education-intensive sector reinforces the resources to a certain ection of the society. How has the Reforms been successful in creating a widely shared developmental process? The issue as to whether the reforms have been successful in eliminating poverty to a greater extent than say in the 80’s is a contentious issue. Experience prior to the 1990s suggests that economic growth in India has typically reduced poverty. Using data from 1958 to 1991, Ravallion and Datt (1996) find that the elasticity of the incidence of poverty with respect to net domestic product per capita was –0. 5 and that with respect to private consumption per capita it was –0. 9. However, the 1990s are more contentious. Some observers have argued that poverty has fallen far more rapidly in the 1990s than previously (for example, Bhalla, 2000). Others have argued that poverty reduction has stalled and that the poverty rate may even have risen (for example, Sen, 2001). The basic question of measuring India’s poverty rate has turned out to be harder to answer than it needed to be because of difficulties with coverage and comparability of the survey data.Correcting for all those, Datt and Ravallion in a recent study find that India has probably maintained its 1980s rate of poverty reduction in the 1990s, though they do not find any convincing evidence of an acceleration in the decline of poverty. It is probably apt to remark here that oft entimes the public rhetoric fails to take the incomparability problems in the surveys from which the poverty estimates are calculated and try to interpret the estimates to reinforce their particular arguments.Even the Finance Minister’s ‘budget speech’ of Feb 2001 coincluded firmly that ‘poverty has fallen from 36 ppercent in 1993-4 to 26 ppercent or less now’. It is worth noting that even if one were to endorse the official 1999-2000 headcount ratio of 26 per cent, which is known to be biased downwards, one would find that poverty reduction in the 90s has proceeded at a ssimilar rates as in the earlier decades, in spite of a significant acceleration in the economic growth rate. As things stand, this is the most optimistic reading of the available evidence.All of the estimates were made with respect to head-count indexes. I think it is necessary to move away from this narrow index and to consider a broader range of social 13 indicators. Much of the d ebate in this area has focused on what has happened to expenditure on social sector development in the post reform period. Dev and Mooji (2002) find that central

Detective stories Essay

The Sherlock Holmes stories are the work of Arthur Conan Doyle. Holmes first appeared in the story ‘The Study of Scarlet’, which was published in the magazine ‘Beetons Christmas Annual of 1887†². Over the 40 years that followed Holmes appeared in 5 collections of short stories, and 3 other novels. Holmes’ popularity was partly due to the fact that his books were the detective fiction books ever to be published. These books are still read today all over the world, because of the brilliant way in which they are written. The Sherlock Holmes books are written from the point of view of his sidekick Watson. Many modern day detective fiction books and television programmes follow the pattern of a detective and sidekick. The stories of Sherlock Holmes caused him to become a national hero. In all the Sherlock Holmes stories we are given a detailed description of what he looks like, that is a tall gaunt person with long thin legs, a thin hawk like nose a square chin, and of course a pipe and deerstalker hat. The first story I read of Sherlock Holmes was ‘The Red Headed League’. In this story the reader is immedietely introduced to problematic situation, where a pawnbroker has come for advice. The pawnbroker goes on to tell Holmes and Watson his situation. Whilst the man is in the company of Holmes, Holmes begins to deduce things from just looking at the ware and tare of the mans body and clothes. From a very early point in the story we begin to see characteristics in Holmes, such as habits when he id thinking, ‘†¦ relapsing into his armchair, and putting his finger-tips together, as was his custom when in judicial moods. ‘ I was are given the impression that Holmes is the type of person who tries to treat people with respect, but his tolerance level for people who are not as intellectual as him is very low. Holmes becomes agitated at people’s incompetence to understand his complex ideas. I think Holmes pretends to expect every one to be as clever as him to make him look more intelligent and therefore superior. So in that respect I see Holmes as quite an ignorant person. However he does not treat Watson this way, I think he treats Watson more like a younger brother. I think that Holmes would like Watson to be like Holmes. From ‘he Red Headed League’ we can see that Holmes has a short temper, for example when he is in the cellar of the bank and Mr. Merryweather struck rather loudly his stick upon the flags which lined the floor, ‘ I must really ask you to be a little more quiet†¦ You have already imperilled the whole of our expedition. ‘

Saturday, September 28, 2019

American Education Vs. The World Essay

United States public schools were regarded as some of the best public schools in the world up to the 1970’s. Compared to the educational development of Asian and European students, American students and the education system are ranked embarrassingly low. â€Å"In a 2003 study conducted by UNICEF that took the averages from five different international education studies, the researchers ranked the United States number 18 out of 24 nations in terms of the relative effectiveness of its educational system† (Wu 2). Some students are graduating from high school with little or no knowledge about the core classes, while other students are dropping out and not graduating at all due to lack of discipline. Recently, colleges and universities have stopped using diplomas and grade point averages as a basis of admission because American high school curriculums have consistently simplified over the years and do not come close to compare to other schools around the world. The structure of American public schools compared to European and Asian schools are greatly disorganized, and many American teachers hold no credential and are extremely unqualified. Another major issue facing students today is the number of students per classroom, which differs greatly between competing countries. The American students are floundering in comparison with their counterparts around the world educationally due to lack of educational preparation, simpler curriculums, and unqualified teachers. When it comes to international education rankings, recent studies show that other nations in the developed world have a higher caliber of student success than that of the United States. In 2003, the United Nations Children’s Fund conducted an educational study that took the averages of five different tests and ranked the 24 participating nations; the United States was ranked 18. In that same year a similar study, the Tends in International Mathematics and Science Study, showed that the United States students tended to decline in performance from grades 4 to 12 more than any other nation. In both studies, South Korea, Japan, and Singapore ranked the highest respectively, followed by the United Kingdom, Finland, Australia, and Netherlands. The United States preceded all of these. â€Å"In fourth grade, American kids do above average internationally. By eighth grade, they slip a bit, and by 12th-grade, they’ve slipped a lot, we’re the only country that slides down that much from fourth to 12th grade† (Marsh). Because of this  recent slip in rank, the educators of the United States have somewhat resorted to teaching the material that will only be on the test rather than material that actually needs to be learned by the students. The United States teachers focus more on procedure, and try to teach multiple topics quickly while other countries tend to break up the topics and go more in-depth. Other countries’ teachers work on the concept and the background of the material instead of just teaching the procedure. For example, teachers in the United States tend to teach mathematics in whole numbers, while other countries use rulers to teach mathematics to show that there are numbers between whole numbers, implying infinite many numbers. Students are then taught the concept behind mathematics at an early age, making progression more productive. When students are taught the procedure and not the concept behind the procedure, there is a higher chance of forgetting the subject all together. It’s not just the international education rankings that American students are lagging in, but also the high school’s curriculum for their students, which differs greatly from other developed nations. American students are graduating from high school with little or no knowledge about the core classes. In other nations, the curriculum and requirements for progression are extremely strict, and if the requirements aren’t fulfilled by the student within a certain time period, the student will not continue on the educational ladder. In America, this idea is altered greatly by letting unqualified students consistently progress towards the next level in their education without any repercussions. â€Å"Between 1995 and 2004, the percentage of youth ages 16-19 who had ever been retained decreased; high school dropouts were more likely than high school completers to have been retained in a grade at some point in their school career† (Indicator 1). This decrease in the grade retention rate is physical evidence that school systems are letting students that do not fulfill the curriculum for a certain grade continue onto the next grade without any repercussions. The decrease is also evidence that the school systems concentrate more on other issues than education. In America, it has basically become custom to put education behind everything; school administrators believe that self-esteem and even religion are more important than the education of the children. In other countries, this isn’t so, and almost nothing comes prior to education.  When comparing the mission statements from that of an American public school to one of a European public school, one can see how the educational goals differ. For example, the mission statement for City High School in Tucson, Arizona is as follows:City High School strives to be a community of learners in which all members use their minds well and care about one another. We engage with challenging academics and the unique resources of our city and region in order to become active citizens and responsible stewards of our world. City High School’s mission statement doesn’t differ greatly from those of other American public schools. The schools want their students to â€Å"care about one another† and â€Å"become active citizens and responsible stewards†. Only a few words have anything to do with education, which proves the point that American public schools believe that good citizenship should come before good scholarship, also providing evidence that the curriculum is not as competitive as other nations’. Another major setback for the students in America compared to students around the world is the increasing amount of unqualified instructors. â€Å"One out of every seven teachers holds no credential and has little or no training in how to teach or manage a classroom† (SF Gate 1). The Center for the Future of Teaching and Learning conducted a report on unqualified instructors and found that the number of teachers holding no credential rose 11% between 2005 and 2006 and rose 13% between 2006 and 2007. Each year, more than 40,000 teachers work under an â€Å"emergency permit†, allowing them to instruct classrooms for the school year. This is a major flaw in the United State’s educational system because these â€Å"teachers† know just about as much of a subject as their students do, thus creating a stagnation in educational progression. In 2001, the â€Å"No Child Left Behind† act was passed and forced teachers to demonstrate that they are qualified in the subject(s) that they are instructing by passing certification exams and completing graduate coursework. This act obviously did no good to the education system, for it allowed even more teachers to teach without credentials. American student’s poorer educational rankings cannot entirely be blamed upon American students, but upon American instructors.

Friday, September 27, 2019

WEEK 4 DISCUSSION PART 1 Essay Example | Topics and Well Written Essays - 500 words - 1

WEEK 4 DISCUSSION PART 1 - Essay Example In order to quantify the magnitude of Medicaid fraud in the US, the study will be conducted across a wide range of participants. The study will target people who have participated in Medicaid fraud or who have been affected by the scheme. Throughout the study, anonymity of the participants will be maintained unless instructed otherwise. In addition, the study will also target professional in the medical and legislative sectors who have facilitated the scheme either directly or indirectly. The study will be conducted through both open ended and closed ended questionnaires. Research assistant will conduct manual interviews and filling of questionnaires, however, electronic (online) questionnaires will be available. In either of the data collection methods, anonymity of the participants will be maintained unless they recommend otherwise. The research site will be identified through a random process that will be highly inclusive and conclusive. The selection will target both upper and mi ddle class Americans. In addition, the study will have a professional phase whereby the participants will be drawn from both the medical and legal professionals. The study will have a quantitative segment that will capture the magnitude of the phenomenon. On the other hand, the qualitative segment of the study will focus on the reasons or factors that contribute to Medicaid fraud among Americans. In the quantitative segment of the study, the researcher will be interested in identifying the magnitude of losses incurred in the health sector due to the scheme. Financial loses will be identified as the dependent variable while time will be identified as an independent variable. The quantitative segment will also be concerned with the variation of fraud across different social classes. Thus, different income classes will be identified as independent variables while financial losses will

Thursday, September 26, 2019

Reflective Practitioner Assessment Research Proposal

Reflective Practitioner Assessment - Research Proposal Example Even though endorsements have taken on a quasi-industry stature, there is hardly any hugely successful collaboration as those of Nike" Background: Over the past fifty years, the association of brands with celebrities has been used as a widely accepted and popular means of branding any product. Irrespective of the industry diversity celebrity endorsement is being used to appeal customers in the pharmaceutical, watches & accessories, personal use items, cosmetics, cola drinks and virtually everything. This method of branding has not lost its popularity over the years and the potential in this sector is enough to have sustained the emergence of brand consultancy firms specialized in providing celebrity matching and celebrity endorsement services. Considering the above, the purpose of this research is whether celebrity branding is actually beneficial for the product or are their some hidden, spillover negative effects Is celebrity-branding effective in generating sales by influencing the decision making process Is celebrity endorsement as successful a tool in all the industries or whether its impact differs form industry to industry Is the celebrity appeal the same across different geographical regions and cultures And the focal question is celebrity endorsement really necessary for all products or is it a marketing myth phrased as " Is Celebrity Endorsement beneficial for the product or is it a li... Celebrity Endorsement is a part of the branding strategy. Considering this the 22 immutable laws relate to this specifically the 18th Law the law of borders which states that a brand should not have any borders and should be global and la 13 which states that the brand name should take precedence over the name of the company to avoid confusion. Considering this, the question that arises is that since singularity is important to leave an impact on the mind of the customers, is it really prudent to have a celebrity endorsement, which might confuse the customer by removing the singularity or overshadowing the brand in itself The general background to branding and advantages and disadvantages of labeling, branding and other functional aspects are well explained by Kotler in the Book Marketing Management. Literature Review: To conduct this elementary level research, academic journals, articles available on the internet and case studies pertaining to well-known brands have been reviewed and they provide useful insight onto the different circumstances that the brands face, the way they adapt, how celebrity branding has helped them boost their sales or bring it to low levels. A study conducted at University of Columbia by Christina Schlecht "Celebrities impact on Branding" provides good reading material for this subject. For an insight into the current topics, the articles available at www.about.com were very insightful especially "Celebrity Endorsement Deals Gone Astray" by Apryl Duncan. For the case study analysis, www.fultable.com and www.celebrityendorsements.co.uk are particularly useful. From the above it can be seen that a lot has been written on this subject. However, as observed by on

Religious Perspectives in Nation Building Essay

Religious Perspectives in Nation Building - Essay Example Benjamin Franklin is considered as one of the founding fathers of the United States of America. He is credited as foundational to the roots of American values and character, which combines the practical and democratic Puritan values of thrift, hard work, education, community spirit, self-governing institutions, and opposition to authoritarianism both political and religious, with the scientific and tolerant values of the Enlightenment (Isaacson p. 491). Thomas Jefferson is the principal author of the Declaration of Independence and became the third president of the United States in 1801. He is considered as one of the most influential founding fathers with his espousing the ideals of republicanism (Wiki). William Bradford was already committed to the radical idea of the separatist movement since the age of 17 (Kelso). "The one side [the Reformers] laboured to have ye right worship of God & discipline of Christ established in ye church, according to ye simplicitie of ye gospell, without the mixture of mens inventions, and to have & to be ruled by ye laws of Gods word, dispensed in those offices, & by those officers of Pastors, Teachers, & Elders, &c. according to ye Scripturs. The other partie [the Church of England], though under many colours & pretences, endevored to have ye episcopall dignitie (affter ye popish maner) with their large power & jurisdiction still retained; with all those courts, cannons, & ceremonies, togeather with all such livings, revenues, & subordinate officers, with other such means as formerly upheld their antichristian greatnes, and enabled them with lordly & tyranous power to persecute ye poore servants of God" (Bradford). John Carver, a deacon of the separa tist congregation and one of the prime organizers of the Mayflower voyage was very influential in Bradford's religious beliefs. Bradford also succeeded Carver as the governor of the Plymouth colony when the latter died (Kelso). Benjamin Franklin was an important conservative figure in the American restoration movement, which sought to renew the entire Christian church based on the New Testament and disregarding the creeds of Catholicism, Orthodoxy, or Reformed Protestantism, which allegedly divided Christianity (Wiki). "I never doubted, for instance, the existence of the Deity; that he made the world, and govern'd it by his Providence; that the most acceptable service of God was the doing good to man; that our souls are immortal; and that all crime will be punished, and virtue rewarded, either here or hereafter. These I esteem'd the essentials of every religion; and, being to be found in all the religions we had in our country, I respected them all, tho' with different degrees of respect, as I found them more or less mix'd with other articles, which, without any tendency to inspire, promote, or confirm morality, serv'd principally to divide us, and make us unfriendly to one another" (Franklin). Samuel Roge rs, noted Restoration movement preacher and elder moved to the community of the Franklin family and became influential in Benjamin Franklin's religious perspectives. Franklin, himself began preaching after Rogers' arrival, as well as began publishing religious publications such as the Reformer, Christian Age and

Wednesday, September 25, 2019

Political Ideologies Essay Example | Topics and Well Written Essays - 750 words - 1

Political Ideologies - Essay Example Ideals such as ethnic diveristy, equal opportunity and accurate representation are great ideals but it cannot be imposed that will impede free speech which is inherent in the artistic expression of film making. Film making just like any art form, are not subjected to any absolute prescription of how it should be done but depends on the film maker’s interpretation of the universe and the artistic manner it is conveyed. ACE’s advocacy to impose ethic diversity, equal opportunity, and accurate representation of a movie is a casting decision with expressive element because such advocacy also entails the expression of a particular political persuasion. Such political persuasion (ethnic diversity, equal opportunity and accuracy of representation) will not take any material form if not explicitly expressed and thus the validity of such expressive element should be subjected to the O’Brien and Cohen tests. To pass the O’Brien test, ACE must satisfy all four regula tions which are; 1. The law must be within the constitutional power of government.   2. The law must further important or substantial government interest.   3. The interest must be unrelated to the suppressing expressions.   4. Any incidental limit on expression is no greater than necessary.  ... quality and diversity are one of many the bedrocks of this country.   Indeed the political persuasion or advocacy of ACE is important or substantial to government interest. ACE however did not meet the criteria in the third element of O’Brien test that stipulates that such expressive element must be unrelated to the suppressing expressions in the same manner that â€Å"the constitutional power of Congress to raise and support armies and to make all laws necessary and proper to that end is broad and sweeping† (UNITED STATES v. O'BRIEN, 1968) has no relation with the suppression of speech. In Ace’s case, it is DIRECTLY RELATED to suppressing another’s expression which is that of Donalds interpretation of rendering a particular movie. ACE, in pushing for its advocacy for equality, diversity and accurate representation of film albeit laudable, trampled on another right to free speech thus directly relates to suppression of another’s right to free spee ch. It follows that the exercise of ACE’s advocacy directly suppress another’s right to artistic expression and freedom of speech and places not only an incidental limit on expression that is not only greater than necessary but unnecessary in the first place because no attempt should be made to abridge artistic vision. Unlike in O’Brien case where the Supreme Court ruled that â€Å"we perceive no alternative means that would more precisely and narrowly assure the continuing availability of issued Selective Service certificates than a law which prohibits their wilful mutilation or destruction† (United States vs. O’Brien, 1968) ACE has other avenues where the advocacy of ethnic diversity and equality is appropriate. Thus, ACE may have passed the first two elements of the O’Brien test but grossly failed on the

Tuesday, September 24, 2019

Final exam Assignment Example | Topics and Well Written Essays - 1000 words - 2

Final exam - Assignment Example The license fee for the facility proposed is $200 plus $50 for each bed up to a maximum of $1,500 upon application for one year and $100 plus $50 for each bed for renewal each year to a maximum of $750 (Texas Department of Aging and Disability Services (n.d.). Therefore, a total of $1,500 would be paid in the first year and $750 each year thereafter until an adjustment is made. The revenue per unit/person is in keeping with rates obtained from Genworth Cost of Care Survey for 2013 which suggest that the minimum, median, and maximum monthly rates are $933, $3,710 and $8,445 respectively for a one bedroom single occupancy assisted living facility in California (Genworth 2013). The information in Table 1 in the Appendix indicates that these rates would result in minimum, median and maximum income per person per annum of $$11,196, $44520 and $101,340 respectively. Table 1 also indicates that 53 single occupancy units would be available for assisted living over the ten year period. If all units were occupied during the year the total annual revenue from this would be $593,388 at its lowest, $2,359,560 at the median level and $5,371,020 – the maximum per annum. An occupancy level of 93% is assumed for year 2 to year 10 for assisted living. ... This could lead to less than expected occupancy levels for the company and thus lead to a significant reduction in projected revenues (See Table 2 in the Appendix for income at various occupancy levels). In terms of wages, the employees of the facility should not be paid lower than Federal of State minimum wage which is $7.25 and $8 respectively (Minimum-Wage.org 2013). The lowest wage rate to be paid by Blueberry Acres LLC is $7.25. Both the Federal and State Minimum wage are expected to increase to $9 by 2014 (Hicken 2013) – a 24% increase in the Federal minimum wage rate projected. In 2016 the State minimum wage is expected to increase to $10 and it is also hoped that the Federal rate will be tied to the inflation rate (Hicken 2013). The interest rate on the construction loan appears to be too high at 7.5%. The prime lending rate is currently 3.25 and is projected to be the same at the end of March 2014 (Financial Forecast Center 2013). The 7.5% projected is more than twice the prime lending rate. Additionally, the projected length of time for the loan may not be allowed to exceed 20 years. This would change the annual payments to $488,746 representing interest payments of $410,787 and principal repayments of $78,051 in the first 12 months. With the exception of vehicle costs and expenses which are assumed to remain constant, the depreciation rate and other expenses appear reasonable. Vehicle costs should increase by at least 3% - the level of inflation anticipated each year. The budget needs to be adjusted for this error. Assessing the reasonability of the construction contract The assisted care facility is a profit making venture and should be

Monday, September 23, 2019

Critically evaluate the claim that Marxism offers a coherent account Essay

Critically evaluate the claim that Marxism offers a coherent account of the modern international political system - Essay Example The most notable example of its failure to build up a fruitful political system is the Soviet Union whose eventual collapse was engendered by the flaws and paradoxes lying at its heart. In the first place, Marxism fails to perceive the corruption of absolute power. Secondly, it fails to perceive that ruling proletariats in the power of a socialist state are bound to assume the behaviors of capitalist elite class. The third flaw lies in its attempt to impose equality and to steal away freedom in the name of a classless society. Such socialist attempt to maintain equality by robbing a man’s freedom is essentially Totalitarianism which inspires corruption and discourages people’s protest against this corruption. Further the paradox of an equal society does not permit a congenially productive economic system. When the equal distribution of wealth among the citizens theoretically seems to contribute to an equal and just society, this distribution ultimately discourages the i ndividual to involve in production activities. Thus a socialist economy goes futile. Again, a Marxist state continually aims at establishing the working class at the power of a country. Therefore, a socialist political system poses threat to other states which do not hold a socialist view. A Brief Overview of Marxism and a Marxist State Being commissioned by the Communist League, Karl Marx and Frederick Engels jointly wrote the book, â€Å"The Communist Manifesto†, in 1848, which is often accepted as one of the most influential political documents around the world. Indeed, Marxism is regarded as a social theory which foretells and philosophizes about the future of modern capitalist society as well as human history. But it is also true that the Communist Manifesto can be considered as the political guidance for those who are involved in the production system of the modern capitalist society. For these authors, modern society is essentially the latest one of those changes in th e mode of production throughout the evolution of human society. At the same time, since Marx and Engels envisaged that the stage of human society, next to Capitalism, is a world of the working class people, in Marx and Engel’s word the proletariats, that is based on the socialist modes of labor as well as production, the proletariat or the working class people of the world should unite themselves to take the society to this stage. Theory of Class-struggle and Marx’s View of a Socialist Political System Marx and Engels have assumed that the Capitalist society has evolved from the breakdown of the previous feudalist society through the conflicts between the feudal landowners and their subjects. Through this dissolution of the feudal society two more incompatible classes emerged: the bourgeoisie and the working class. While the bourgeoisies, occupying a country’s political system, control the instruments of production, the working-class are economically subjugated by those in a capitalist political system. These bourgeoisies are exclusively profit-driven. Though they lack any morals, they continue to support any moral system which is congenial to

Sunday, September 22, 2019

Technology Review and Integration Plan Essay Example for Free

Technology Review and Integration Plan Essay In the 80’s children normally depended on flash card, heavy encyclopedia, libraries and their parents to learn. When the age of personal computers (PC) started to gain momentum in the 90’s, children started to experience computer programs that help them learn. In the late 90’s multimedia application distributed in Compact Disc (CD) can be installed into computer to provide an interactive learning for children. Just recently with the dramatic growth of the internet, nearly all children have access to an internet connected PC. This also boosts the demand for educational websites that provide learning tools either for a fee or completely free. There are probably millions of websites that can be classified as educational but only few studies that directs us which website are best for our teachers, parents and students. This paper examines educational websites for children by going through at least five educational websites. Then from these five, detailed comparison is done on three best educational website found. Technology Review and Integration Plan The internet which connects computers and servers around the world is now the leading source of information. Vast amount of data can be gathered from internet. One of the very useful websites for teachers, parents and students are educational websites. They come either for free or for a certain fee. Free educational websites normally earn through advertisement. Five of the most notable children’s educational website are http://funschool. kaboose. com/, http://www. kidsknowit. com/, http://www. teach-nology. com/, http://www. kbears. com/, and http://nobelprize. org/. The best characteristic of the Knowledge Bears website is the futuristic submarine like graphical interface. This feature makes the site very attractive and easy to use. Although on the other side, since it is full of flash interactive animation, it loads relatively slower than a more text oriented website like the KidsKnowIt. com website. One of the most interesting features of the Funschool Kaboose Website is the kid oriented look of the graphical user interface like the one shown in Figure 2. This makes it easy for kids to understand the usage of the site. The negative side of this site is that it is more of games than information. Each link are consists of educational games. This makes it less informative than the two other educational websites. The KidsKnowit website contains a simple interface that allows it to be loaded quickly in any internet browser. It has more topics and more information than the Knowledge Bears and Funschool Kaboose website. Although it has some disadvantages compared to other sites, but overall, from cost, ease of use, availability, reliability and learning outcome this site proves to be better. Even with the distracting Google ads, the KidsKnowIt website still gives the best experience. It would be easy to integrate these educational websites into the classroom. First, time must be allotted in every subject to learn from these sites. Then initially, an overview should be given to all the children how to navigate through these website’s educational tools. Then some rules must be set as to how long, what areas and what objectives must be met every time the students visit this site. Finally, an evaluation procedure such as how fast they accomplish the games or an exam about the subject matter must be provided to check the progress of learning.With all of these put in place, then there is no doubt that these sites will be very helpful in teaching students. References About us: Kidsknowit Network Outreach Program. Retrieved August 15, 2008, from Kidsknowit Web site: http://www. kidsknowit. com/about. php About us: Kbears. com . Retrieved August 16, 2008, from Knowledge Bears Website http://www. kbears. com/about. html Main Page: Kidsknowit. com. THE TOTALLY FREE CHILDREN’S LEARNING NETWORK. Retrieved August 16, 2008, from KidsKnowIt Website http://www. kidsknowit. com/

Saturday, September 21, 2019

Dormancy-breaking Treatment on Spices of Fennel

Dormancy-breaking Treatment on Spices of Fennel Effect of moist chilling and application of dormancy-breaking treatment on two spices of Foeniculum vulgare Mill Sayyid Amirreza Tahaei, Ali Soleimani Abstract One of the problems of planting of fennel (Foeniculum vulgare Mill) is seeds dormancy of this plant’s seed. We designed an experiment and investigated effect of wet chilling levels including 0, 15, 30 and 45 days of chilling and different breaking dormancy treatments on breaking dormancy of seeds of two fennel species which are named Nahavand and Malayer. Dormancy-breaking treatments were including gibberellic acid (100 mg L-1), benzyl adenine (10-5 M), chitin (10-5 M), mixture of gibberellic acid + benzyl adenine, mixture of gibberellic acid + chitin, mixture of benzyl adenine + chitin, mixture of gibberellic acid + benzyl adenine + chitin (with the mentioned concentrations(, concentrated sulfuric acid (90%) for 15 second, potassium nitrate 0.4%, distilled water, aminol forte 0.4%, kadostim 0.4%, fosnutren 0.4% and humiforte 0.4%. The results were indicated that chilling have significant effect on increasing of the germination percentage, coleoptile length, radical length, se edling length, rate of germination and vigor index. The treatments of benzyl adenine + chitin, gibberellic acid + chitin, aminol forte and distilled water caused a significant increase in characteristics of fennel germination in compare to other treatments. Varieties of Malayer has significant percentage of germination, coleoptile length, radical length, seedling length and vigor index in compared to varieties of Nahavand. The results indicated that in the condition of this study moist chilling treatment (stratification) for 15 days convey with application of treatments of benzyl adenine + chitin and aminol forte is suitable to breaking dormancy of fennel seeds. Keywords: Chilling, dormancy of seed, dormancy-breaking treatments, fennel Introduction Fennel (Foeniculum vulgare Mill.) is an aromatic, Herbaceous and biennial plant which grows to a height of 2 m. Fennel fruit is small with the length of about 8 mm and width of 3 mm, aromatic and sweet (Rchinger1987). One of the main characteristics of seed is the power of germination and vigor that is important for farmers (Copeland L. O. and M. B. McDonald, 1375). Studies have shown that many of the family Apiaceae plants are produced seeds with lots of endosperm and small embryos (Baskin, 1992). Percentage of seed germination in many plants of this family are low and their germination standards is lower than other plants. Lack of embryo, incompletely and dormant embryo in seed are the most important factors of seed dormancy and reducing of germination in this plants category (Robinson, 1954). The results of the studies of (Hedayati, 2000 and Nichols; ,1934Baskin, 1984, 1991 and , 1999 Troy, 1993; Widrlechner, 2000) indicate that a variety types of the Saubacus genus, Umbelliferus seeds and Dioscora and Cuphea species also show different degrees of physiological dormancy patterns and chilling can greatly help to resolve this type of dormancy. (Slater, 1982). Soltani Pour (1388) showed that the maximum and minimum rate of germination of fennel were 34.1 and zero per day related to cold treatment and hot water treatment. The maximum index of vigor seed was 365.75 related to cold treatment and the minimum index of vigor seed was zero related to hot water treatment. Moist cold causes permeability of seed, materials washing and prevents germination, also cold resolves physiological dormancy and overcomes internal abscisic acid. Plant hormones or growth regulators are involved in many aspects of plant growth. Investigations showed that most of the plant hormones such as auxins, gibberellin, cytokinin, ethylene and abscisic acid affect on the stimulating germination or dormancy of seed via specific ways that cause controlling functions of nucleic acids (Cournf et al., 2002). The results of the interaction of different concentrations of gibberellin hormone (0, 100, 250, 500 and 1000 ppm) and the time of dipping in this hormone (24 and 48 hours) of the rhubarb indicated significant differences between treatments in terms of the evaluated characteristics (percentage and rate of Fecula gummosa). The maximum a mount (65%) was obtained by increasing concentrations of this hormone from 100 to 500 ppm. However it was reduced by increasing concentrations of hormone to 1000 ppm. Bannayan and Najafi (1383) stated that germination of Fercula gummosa was increased at concentrations above 50 ppm of gibberellic acid. They observed that low concentration of gibberellic acid had no effect on breaking dormancy of Fecula gummosa, but the concentration of 50 ppm and increasing the soaking time from 48 to 72 hours improved percentage of germination. As the maximum percentage of Fercula gummosa seed germination was obtained at concentrations of 1000 and 2500 ppm. Materials and Methods Seed Characterization Seed samples of two species namely Malayer and Nahavan were collected from Sarsir area of Hamadan in 1390. In order to preparation seeds, at first the same size and apparently healthy seeds were separated by a loop. Average weight of 1000 seeds was 3 g. After stratification period, the seeds were sterilized and breaking dormancy treatments were applied on them and they were placed in germinator machine with photoperiod of 8 hours of light at 30  ° C and 16 hours of darkness at 20  ° C (ISTA, 1985). Characteristics of Experiment Location To evaluate the effects of moist chilling and application of hormonal, chemical treatments and biological stimuli to break dormancy and induce germination of two varieties of fennel, an experiment was performed in laboratory of seed technology of the Agriculture faculty of Islamic Azad University khorasgan brunch Isfahan in 91-92 years. The research was performed based on a split factorial experiment in a completely randomized design (CRD) with four replications. Levels of stratification set in the main plot and variety factorial and break dormancy treatment set in sub-plots. Effect of moist chilling on breaking dormancy of seed Moist chilling levels include 0, 15, 30 and 45 days. This means that the seed is placed in distilled water for 24 hours. Then the moist seeds were placed inside sterilized and humid cotton bags separately. These bags were kept in isolated and dark environment at 4  ° C in the refrigerator. Effect of treatments on breaking dormancy of seed After the chilling period, 14 pregermination treatments were applied on the seeds. 14 braking dormancy treatments were as follow: 1-Gibberellic acid (100 mg L-1). 2- Benzyl adenine (10-5 M). 3- Chitin (10-5 M). 4- Mixture of gibberellic acid + benzyl adenine. 5- Mixture of gibberellic acid + chitin. 6- Mixture of benzyl adenine + chitin. 7- Mixture of gibberellic acid + benzyl adenine + chitin. 8- Concentrated sulfuric acid (90%) for 15 second. 9- Potassium nitrate 0.4%. 10- Distilled water. 11- Aminol forte 0.4% (Growth Stimulus). 12- Kadostim 0.4% (Growth Stimulus). Fosnutren 0.4% and Humiforte 0.4% (Growth Stimulus). Concentrations used for treatments 4, 5, 6 and 7 were same as the concentrations of treatments 1, 2 and 3. Used hormones in these experiments were manufactured by Sigma Company and biological stimuli, including 19 kinds of amino acids, low molecular weight polypeptides and also nutrients were manufactured by Inagrosa Company. Statistical population and sampling In order to determine the percentage and rate of germination germinated seeds were counted once per 24 hours according to the ISTA instruction. Root were considered germinated when they exhibited a radical extension of 2 to 3 mm (Bahadori et al., 1386). Evaluation of germination will be end when amount of germinated seeds in consecutive counts are same and this time was considered as the end of the germination period. After this period, the following parameters were measured: 1-Germination percentage 2- Coleoptile length 3-Radicle length 4- Germination rate 5- Uniformity of germination 6- Seed vigor index Percentage of germination according to equation of (Jefferson and Pennacchio, 2003) G=(n/N)Ãâ€"100 G = germination percentage n = amount of germinated seeds N = amount of the seeds in each petri dish container Germination rate according to equation of (Bahadori and Javanbakht, 1385) RG =1/MTG RG = rate of germination MTG = Mean Time of Germination Uniformity of germination was calculated according follow equation (Karam and Al-Salem, 2001) Uniformity of germination = (4) D = number of days from the beginning of germination ∑n = total number of germinated seeds N = number of germinated seeds at day = average number of days from the beginning of germination Data Analysis: Analyses of variance was performed on the data by MSTATC software. The means of parameters levels was compared using Duncan’s multiple range tests at %5 level of probability. Excel software was used to plot diagrams. Results Stratification It was observed that moist chilling is the most important treatment on seed germination of Fennel like many other members of Apiaceae family. However, Stratification have a significant effect on percentage of germination, coleoptile length, radical length, seedling length, average time of germination, germination rate and seed vigor index at %1 level of probability but the uniformity of germination was not significant (Table 1). The results indicated that increasing cold stratification duration for 15 days increased seed germination. Comparison of means using Duncan method indicated that the maximum percentage was obtained with 15 days cold stratification treatment resulting in 36.86% and it was decreased by increasing moist cold stratification duration as 45 days cold stratification treatment resulting in 11.18% stated that there was no significant difference in germination practically (Table 2). Results indicated that the maximum average time of germination was obtained by using no stratification treatment and cold stratification of 15 days but average time of germination was decreased significantly by increasing cold stratification duration from 15 to 30 and 45 days (Table 2). Stratification treatment have a significant effect on seed vigor index at %1 level of probability (Table 1). the maximum seed vigor index was obtained by using cold stratification treatment of 15 days and there was a significant difference between this treatment and other treatments. the minimum seed vigor index was obtained by using cold stratification treatment of 45 days and there was a significant difference between this treatment and other treatments (Table 2). Breaking dormancy treatments The results of variance analyses table of breaking dormancy treatments application indicated that applied of hormone- amino acid- chemical treatment caused a significant difference between germination parameters such as percentage of germination, coleoptile length, radical length, seedling length, average time of germination, germination uniformity, germination rate and seed vigor index (Table 1 and 2). Comparision of means indicated that the maximum affect on germination of fennel was obtained by using aminol forte, benzyl adenine + chitin treatments (Table 2). Interactions of moist chilling and breaking dormancy treatments indicated that bio-stimulant treatments of kadostim and aminol forte and hormonal treatments of benzyl adenine + chitin had the highest percentage of germination in the absence of cold. An increased in germination percentage was observed by increasing cold stratification duration of mentioned treatments to 15 days but increasing cold stratification duration up to 15 days reduced germination percentage with the same ratio. However, increasing cold stratification duration from 0 to 15 days for nitrate potassium treatment increased germination percentage of 26% that is maximum percentage of germination but increasing stratification duration to 30 and 45 days reduce germination percentage to a lesser extent compared with that (Table 2). The results of applied treatments revealed that the concentrations of used materials and the duration of these treatments are effective in stimulating germination. Varieties The results of varieties reaction indicate that increasing percentage of germination, coleoptile length, radical length, seedling length and seed vigor index of Malayer variety is significantly higher than nahavand variety. The seeds of Malayer variety with 32.29% of germination is ranked higher than the seeds of Nahavand variety with 13.11% of germination (Table 2). The interaction between the moist chilling with break dormancy treatments and varieties Study of interactions of moist chilling, varieties and breaking dormancy treatments indicated that cytokinins convey with increasing cold stratification duration to 15 days increased some parameters such as percentage of germination, coleoptile length, radical length, seedling length and etc. in Malayer and Nahavand varieties (Table 3). The highest average time of germination was obtained by using 15 days cold stratification treatment covey with distilled water treatment in Nahavand variety while the lowest average time of germination was obtained using 30 days cold stratification treatment covey with sulfuric acid treatment in Nahavand and Malayer varieties and 30 days cold stratification convey with kadostim in Malayer variety. Interactions of variety, chilling and breaking dormancy treatments have a significant effect on seed vigor index at %1 level of probability (Table 3). The maximum seed vigor index was obtained by using humiforte treatments convey with cold stratification tre atment of 15 days in Malayer variety and there was a significant difference between this treatment and other treatments. The minimum seed vigor index was obtained by using sulfuric acid treatment convey with cold stratification treatment of 30 days in Nahavand and Malayer varieties and there was a significant difference between this treatment and other treatments except kadostim treatment convey with cold stratification treatment of 45 days in Nahavand variety and sulfuric acid treatment convey with cold stratification treatment of 15 days in Nahavand variety (Table 3). Discussion Our data indicated that improving moist chilling to 15 days can be caused breaking dormancy of seeds as the maximum amount was obtained with 15 days cold stratification treatment resulting in 36.86% and it was decreased by increasing moist cold stratification duration as 45 days cold stratification treatment resulting in 11.18% stated that there was no significant difference in germination practically. There is continuity between seed dormancy and germination (Choun, 1996). This reaction can be due to growth of stratified embryos. Similarly, number of cell, dry weight and final length were increased in the embryonic axis of cherries seed which had been stratified (Eleni and Pelak, 1960). Also absorption of oxygen and energy were provided at the surface of cellular that the enzymes catalase, phosphatase, alkaline lipase and peroxidase were increased (Zarska-Maciejewska and Lewak, 1976). Thus it becomes clear that many of fetal development and metabolism are affected by stratification. In addition amount of hormone changes in stratified seeds. For example, ABA level is reduced due to seeds stratification of apple, pine, walnut and hazelnut. Adding gibberellin can be substituted stratification in some seeds (Pinà ¯Ã‚ ¬Ã‚ eld, 1968) due to the stimulatory role of this hormone. During the stratification procedure observed an increase in the amount of internal gibberellin which can be confirmed previous hypothesis )Frankland and Virnik, 1966). Therefore, in addition of the increase in growth rate and metabolic activity of the stratified seeds, changing of the inhibitor and stimulator levels can also be effective in breaking of the seed dormancy. The stratification time varies depending on the species. Seeds of Wild Rose needs to have a two-month period of stratification (Crocker and Barton, 1931). The stratification time depends on the age of the seeds. The seeds of Malayer variety with 32.29% of germination is ranked higher than the seeds of Nahavand variety with 13.11% of germination. The difference between seeds collection areas or the condition of the seed on the mother plant and strength of seed dormancy could be the reasons of this observation. On the other hand, it seems that induction of secondary dormancy is provided by the development of chilling on the studied seeds of the 1390. Study of breaking dormancy treatments effect indicated that the maximum affect on germination of fennel was obtained by using aminol forte, benzyl adenine + chitin treatments (Table 2-4). These reactions can probably be accrued due to the effect of cytokinins such as kinetin on increase activity of alpha-amylase that finally lead to the breakdown of starch molecules. In the other hand it seems that the resolve of seed dormancy by cytokinins may be related to increasing membrane permeability and exchanging storage materials. In addition cytokinins increase the process of cell division in the embryo by stimulating the synthesis of RNA and DNA and thereby facilitates seed germination. Thus the cytokinins are required to complete induction of germination by GA and reduce the effect of growth inhibitor (such as ABA) indirectly. Agrol and Dalani (2004) also proposed a model to study the effect of the germination stimulating and inhibitory hormones and they concluded that 1ibberellins can n ot break seed dormancy when there are high levels of ABA in the seed and in this case, presence of 1ibberellins besides the cytokinin leads to overcome ABA and seed dormancy will be beaked. Totally, it is believed that seed dormancy in excellent plants will be occurred by the balance of growth internal inhibitory and stimulating. Therefore, dormancy may be occurred due to the presence of growth inhibiting substances or lack of growth stimulating substances or a combination of both them. The amount of these endogenous compounds are controlled by environmental factors such as light and temperature. Study of interactions of moist chilling, varieties and breaking dormancy treatments indicated that cytokinins convey with increasing cold stratification duration to 15 days increased some parameters such as percentage of germination, coleoptile length, radical length, seedling length and etc. in Malayer and Nahavand varieties. The increase of mentioned parameters by cytokinins probability r elated to increasing membrane permeability and exchanging storage materials. However, chilling is effective due to elimination of germination inhibitory factors. Moist chilling increases effects of breaking dormancy treatments and as a result resolve dormancy and Induces germination. In the other hand, bio-stimulant treatments of kadostim and aminol forte and chemical treatments of nitrate potassium convey with increasing chilling duration improved degree of their germination. Acknowledgements This study was part of a MS thesis of Sayyid Amirreza Tahaei that was defended in Faculty of Agriculture of Islamic Azad University Isfahan brunch. Therefore, I would like to express my special appreciation and thanks to manager of Faculty of Agriculture of Islamic Azad University Isfahan brunch, my supervisor Dr. Ali Soleimani and my advisor.

Friday, September 20, 2019

Strains of ESBL Producing E. Coli | Investigation

Strains of ESBL Producing E. Coli | Investigation Introduction Background of Study Extended Spectrum Beta- Lactamases (ESBL) are beta lactamases which are mainly produced by family members of Enterobacteriaceae derived from mutations of the previous broad-spectrum beta-lactamase (Sharma et al., 2010). This enzyme works by hydrolysing and destroying the ÃŽ ²- Lactam ring of all cephalosporins, penicillins and monobactams (Sharma et al., 2010). In recent years, the emergence of ESBL producing Escherichia coli has posed a very serious problem to the management of diseases caused by this organism as only limited choice antibiotics can be given to patients. Carbapenems are the drugs of choice for the treatment of ESBL producing E.coli, however, carbapenamase resistance has recently been reported (Paterson and Bonomo, 2005). Prolonged use of antibiotics was suggested as the main cause of the emergence of ESBL E.coli and the fact that the genes coding for ESBLs are easily transferred from one organism to another organism via conjugation, transduction and transformation ma ke the spread even quicker (Vaidya et al., 2011). ESBL producing organisms were first reported from a patient in Germany in 1983 and since then , several outbreaks have been reported worldwide usually one particular â€Å"super† strain has been involved presumably combining not only the capability to produce ESBLs but also possessing various other virulence factors that contribute to their pathogenic success. (Harada et al., 2013). These pathogenic ESBL producing Escherichia coli in recent years have become a major concern and their emergence is now become alarming in clinical fields and subjected to comprehensive studies worldwide. The most common infections caused by pathogenic ESBL producing E.coli are urinary tract infections (UTI), bloodstream infections, gastrointestinal infections (Fatima et al., 2012; Bekat et al., 2002). According to Petty et al., (2013), globally, E.coli sequence type ST131 is the multidrug resistant clone strain which is responsible for ESBL CTX-M15 bearing genes, and it is the most alarming pathogenic ESBL producing E.coli associated in causing UTIs and septicaemia in hospital community acquired infections. ? in UK or worldwide? As genes coding for ESBL in Escherichia coli are known to be transferable this raises further fear of the spread of these genes to other strains, continuous monitoring of the predominant strains of E.coli which carry the ESBL genes is therefore important. Problem statement Studies of ESBL producing Escherichia coli in the South Manchester population have been carried out previously. This study will investigate strains of ESBL producing E. coli currently circulating in the Stockport Population of South Manchester and compare them to those delineated in the previous studies using a molecular typing and pulse-field gel electrophoresis. Objectives The objectives of the project are: Screen for ESBL Escherichia coli clinical isolates Identify strain using PFGE Assess the relatedness of the strains by PFGE analysis Determine Escherichia coli plasmid profile Identify Escherichia coli phylotyping group 1.0.4. Significance of study Finding from this study will contribute to the existing data and the body of knowledge on the molecular relationship of predominating of E.coli isolates from South Manchester populations. 1.0.5. Scope and Limitations There are no data on the antibiotics consumed by the patients in which the clinical isolates originates from. The availability of this data might help in understanding relationship between an exposures of certain antibiotics to the emergence of ESBL producing E.coli strain. PFGE also has several limitations in which the method assess visual relatedness of an isolates and not using a phylogeny relationship which provide more accurate molecular relationship between an isolates. Escherichia coli Escherichia coli is a motile gram negative rod, facultative anaerobe, non- spore forming bacteria taxonomically belong to the family of Enterobacteriaceae. It is considered as a normal inhabitants of gut and intestine in almost all warm blooded mammals and found as a faecal contaminant in the environment (Brennan et al., 2010; Darnton et al., 2007; Diniz et al., 2005). Most varieties of E.coli are harmless and do in the most part contribute to the normal and healthy intestine condition, while a few cause limiting abdominal cramp associated with diarrhoea. However, there are some serotypes that becoming a major threat to the human health, because they have acquired certain genetic material and virulence factors which enabling them transformed into pathogenic E.coli causing broad spectrum of disease (Clarke et al., 2003; Kaper et al., 2004). Pathotypes of E.coli are classified by specific mechanism in which they causing a disease, presence of certain virulence genes and their clinical manifestations (Chang et al., 2004). Growth requirements E.coli are non- fastidious bacteria, thus it can be cultured in artificial media with various altered physical and nutritional growth factors. It can be isolated easily from clinical samples by culturing into culture media and incubated at optimum temperature of 37 ºC anaerobically or aerobically as it is a facultative organisms (Yunlin et al., 2004) Uropathogenic Escherichia coli According to Pitout et al., (2005) E. coli is a frequent cause of the urinary tract infections (UTIs) of a hospitalised and non- hospitalised patients. UTIs are usually self- limiting but untreated lower urinary tract infections such as simple cystitis (bladder infection) can lead to much more severe illness known as pyelonephritis (renal infections) mainly among adult women (James et al., 2011). Infections occur by ascending movement of E. coli up the periurethral area colonising the bladder or infections by movement down from the intestinal tract. Due to anatomical complexities in women, they are more prone to be diagnosed with UTIs for at least once in their lifetime (James et al., 2001) 1.3  Escherichia coli typing 1.3.1  Plasmid profiling Multidrug resistant bacteria including ESBL producing Escherichia coli acquire their resistance by various gene transfer mechanisms which include transformation, horizontal transfer either by transduction, and conjugation, transposon and most often, are plasmid mediated (Carattoli et al., 2005) Plasmids are an extra chromosomal fragments of self- replicating DNA present in most of the bacterial species. Plasmids contain genes that are an essential for the replication of genes that promotes resistance to agents such as antibiotics, ultraviolet radiation, metals and bacteriophages. 1.3.2  Pulse-field gel electrophoresis PFGE was developed and described first by Schwartz and Cantor (1984). It is a molecular technique of typing a bacteria especially pathogenic Escherichia coli 0157:H7, non 0157: H7, Salmonella serotypes, Shigella sonnei and Shigella flexneri. PFGE uses a gel electrophoresis- based technique that allows separation of large molecular weight DNA up to 2Mb- 10Mb using a standard PFGE method (CDC, 2013; Hansen et al., 2002; Vimonet et al., 2008) PFGE is different to conventional gel electrophoresis as the large genomic DNA is digested with restriction enzyme that recognise and cleave specific sequences of DNA known as restriction site in an organism to produce a multiple DNA fragments which differ in size of their molecular weight (Van der Ploeg et al., 1984). The fragments are then run through constant changing electric field of PFGE resulting in a formation of DNA at various discrete size bands. This typing method has also been shown to have more discriminating power and reproducibility between laboratories than the newer molecular typing method such as ribotyping and multi- locus sequence typing (MLST) which confer more on the global epidemiology and revolutionary relationship between bacterial species (Vimonet et al., 2008) 1.3.3.  Escherichia coli phylogenetic group 2.0  Materials and Methods 2.0.1  Bacterial Isolates Bacterial isolates used in this study were Escherichia coli clinical isolates which was collected from Stepping Hill Hospital. Isolates undergo an anonymisation numbering of 1 to 20. 2.0.2.  Bacteriological Media The media used in the study were a selective differential medium for UTI Escherichia coli which is Chromogenic agar and nutrient agar which was used as a medium for growth and maintenance of isolates. 2.0.3  Antibiotic disks Table 1: Antibiotic disks used in this study was obtained from Oxoid.Ltd. Antibiotics Antibiotic Group Gentamicin (10 µg) Aminoglycosides Ciprofloxacin (5 µg) Quinolone Amoxicillin (25 µg) Penicillin Cefpodozime (10 µg) Cephalosporin Mecillinam (10 µg) Beta lactam Trimetophrim (2.5 µg) Bacteriostatic ESBL Disk kit (Mast Diagnostics) 2.0.4  Buffers and solutions Tris Borate EDTA (TBE X1 and X0.5) (Sigma) pH 8.2 was used as a running buffer in agarose gel electrophoresis. 2.0.5  Commercial kits The commercial kit used in this study was QIAprep Spin Miniprep Kit (Qiagen) and DNeasy Blood and Tissue Kit (Qiagen) 2.1.  Screening for multidrug resistance and potential ESBL producers in Escherichia coli clinical isolates Antibiotic susceptibility of Escherichia coli to six antibiotics (Table 1) were tested using the Kirby Bauer disk diffusion method. A 24 hour cultures from Nutrient agar was used. Then, a single colony was taken and transferred into 5ml Mueller Hinton Broth. It was then incubated at 37 °C to develop a heavy suspension of overnight cultures. A sterile cotton swabs were used to streak onto the Mueller Hinton agar and the rotation were repeated for three times. A final sweep was made around the rim of the agar. The plates were allowed to dry for several minutes. Using antibiotic dispenser, the disk that has been impregnated with a fixed antibiotic concentration was placed on the surface of the agar surface. After 24hr of an incubation period, the plates were checked for the presence of inhibition zone. Each recorded inhibition zone was compared with antimicrobial susceptibility testing disc chart provided by The British Society for Antimicrobial Chemotherapy (BSAC). The inhibition zon e of each antibiotic was reported as ‘sensitive’, ‘intermediate’ or ‘resistance’. Isolates showing resistance to three or more classes of antibiotics were considered as multidrug resistance (Falagas, 2007). ESBL producers were detected by testing sensitivity of isolates against a pair discs (cefpodoxime 10 µg and cefepime 10 µg) with and without clavulanic acid placed oppositely on an agar. According to manufacturer (Mast diagnostics), isolates were considered as an ESBL if there is a presence of 5mm larger inhibition zone in disks with clavulanic acid rather than the disks without the clavulanic acid. 2.2. Determination of plasmid profiles in MDR and ESBL Escherichia coli 2.2.1  Plasmid Extraction Prior to Plasmid DNA extraction, a fresh overnight cultures of E.coli after an incubation at 37 ºC in a Mueller Hinton broth were harvested. Plasmid DNA extraction was carried out using QIAprep Spin Miniprep Kit (Qiagen) following the manufacturer’s instructions. Extracted plasmid DNA was stored at -20 ºC until use. 2.2.2  Detection of plasmid by agarose gel electrophoresis The profiles of the plasmid DNA was determined on a 0.7% agarose gel electrophoresis which has been carried out at 70 Vcm-1 for 120 minutes. The size of DNA bands was estimated using Hyper ladder 1 (Bioline) as a reference molecular weights marker. The bands were visualized under UV transilluminator and photographed with digital camera connected to visualisation unit (Alpha Innotech) and the size of the plasmid were measured by visual comparison to the reference marker. 2.3  Escherichia coli pathotypes determination 2.3.1.  Genomic DNA extraction Primary cultures on the nutrient agar was inoculated into 3ml Mueller Hinton broth for 24 hours at 37 ºC. The cells was then harvested by centrifugation at 12, 000 for 3 minutes. Genomic DNA extraction was carried out using DNeasy Blood and Tissue (Qiagen) kit following the manufacturer’s instructions. Final volume of 150 µl genomic DNA were collected and kept at -20 ºC until needed. 2.3.2  Multiplex PCR for Escherichia coli phylotyping PCR reaction mix preparation must be carried out on ice. PCR was performed in 0.2ml PCR tubes on a GeneAmp PCR System 9700 thermocycler (Applied Biosystems ®) with a total 25 µl of reaction volume as described in Table 2 and PCR condition according to Table 3. The negative control reaction lacking the DNA was included. Table 2:  PCR reaction mix Components Required concentrations Volume ( µl) per reaction Biomix Red 2X 12.5 Primer (forward) chuA yjaA tspE4.c2 20pmol 20pmol 20pmol 1 1 1 Primer (reverse) chuA yjaA tspE4.c2 20pmol 20pmol 20pmol 1 1 1 DNA 2 Ultrapure sterile water 4.5 Total volume per reaction 25 Table 3: Conditions for PCR gene amplification Genes Primer sequence PCR condition chuA Forward 5’-GACGAACCAACGGTCAGGAT-3’ Reverse 5’-TGCCGCCAGTACCAAAGACA-3’ Initial denaturation: 94 °C for 4 mins Denaturation: 94 °C for 25 secs Annealing: 52 °C for 40 secs 30 cycles Extension: 72 °C for 50sec Final extension: 72 °C for 6 mins yjaA Forward 5’-TGAAGTGTCAGGAGACGCTG-3’ Reverse 5’-ATGGAGAATCGGTTCCTCAAC-3’ tspE4.c2 Forward 5’-GAGTAATGTCGGGGCATTCA-3’ Reverse 5’-CGCGCCAACAAAGTATTACG-3’ 2.3.3  Detection of by agarose gel electrophoresis After completion of the multiplex PCR, the amplification product were separated by dry electrophoresis system. 15 µl of amplified product was mixed with 5 µ 5X DNA loading buffer (Bioline) and loaded onto 2% agarose gel incorporated with SYBR green dye. After electrophoresis, the gel was visualised by exposing the gel under UV light and was photographed with a digital UV camera connected together with the visualisation unit (AlphaInnotech). The size of the amplicon were measured by visual comparison to the 1kb DNA marker (Bioline). Phylogenetic typing analysis were carried on the basis of the presence or absence of an amplicon sized 279bp, 211bp and 152bp which belong to chuaA, yjaA and tspE4.c2 genes respectively. 2.4.  Pulse- field gel electrophoresis (PFGE) 2.4.1.  DNA extraction Each isolates was inoculated into 5ml Mueller Hinton Broth and incubated overnight at 37 ºC with gentle agitation. Cells were then harvested by placing 1ml of culture into 1.5ml microcentrifuge tube and was centrifuged at 13, 000 rpm for one minutes. The supernatant was discarded and the process was repeated until all the 5ml of culture finished. The supernatant was again discarded and pellet of cells was resuspended in 500 µl of 0.5M EDTA buffer (see appendix) and was centrifuged at 13, 000rpm for one minutes to removes broth debris that might be interfering with the extraction processes. The washing step was repeated twice to ensure complete removal of debris. The supernatant was discarded once again and pellet was resuspended in 500 µl of suspension buffer. 2.4.2.  Preparation of low melting point (LMP) agarose To prepare the LMP agarose, 3g of SeaKem PFGE agarose (BioRad) were dispensed into 100ml of TE buffer (see appendix) in a universal bottle. It were then heated to dissolve. Agarose was transferred to a 56 ºC waterbath until needed. 2.4.3.  Preparation of the bacterial plugs The wells of PFGE plug molds were numbered. 3 plugs was prepared for each isolates. Then, 750 µl of LMP agarose was added immediately into each cell- buffer suspension and carefully mixed by pipetting up and down several times and be careful not to induce any formation of bubbles. The mixture of cells and agarose was quickly pipetted into the well of a plastic PFGE plug molds (BioRad). The wells was filled to the rim and plugs were allowed to solidify at room temperature or chilled for 5 minutes in the refrigerator. 2.4.4.  Lysis of the cells The cells were lysed by adding a mixture of 1ml of proteolysis buffer with 10 µl of Proteinase K stock solution (50mg/ml) (see appendix) into a 1.5ml new labelled microcentrifuge tube. The plugs were removed from the plug molds by peeling the sealant tape below the wells until all tape was removed. The PFGE plastic arm was used to push the plugs out of the molds into the microcentrifuge containing the mix of proteolysis buffer-proteinase K solution. All plugs for one isolates were transferred into the same tubes. Care was taken while pushing the plugs out of the molds as not to tear the fragile plugs. Tubes was then incubated in a heating block at 50 ºC for 24 hours for digestion to take place. 2.4.5.  Washing of the plugs After completion of an overnight incubation, the proteolysis buffer and Proteinase K activity were eliminated by carefully pipetting out the volume, care taken not to tear the plugs. The plugs were then washed with TE buffer. The washing steps was repeated three times, for every half an hour and were held at room temperature to equilibrate the plugs. 2.4.6.  Restriction enzyme digestion After completion of the washing steps, wash buffer was removed in the final wash leaving only agarose gel in the tubes. Then, 300 µl of 1X restriction enzyme buffer specific to the enzyme used was pipetted in each tubes containing the agarose plugs and was let to equilibrate at room temperature for 10 minutes. The restriction buffer was then discarded, taking care not to tear the plugs. Next, 300 µl of restriction buffer containing 50U of Xbal enzyme was added into the tubes and was incubated in an incubator for 24 hours at 37 ºC specific to the optimal temperature for Xbal enzyme. 2.4.7.  Pulse- field gel electrophoresis 2.4.7.1.  Electrophoresis gel preparation. After incubation, restriction enzyme reaction was stopped by addition of 200 µl of 50mM EDTA. Plugs were cooled at 4 ºC until needed. Then, a (1%) agarose gel was prepared by heated to dissolved 3g of PFGE grade agarose (BioRad) into 300ml of 0.5X TBE buffer over magnetic hot plate with constant stirring or in the microwave and swirl to dissolved. The agarose was then poured into a casting tray that has been placed with PFGE comb and let to solidify at room temperature. The enzyme- buffer was aspirated and one plug of each isolates was loaded into the gel. Care was taken not to tear the plugs. Then, a thin slice high range and mid- range lambda molecular weight marker (New England Biolabs) was loaded into the wells next to each other. After all samples was loaded into wells, the wells were sealed with melted LMP agarose. 2.4.7.2.  Electrophoresis Run The electrophoresis was performed by using a CHEF mapper (BioRad) which subsequently was filled with approximately 3 liters of 0.5ml TBE buffer. The running buffer was let to cool approximately at 14 ºC before turning on the pump. The run time was set for 24 hours at 6 Vcm-1 with 120 º angle using switch time of 2.16 sec to 54.17 sec. 2.4.7.3.  Gel staining Once the run was complete, the gel was stained with 3X Gel red nucleic acid stain (Biotium) with approximately 200ml distilled water and was gently agitated on rotary shaker for 20 minutes. The gel was then visualised under UV transilluminator and a picture was taken once optimal image obtained.