Respondents’ occupation and monthly income (n = 120).
\\n\\n
More than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\\n\\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\\n\\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\\n\\nAdditionally, each book published by IntechOpen contains original content and research findings.
\\n\\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\\n\\n\\n\\n
\\n"}]',published:!0,mainMedia:null},components:[{type:"htmlEditorComponent",content:'
Simba Information has released its Open Access Book Publishing 2020 - 2024 report and has again identified IntechOpen as the world’s largest Open Access book publisher by title count.
\n\nSimba Information is a leading provider for market intelligence and forecasts in the media and publishing industry. The report, published every year, provides an overview and financial outlook for the global professional e-book publishing market.
\n\nIntechOpen, De Gruyter, and Frontiers are the largest OA book publishers by title count, with IntechOpen coming in at first place with 5,101 OA books published, a good 1,782 titles ahead of the nearest competitor.
\n\nSince the first Open Access Book Publishing report published in 2016, IntechOpen has held the top stop each year.
\n\n\n\nMore than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\n\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\n\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\n\nAdditionally, each book published by IntechOpen contains original content and research findings.
\n\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\n\n\n\n
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Peri-urban land is conceptualized as a third space between the urban and rural hinterlands where urban land development processes meet, mix and interact on edge of the cities [1]. It has ever changing characteristics of land uses and land ownership with time and investments changes [2]. It is also a zone where peri-urban dwellers and other actors are confronted with both urban and rural laws and institutions [3]. It is a space crying out for attention [4].
Peri-urban land is of capital importance in any society. It is place where transformations resulting from the dynamics of rapid urbanization are concentrated [5]. Much of the current urban growth is taking place at the peri-urban areas. Similarly, the competition for land between agriculture and non-agriculture (urban housing) is intense in the peri-urban areas [6]. Thus, peri-urban areas are centers for almost all new land developments and changes in property right systems both formally and informally [7].
From property rights perspective, peri-urban areas are places where new urban property rights emerge to replace traditional or customary rights [8, 9]. As urbanization is penetrating into the countryside, agricultural lands are increasingly converted into non-agricultural uses or urban built-up properties. Moreover, urbanization in sub-Saharan Africa is often accompanied by the termination of existing local land rights in the peri-urban areas and the birth of new and urbanized form of land/property rights [10].
In the transitional peri-urban areas, competition for land between local rural settlers engaged in agricultural practices and urban actors interested in this land for residential purposes is fierce. The competitions for land during transition could instigate institutional changes need to regulate the emerging land rights. It is obvious that peri-urban areas are largely characterized by a wide range coexistence of formal and informal land transaction practices. Consequently, they serve as a breeding ground for new types of land tenure systems (which can be either semi-legal such as occupation of state land or non-legitimated totally) and land transaction processes which exist side by side with formal and customary systems [5].
Peri-urban land from the Ethiopian context involves an agricultural rural land adjacent to municipal boundaries, often held by local peri-urban farming communities. It is also a land under constant threat of expropriation of by the government agents and with a very high possibility of being converted into urban built-up property [11]. Peri-urban areas in Ethiopia can also be described as mushrooming place for new unauthorized/informal settlement without basic utilities. Therefore, peri-urban land in Ethiopia is a fascinating arena displaying complementary and conflicting interests.
Moreover, land in the peri-urban areas in Ethiopia is in a rapid process of change from rural agricultural land to urban built up [11]. New urban built-up properties and associated rights are being evolved at the expense of rural agricultural land rights [12]. The process of new property and property right formation in Ethiopia is not well studied and explored. This book chapter primarily explores the process of new urban built-up property formation in the peri-urban areas of Ethiopia. Details on the peri-urbanization process are discussed in this chapter under six sections. The first section details with the conceptualization of peri-urban land. The second section discusses the property right system of Ethiopia focusing on the strength and limitations. The third section focuses on how the research was conducted. The fourth section covers about urban built-up property formation based on empirical analysis and debates. The reason for why the formal and informal channel of land development co-existence in the peri-urban areas is also explored in the fifth section of the book chapter. The final section covers the conclusion of the study.
As it is clearly defined in the federal constitution of Ethiopia, land is the property of state the peoples of Ethiopia and accordingly sale and as a means of exchange is prohibited [13]. Under the umbrella of exclusive state ownership of all land, the property system is bifurcated into rural and urban landholding systems due to the bifurcation of legal and institutional frameworks for rural and urban land (see Figure 1). Land located in the rural territory is governed by a rural land holding system by which rural landholders are allowed to exercise usufruct right only without the right to permanent transfer through sale or without the right to convert it into non-agricultural use by themselves [14]. On the other hand, land in the urban jurisdiction is governed by a leasehold system by which allocation of urban land is carried out through auction and government allotment on the basis of annual rent for a specified lease period and allows development rights according to a land use plan [15].
The bifurcated property rights system in Ethiopia. Source: author produced.
State as sole supplier and owner of land is responsible to allocate all types of land use rights to the citizens. In the process of land allocation different requirements and procedures are being employed to allocate rural and urban land use rights. According to both federal and regional legislations, rural land can be granted to the people with usufruct right free of charge without time limit for those citizens who want to engage in agricultural activities for their livelihood [13, 14]. These two legal frameworks allow every citizen from 18 years of age whose main residence is in rural areas and who wants to make a living from agriculture to be accorded free access to rural land and permitted to exercise usufruct/holding rights for an indefinite period (FDRE, Proc. No. 456/2005, Art.5). Rural land in Ethiopia can be acquired by reallocation of communal or other unoccupied lands [16]. The landholders of rural land in Ethiopia have the authority to use and harvest on it, to rent it, to donate it, to bequeath and sharecropping except selling and mortgaging it.
On the other hand, the land within the administrative boundary of urban areas in Ethiopia is governed by the urban lease holding system. The lease system is proclaimed as a sole means of accessing urban land in the country since the 1990s. The system allows that all land in urban areas to be transferred in to lease system and calls for conversion of old possessions to leasehold [15]. The current lease law recognizes tender (auction) and allotment as the basic means of lease transfer from government to citizens. As a matter of principle, land needed for residential, commercial (urban agriculture, industry, or service), and other uses are transferred by competitive tender. As an exception, city municipality may give land by allotment to selected areas of paramount importance to government institutions, religious institutions, public residential housing programs, diplomatic mission, displaced persons for urban renewal. The lease system serves as a hot political and legal agenda and has been criticized for a number of reasons including rent seeking, corruption, inefficiency, price hike, etc. that made the system unable to satisfy the growing demand of the urban poor and investors [17].
Land governance and institutional setup is derived from the constitutional provision and are a reflection of the property system of the country. The responsible authorities managing land are divided into municipal and rural land administration for urban and rural land respectively both at federal government and regional government level. The division of land governance institutions into urban and rural has been creating confusion in the transitional peri-urban areas where the land is highly demanded for urbanization and being converted into urban built-up property rapidly. The authority and the land tenure system in the transitional peri-urban areas lack clarity. Sometimes this transitional area may also fall under neither rural nor urban jurisdictions.
Moreover, the urban boundaries are always expanding into the surrounding peri-urban agricultural and rural areas and this in turn has been affecting the rural jurisdiction. As urban growth strategy, the government has been implementing expropriation of land from peri-urban areas as a response to the growing demand of land for rapid urbanization and the need for modernized infrastructures.
The primary objective of this study was to create an understanding of the process of new urban property and property right formation in the transitional peri-urban areas of Ethiopia. To attain the objective a mixture of desk review and case study research approaches were employed. As a case study area, Bahir Dar city located in the north western part of Ethiopia was chosen (see Figure 2). Bahir Dar city is one of the fastest growing metropolitan areas in Ethiopia where a lot of rural agricultural land has already been converted into urban built-up properties. It is also the area where there is a huge land demand for urban development purposes.
Location map of Bahir Dar city. Source: author produced.
Focus group discussion (FGD) was undertaken with urban and rural land agency officials and experts. The officials and experts shared their views on how new urban built-up properties and property rights are being evolving. In addition, the discussants shared their views on how rural land rights are disappearing in the process of urban expansion. The focus group discussion was complemented by key informant interviews and site observations in the peri-urban areas. Key informant interview was held with key stakeholder such as planners, academia, local and regional government officials and the community leaders in the peri-urban areas. In addition to primary sources of data, an intensive desk review was conducted with a purpose to get a clear picture on the process of peri-urbanization. In addition, reviews on legal and policy frameworks and urban development strategies been conducted.
Urban areas in Ethiopia are growing and over spilling into the peri-urban areas in terms of space and population [12]. Peri-urban areas located adjacent to the municipal boundaries have become the most dynamic areas in Ethiopia. They are places where all forms of lively competitions for land are fierce. Due to the rural-urban dichotomy of land holding systems in Ethiopia, urbanization and urban development in peri-urban areas involves land holding right acquisition and transfer issues. In the process of urban expansion and development in Ethiopia, peri-urban landholders or farmers’ land rights are forcibly taken by the state and thereafter reallocated to urban residents and private developers through lease agreement. At the same time the informal acquisition and development of land is a commonly seen phenomenon in the transitional peri-urban areas of Ethiopia. This section deals with the formal and informal ways of built-up property formation process in the peri-urban areas of Ethiopia.
The formal urban development strategy of Ethiopia is solely dependent on compulsory expropriation and re-allocation of land. Expropriation measures as a mechanism to supply urban land are largely implemented in the transitional peri-urban areas and it is the single formal way of trading between the dichotomized rural and urban tenure systems where by the output would be the formation of urban built-up property. The formal urban built-up property formation process by expropriation decisions of the government involves a three step process of land acquisition and delivery [11]. First, the peri-urban area must be included within the city’s master plan; in the second step, expropriation decisions should have to be made; and third, the expropriated land need to be reallocated to different private and public users through lease contract on the basis of annual ground rent for the period specified in lease contract. As soon as previously peri-urban and rural territory is planned for urban expansion, its inhabitants become the subject of “default” expropriation, with compensation only in cases where they have legal rights to the land, and at prices that are many times lower than those farmers can fetch on the informal market.
The process of urban built-up property formation through the formal channel also reveals that the mechanism to convert peri-urban/rural land rights into urban rights is non-existent. The use right of the local peri-urban landholders supposed to be exercised for lifetime can be terminated at any time by expropriation decisions of the government. As urban boundary approaches to the peri-urban territory, local landholders at this territory are assumed to be subjects of expropriation. As a consequence, sense of land tenure insecurity is very high in the transitional peri-urban areas than any other places in Ethiopia and about 94% of the local peri-urban landholders in one way or the other feel insecure for their land right [11]. The local landholders in the peri-urban areas expect that their land shall be taken by the municipality at any time when the land is needed for urban expansion programs.
Another deficit of the urban built-up property formation through expropriation in Ethiopia is related to the wrong interpretation and assumption that all land belongs to the state which has resulted in unjustifiable disregard of land value in the amount of compensation paid to those people expropriated form their land. In this regard, the practice shows that local peri-urban landholders can be paid compensation only for improvements on land, i.e., buildings and structures on the land [18], without taking into consideration the land value itself. The practice also shows that there is a considerable variation in the rate and amount of compensation paid to the expropriated landholders [19, 20]. For example, if the land is taken for a federal purpose, landholders would be compensated at a rate determined by the federal government; if the land is taken by regional or local authorities, the rate would be determined by regional or local authorities. Therefore, the amount of compensation paid by the federal government is much higher than local or regional government. As a result landholders in the peri-urban areas prefer their land to be taken by federal government.
Moreover, the urban land development process and a new built-up property formation process in particular is not built on participatory approaches. The stakeholders inability to make a direct involvement and negotiation due to the wrong assumption that land is the exclusive property of the state and can never be definitely negotiated by any one has created uncertainty on peri-urban landholders located adjacent to the cities [11]. Experiences show that expropriation decisions made by municipal authorities are most commonly top-down without considering the interests of the peri-urban communities including the preferences in the type of compensation. For instance, the great majority of the local landholders (91%) in the peri-urban areas prefer to have either land-to-land compensation from other areas or preserving reasonable portion of land within the urban boundary [21]. Therefore, the landholders in the area would like to preserve their land use rights and stay in farming activities. In addition they want to transfer their agricultural land to their sons and daughters as they have received it from their parents. The practice shows that reaching consensus and agreement with the affected local landholders before the final decision of land acquisition is most often non-existent. As a result objection and resistance against government’s expropriation measure is a common phenomenon in the peri-urban areas of Ethiopia. The overall reflection from sample respondents and previous research results on the current urban development process in Ethiopia seems to be ignoring the land rights and livelihoods of the local peri-urban communities and skewed to the urban people [22].
Informal developments and settlements mushroom in the peri-urban areas than any other geographic place. Informal acquisition and transaction is the second mechanism for new built-up property formation in the peri-urban areas The expectation of peri-urban local landholders that their land shall be taken by urban administration compulsorily, on the one hand, and the inefficiency to provide affordable houses to the low income people in the inner-city have created an increasing pressure upon peri-urban land to be sold in the informal market [11]. In the following sub-sections an emphasis is to given explain how new urban built-up property be formed through the informal channel of land subdivision, transaction and development in the peri-urban areas.
The profile of actors involved in acquiring a plot of land through the informal channel from the peri-urban areas is examined with a purpose to provide a clear structure and insight about the characteristics of actors and institutional arrangements governing informal transaction and development of land in the peri-urban areas. The actors interested in acquiring a plot of land from peri-urban areas have multifaceted and engaged in various occupations to earn income for their livelihood (see Table 1). Self-employed households engaged in small scale commerce and daily laborers working mainly in the construction sector account more than 75% of the informal settlers in the case study area. The role of government employees and local residents in the process of new built-up property is significant as well [23]. Again, from the income perspective, the great majority of actors interested in the informal acquisition of land and formation of new built-up property earn very low monthly income.
Respondents’ occupation | Monthly income | Total (%) | |||
---|---|---|---|---|---|
<500 Eth. Birr (%) | 501–1000 Eth. Birr (%) | 1001–2500 Eth. Birr (%) | >2500 Eth. Birr (%) | ||
Low income government employees | 0 | 9 | 7.00 | 0 | 16 |
Peri-urban local residents | 1 | 6 | 0 | 1 | 8 |
Daily laborers | 19 | 23 | 0 | 42 | |
Small scale business | 7 | 24 | 2 | 1 | 34 |
Total | 27 | 62 | 9 | 2 | 100 |
Respondents’ occupation and monthly income (n = 120).
In this study, an attempt was also made to track where the informal settlers are coming form. The largest proportion respondents which account about 92% of informal settlers used to live in the inner-city in rental houses. They informal settlers justify why they preferred to come to the inaccessible and unplanned peri-urban areas to live. According to their views unregulated rising of housing rent and inability to buy a condominium flat in the inner city are the most significant pushing factors that expelled them to the peri-urban areas in search of shelter. On the other hand, availability of informally subdivided parcels in cheap prices relatively attracted them to leave their original place of residence. Some of the housing units are also constructed by local residents themselves whose prior residence is from the same locality. Local residents or local informal settlers are those actors who built muddy house either on their own farm land or on a plot received as a gift, or on a plot bought from other local peri-urban landholder. The housing units constructed by local residents account about 7% of the houses in the area. Thus, the analysis of actors involved in the formation of informal urban built-up properties (construction of informal housing units) and the nature of the houses built in the study area reveals the infancy stage of settlement and land development where the area is inhabited mainly by low income households. Moreover, all house structures built in the area are sub-standard and temporary shelters constructed of muddy and wooden walls and roof of iron sheet scattered on the agricultural land and inhabited by low income households. Moreover, basic amenities such as school for children and other basic facilities such as electricity, road and sanitation services are not available.
Peri-urban areas which used to be prime agricultural areas have also rapidly being converted into urban built-up property informally outside the official and the formal law. The actors in the informal settlement process use different modes of acquiring an informal plot of land from the informal market. The great majority of plots which is about 78% of the informal plats were bought illegally from the local peri-urban smallholding farmers who received the land for agricultural purposes. However, according to the land policy and legislations of Ethiopia, land is not salable property [13]. Some informal settlers also claim as if they have received the plot as a gift from relatives but in reality it was bought illegally. Speculators are also involving in the transaction and construction of informal houses with expectation of profit in the future and they involve in selling and buying of agricultural lands and then they convert into urban built-up property. So informal houses or urban built-up properties can also be built on plots bought from previous informal buyers with some profit. This study also shows that sub-standard houses or informal houses can be constructed by local peri-urban landholders themselves on their own agricultural field [23].
The analysis of the modes of acquisition of informal plots in the peri-urban areas shows that the largest proportion of the land occupied by informal settlers was previously held by local peri-urban farmers which destined to be used for agricultural purpose only. The action of local peri-urban landholders/farmers is against the constitutional provision that forbids land sale. As indicated above, the federal constitution of Ethiopia clearly states that all land is state property in which citizens enjoy only use rights without the right to sale [13]. Thus, peri-urban land holders play a double role in the informal urban built-up property formation process. On the one hand, they are primary suppliers of land to the informal market and on the other hand they are also playing a key role in the construction of unauthorized and sub-standard residential houses on agricultural fields without permission to do so. Their motive to construct unauthorized houses by the local landholders themselves on agricultural fields is partly due to their interest in capturing future land value increase resulted from the incorporation of the land into urban jurisdiction.
It is also important to see other factors that push local peri-urban landholders to involve in unauthorized subdivision of their agricultural fields into pieces of plots and later on to transfer it through sale and other mechanisms of transfer. The key pushing factor is their expectation that they would not be able to keep their land for long time in the future due to the rapid urban expansion into their area. The largest proportions of local peri-urban landholders feel that sooner or later the city administration would take their land for urban expansion programs through expropriation decisions. They have also a feeling that the compensation is not fair enough to cover what they loss and the whole process is not participatory. They have also a feeling that even the decided amount may not be paid on time. As a result, local peri-urban landholders on their side, by weighing the amount of compensation that they will be paid upon expropriation of the land and the sales price they are receiving by their own, prefer to sell the land.
The rural-urban dichotomy of land governance institutions is another favorable ground for the widespread practice of informal transaction of land in the transitional peri-urban areas. As a result of the urban rural dichotomy, the possibility of a power vacuum peri-urban zone to be formed is very high than any other geographic space. Most commonly power vacuum zone could be able to be formed when the municipalities adopt a revised master plan that includes the peri-urban areas into the urban center without expropriating and putting the land into its land bank. This power vacuum zone refers to a space neither under rural jurisdiction nor in the urban jurisdiction which in other words refers to a space under no one’s jurisdiction. This is an excellent opportunity for local landholders to subdivide and sale their land informally and finally to convert it into a built-up property.
The negotiation process of plot acquisition from the peri-urban areas through the informal channel involves various step by step carefully studied activities and decisions made mainly by the informal buyer so as to make the transaction safe and free from fraud. For the buyer, having accurate and reliable information about plot availability for sale and reliability of the seller are the preconditions for initial negotiation to be started between seller and buyer in the informal market. Focus group discussion with the brokers shows that information about the potential land sellers like reputation for good behavior and reliability are among the important issues that buyers want to know. It is only after the informal buyer has developed trust on the behavior and reliability of the seller that the process of negotiation for transaction would start.
Local peri-urban residents play a key role as information center for the newcomers who want to buy a plot of land from the area and to build unauthorized residential houses. Local residents are either the rightful local landholders themselves or informal settlers who came to the area some time earlier. Local residents also act as sub-brokers who supply information to the main brokers. Evidences from previous study show that the majority of informal settlers which is about 72% gained information about plot availability for sale from local residents while the remaining 28% of the respondents got information from brokers [23]. Therefore, the role of brokers in the negotiation and information diffusion process is so significant in the informal land market. They are the main diffusers of information about availability of plot for sale. Once they obtained information, they disseminate it by talking to everyone they meet. Land brokers, in the area, have a wide range of social interactions through which they can get information about plot availability for sale.
After having sufficient information about plot availability by the buyer, the potential seller and the potential buyer come into a negotiation table by the support of mediators most commonly by brokers. Again after the potential seller and buyer are being introduced each other what follows is a process of bargaining by which each party tries to secure the best deal as much as possible. The central point of the negotiation is usually the price of the plot. In addition, plot size, location, and distance from public facilities like public road networks are some of the key issues considered in the price negotiation process.
When the parties reach an agreement on the price of the plot, they end up by concluding a written agreement signed by both parties as part of documentation process. Traditional letter of agreement (contract of sale), locally known as “yemender wule,” would be concluded between the parties as evidence of transaction at the presence of three witnesses who are locally known as “shemagelewoch.” Traditional letter of agreement could be either contract of sale or loan agreement. The amount of money stated in the contractual agreement is usually greater than the real purchase amount with an intention to capture future increase in land value. The content of agreement also states that if the borrower could not pay back the loan within the agreed time, he/she agreed to convey his/her piece of land to the lender in an exchange to the money borrowed. In addition, the content of the agreement contains a written description of the extent of the plot measured in meter or feet. Usually, elders of the village or leaders of traditional social institutions are often preferred as witnesses of transaction. The buyers and sellers receive copies of the agreement, and a third copy is made and kept by one of the witnesses of transaction.
However, the contractual agreements signed by buyers and sellers either in the form of sale or loan or mortgage transfers have no legal ground due to two main reasons. First of all it is not allowed to transfer land through sale in Ethiopia [13] and second of all, contracts of transaction should follow registration by public body as a formal requirement and recognition of transaction. But land transaction the peri-urban areas through the informal channel do not fulfill the requirements. The main purpose of documentation and contractual agreement in the informal market is to avoid conflicts in the future between buyer and seller. The transaction is mainly built on trust.
Plot demarcation and documentation will follow after the parties have reached in agreement on the price of the plot. The spatial extent of the plot can be demarcated by using visible markers such as poles plants, stone marks and fence. The spatial extent of the plots are demarcated and delineated by different actors involved in the transaction most commonly at the presence of three witnesses. Social norms like trust and reciprocity play a prominent role in the land transaction and regulation of the behavior of transacting parties.
As mentioned above the transacting parties in the informal market have no legal grounds and could not be able to take their cases to courts to seek justice in the formal judiciary system when dispute arises between the two transacting parties. The conflicting parties rather prefer to take their cases to the socially respected elders and leaders of “idir” and “iqub” in the village and the elders and leaders social institutions are quite efficient in solving such conflicts. When conflict arises the affected parties will take their cases to the elders in the village and the elders try to solve the problem by urging the conflicting parties to be governed by their agreement. If the parties could not be able to reach into agreement, they may face problems like being excluded from participating in different social affairs with the community. Moreover, traditional social institutions like “ider and iqub” play a significant role in making negotiations with government bodies claiming for formalization and organizing resistance against forced eviction. These institutions also provide the members (residents) an identity of belongingness to the area. It is only through these associations that the residents in this settlement area are recognized and registered as residents of the village.
The formal and informal approaches of land transaction and development are the two contrasting ways built-up property formation ways that persist to exist in the peri-urban areas of Ethiopia (see Figure 3). The formal and informal channels of land transaction are being equally practiced side by side one influencing the other. Peri-urban land which used to be agricultural land has been rapidly converted into urban built-up property both through formal and informal channels. Most commonly, the inability of the formal land acquisition and delivery system to meet the requirements is main reason for the emergence of informality. As it is elaborated in the earlier sections, all land including peri-urban in Ethiopia is state or public property and private property does not exist [13]. According to the formal law, urban land can be allocated to the individuals largely from peri-urban areas based on 99-year leasehold contracts for residential housing [15]. Individuals may also sale and transfer the leasehold right acquired from the state to the others. Therefore, compulsory and formal conversion of the individualized usufruct right of peri-urban landholders into leasehold right is persistently growing and the end result would also be continuous formation of new urban built-up property due to rapid urbanization process.
Built-up property formation process in the peri-urban areas of Ethiopia. Source: author produced.
However, the administrative allocation of urban land through lease system after expropriation seems to be inefficient and not affordable to the urban poor and even to the middle class. On the other hand, rapid urban population growth has been intensifying a great demand for cheap and easily available urban residential land. This demand is partly indeed met by informal supply and acquisition of land from peri-urban areas. Informal supply and acquisition of land includes activities such as illegitimate occupation, unauthorized construction of substandard houses and unauthorized subdivision and sale of land. Peri-urban land holders/farmers are the key actors in the process. Peri-urban farmers fearing expropriation (revocation of their agricultural use rights/holding rights) by the state without adequate compensation prefer to subdivide their farm land into building pieces and transfer their land in the informal (black) market [23]. In addition, other different groups of actors such as brokers, speculators, corrupt government officials, peri-urban residents have been willing to involve in the process of informal transaction and development of land in the peri-urban areas. This trend result in a rapid development of an informal land market, based partly on extra-legal, partly on not-legitimated, and therefore criminal, land tenure regulations. To legitimatize the transaction the parties involved the informal transaction of land use different mechanisms such declaring the transaction as a gift, inheritance, repayment of debt and the like. Despite all illegality in the informal transaction of land, there is also an increasing trend and chance of formalization or legalization by the state. Therefore, this instance best explains the continuous breeding of informal land rights and then after the emergence of new formal property rights by means of formalization/legalization of informal land rights.
The process of peri-urbanization and urban built-up property formation in the transitional peri-urban areas of Ethiopia is assessed in detail in this chapter. Existing contemporary literatures about peri-urbanization and the resulting emergence of new urban built-up property rights formation process both through the formal (legal) system and informally outside the legal framework are also reviewed. The finding in this chapter has shown that both formal and informal ways are equally important in the process of converting peri-urban agricultural lands into new urban built-up non-agricultural properties. In the process of urban expansion, the former peri-urban landholders are expected to surrender their land to the urbanities through expropriation measures. The expropriated land is expected to be allocated to private developers and business men through lease agreement with the purpose to facilitate urban-based economic growth. This is the general insight how individualized rural usufruct right has been converting into individualized lease holding right formally and the end result of the process would be the formation of new urban built-up property. This study also shows that the urban expansion and development programs of the government into the peri-urban areas seem to seem to favor the urbanities at the expense of the local peri-urban communities.
The inefficiency of the formal land acquisition and delivery system for urban development is found out to be the driving force for the emergence of new urban built-up properties in the peri-urban areas informally. One of the key indications of the inefficiency of the formal system is the termination of agricultural usufruct right held by local peri-urban communities and the replacement of the system by the urban lease system compulsorily by the government. The unparticipatory and top-down decision of the government to expropriate land and to transfer this land to the outsider users through lease system has been pushing the local peri-urban landholders to subdivide and sell their agricultural land illegally before the government expropriates and reallocates their land to the urban developers. This is also aggravated by the bifurcation of the rural and urban land tenure system for urban and rural areas which has resulted in ambiguities on by which system that the transitional peri-urban areas shall be governed.
Finally, this study has proven that the quantity of informal built-up properties will continue to grow unless the government has made accommodative measures are taken to all group of the society. In other words, the dichotomy between the existing “formal” and “informal” city will continue to exist and it will continue to influence one another. The practice shows that unauthorized and illegal houses constructed in the peri-urban areas likely to grow in number and it is accommodating the majority and indeed the poorer section of the population who have no other option. This means it can neither be ignored nor be left to continue to grow and take its own path of development. Therefore, this requires the government to set up a responsive institutional framework that narrows down the rural-urban land governance dichotomy which has been resulting in power vacuum zone in the transitional peri-urban areas. Moreover, the federal government or the local government needs to develop proactive and protective measures for regulating informal land subdivision, use and development in the peri-urban areas.
Microbial biofilms that are sticky exopolymeric substances (EPS) causing adherence of microorganism to biotic surfaces such as host cells or abiotic surfaces such as medical devices cause antimicrobial resistance, due to its molecular contents such as eDNA and exoenzymes (β-lactamase, toxins, etc.), limited diffusion of antimicrobials through the biofilm matrix, persister cell content, and limited nutrient and oxygen. Surface proteins and polysaccharide intercellular adhesions (PIA) play a role in the biofilm production and development. It is hard to treat biofilm-embedded bacteria than planktonic forms. Biofilm producer microorganism causes biofilm-related infections such as indwelling and medical device-related infections such as endocarditis, urinary tract infections, septic arthritis, chronic rhinosinusitis, ocular infections, wound infections, etc. The results of biofilm produced on indwelling medical devices are recurrent, untreatable infections and failure of medical device. To overcome chronic and recurrent infections, it is important to detect biofilms of microorganisms, maturation and dispersion, and determine antibiofilm and antibacterial activity of agents against biofilm and bacteria within biofilm, respectively [1]. Identification of genes involved in biofilm formation and measurement of gene expression as a result of antibiofilm and antibacterial activity of agents can be advantageous with carrying out high-throughput screens using microtiter plate assay system.
The standard antimicrobial susceptibility tests such as broth macrodilution and microdilution methods that are routinely used in laboratories and published by Clinical Laboratory Standards Institute (CLSI), National Committee for Clinical Laboratory Standards (NCCLS), and European Committee on Antimicrobial Susceptibility Testing (EUCAST) could never yield accurate results in biofilm producer microorganisms, due to being appropriate for the detection of antimicrobial activity of agents against planktonic microorganism [2].
There are several methods which have been used by clinical microbiologist for detection and measurement of microbial biofilms in response to agents (Tables 1–3). Several instruments as model system have been improved such as modified Robbins device, Calgary biofilm device, disk reactor, Centers for Disease Control (CDC) biofilm reactor, perfused biofilm fermenter, and model bladder. Model systems help to define susceptibility of antimicrobial agents against biofilm producer microorganisms by providing information about biofilm mechanisms. Substratums of modified Robbins device, Calgary biofilm device, disk reactor, CDC biofilm reactor, and perfused biofilm fermenter are silastic disks, plastic pegs, Teflon coupons, plastic needleless connectors, and cellulose acetate filters, respectively, whereas substratum of model bladder is urinary catheters (UCs). Medical devices of which dimensions are adjusted to appropriate sizes can also be used as a substratum (abiotic surfaces) for biofilm production by adapting and modifying to related methods by some biofilm researchers. The methods of modified Robbins device and Calgary biofilm device are based on viable counting. In Calgary biofilm device, pegs are sonicated before counting. The methods of disk reactor and CDC biofilm reactor based on direct and viable counting, after substratums, are sonicated, vortexed, and homogenized. In perfused biofilm fermenter, viable counting is done, after filters are shaken in sterile distilled water, whereas in model bladder, UCs are examined directly by scanning electron microscopy (SEM) or transmission electron microscopy (TEM) or by chemical analysis [2]. Rate of biofilm formed on model system can be adjusted by parameters such as composition of medium that can contain glucose, iron, antimicrobial agents, multivalent cations such as Ca2+ and Mg2+ supporting adhesion of bacteria by cross-linking anionic groups on bacteria and substratum, shear force, retention time, flow rate, roughness, and chemistry of substratum and species of organisms (Table 4) [2, 3].
Method | Action of application | Aim |
---|---|---|
Roll plate | Extraluminal biofilm detection | Growth of biofilm-embedded bacteria |
Sonication, vortex, and plate counting | Intraluminal and extraluminal biofilm detection | Growth of biofilm-embedded bacteria |
Acridine orange staining | Extraluminal biofilm detection | Direct investigation of biofilm produced on catheter by microscopy |
Streak plating of alginate swab | Investigation of biofilm produced on indwelling catheter | Growth of biofilm-embedded bacteria |
The methods used for detection and measurement of biofilms produced on medical devices.
Method | Aim |
---|---|
Tube method (TM) | Qualitative detection by observing biofilm lined on bottom and walls of tube |
Congo red agar (CRA) | Qualitative detection by observing colony color change |
Microtiter plate (MtP) | Quantitative detection of biofilm by microplate reader (microELISA) |
Real-time PCR | Detection of biofilm genes |
Conventional PCR | |
Multiplex PCR |
The methods used for detection of biofilm.
Method | Application | Target |
---|---|---|
Microtiter plate (MtP) | Measurement of biofilm produced on walls of wells in response to agent | Measures the effect of agents against biofilm production |
Microtiter plate (MtP) (MBEC) | Measurement of biofilm remained on walls of wells in response to agent and detecting MBEC of agents | Measures the effect of agents against mature biofilm formed on walls of wells |
Vortex and plate counting | Plate counting of biofilm-embedded bacteria and detecting bMBC of agents | Screens antimicrobial activity of agents against biofilm-embedded bacteria |
Checkerboard assay | Plate counting of biofilm-embedded bacteria and FIC indexes are calculated | Screens antimicrobial activity of combination of agents |
Sonication, vortex, and plate counting | Plate counting of biofilm-embedded bacteria and detecting bMBC of agents | Screens antimicrobial activity of agents against biofilm-embedded bacteria |
Quantitative PCR | Measurement of specific biofilm gene expression | Monitors expression of biofilm genes in response to agents |
Mass spectrometry (MS) | Measurement of exoenzymes located in biofilm matrix | Monitors expression of bacterial proteins in response to agents |
The screening methods for antibiofilm and antimicrobial activity of agents against biofilm producer bacteria.
Instruments | Culture dynamics | Substratum | Method |
---|---|---|---|
Modified Robbins device | Batch culture | Silastic disks | Viable counting |
Calgary biofilm device | Batch culture | Plastic polycarbonate pegs | Viable counting, after pegs are sonicated |
Disk reactor | Batch culture | Teflon coupons | Direct or viable counting, after coupons are sonicated, vortexed, and homogenized |
CDC biofilm reactor | Continuous culture | Plastic connectors | |
Perfused biofilm fermenter | Continuous culture | Cellulose-acetate filters | Viable counting, after filters are shaken in sterile distilled water |
Model bladder | Continuous culture | Urinary catheters | Examining directly by SEM or TEM or analyzing chemically |
Flow cell | Continuous culture | Chambers with transparent surfaces | Examining by confocal laser scanning microscopy |
Instruments used to produce biofilm and examine biofilm process.
CDC biofilm reactor, Centers for Disease Control biofilm reactor; SEM, scanning electron microscopy; TEM, transmission electron microscopy.
The aim of this chapter is to overview certain methods used by biofilm detection and antibacterial and antibiofilm researches such as tube method (TM), Congo red agar (CRA) method, microtiter plate (MtP) assay, plate counting of biofilm-embedded bacteria (sessile bacteria), PCR, mass spectrometry (MS), confocal laser scanning microscopy (CLSM), etc.
Complexity and dynamics of biofilms can be observed by biofilm imagining optical technology including light microscopy, SEM, TEM, and CLSM. These techniques are used to visualize 3D structure and check the existence of biofilm [4].
Light microscopy is the easiest, cheapest, most simple, convenient and fastest method to quantitatively observe the morphology of microorganisms adhered to surfaces and to semiquantitatively estimate the amount of microorganism attached on surface (exist, absent, abundant, rare, etc.). Microorganisms including Candida albicans, E. coli, Pseudomonas, and Staphylococcus epidermidis adhered on acrylic sheets of polymethacrylate films, glass cover slips, and polystyrene petri dishes have been observed by light microscope, respectively. Observation with light microscopy that requires clear, transparent, and planar surfaces on which microorganisms attach does not create 3D vision of biofilm. Dyes can be used such as epifluorescence and fluorescent to enhance image clarity of microorganisms. The observation with the light microscope enables researchers to compare morphologies of sessile form and planktonic form of microorganism required by making smear and centrifuging of sample, respectively [3].
Images of cells and cell structures such as protein and nucleic acid are obtained by electrons at high magnification and resolution. Monitoring of components of cell can be done directly in TEM by negative staining. Due to photons and electrons penetrating cells poorly, thin section of cell cut is stabilized and stained by certain chemicals with the treatment of osmic acid, permanganate, uranium, lanthanum, or lead salts. These stains contain high atomic weight. Due to stains having high atomic weight, contrast is accelerated by electron dispersion from sample. If observation of outer structure of cells will be done, it is not important whether the section of cell is thin or thick.
Due to inadequate stabilization of polysaccharides is done by the conventional fixatives such as aldehydes, glutaraldehyde, paraformaldehyde, and osmium tetroxide, water content of biofilm is eliminated by graded dehydration with alcohol after this postfixation step. After sample is infiltrated with resin, sample is embedded in gelatin capsule and headed for polymerization. Then, thin section taken is poststained with uranyl acetate and lead citrate.
Exopolysaccharide constituents are not observed with its own electron dense and staining poststains such as uranyl acetate and lead citrate with TEM, due to not only having high electron translucent, but also contrast is not developed by conventional poststains. According to the studies, glycocalyx of Staphylococcus hominis and Staphylococcus epidermidis can be stabilized by the usage of certain cationic reagent combinations including ruthenium red, alcian blue, lysine, lysine monohydrochloride, or lysine acetate and paraformaldehyde [5]. After all these steps are done, sample is observed in TEM (Figure 1).
The SEM image of S. aureus embedded in biofilm colonized on intravenous catheter [6].
To visualize 3D images of cell sample is coated with heavy metals such as gold. Electrons released from metal coating of sample are caught by SEM for image production. The procedure of SEM is similar to TEM except for some additional chemicals (gold), lacking infiltration, embedment in resin, polymerization, and thin section staining with lead citrate and uranyl acetate [5]. As in the steps of TEM method, postfixation and dehydration steps of SEM are similar to TEM. The step is applied after dehydration step is drying and coating sample with gold in the processing for SEM, rather than infiltration with resin, embedment in gelatin capsule, and staining with lead citrate and uranyl acetate in the processing for TEM. After dehydration process with graded alcohol, sample is dried and coated with gold palladium [5]. After all these steps are done, sample is observed in SEM (Figure 2).
The TEM image of Staphylococcus spp. surrounded by glycocalyx [6].
Biofilms formed on flow cells of which surface are transparent can be observed by confocal laser screening microscopy (CLSM). Three-dimensional (3D) morphology and physiology of biofilms can be screened by CLSM [2]. Thick samples such as biofilms and microorganisms localized in the depth such as biofilm-embedded microorganisms need to be observed by CLSM (Figure 3).
Bacterial community embedded in a biofilm matrix visualized by CLSM. Each bacterium observed with a distinct color located at different depths of biofilm [11].
For observation of biofilm with confocal microscopy and related methods, biofilm must be fluorescent as a result of fluorescent molecules such as green fluorescent protein (GFP) that is fluorescent protein expressed by biofilm producer microorganism within biofilm (gene of cell interested is tagged by gene cassette encoding GFP) or staining components of heterogeneous mass of biofilm with fluorescence or fluorescence-labeled dyes [2]. Stains such as lectins target extracellular matrix, whereas certain fluorophores target extracellular DNA (eDNA) to visualize eDNA content of biofilm matrix [2, 7].
By scanning laser light across the sample, deep penetration of excitated energy is provided. As a result of fluorescence of biomolecules such as GFP or chlorophyll that are intrinsic fluorophores or molecules signed by exogenous probes such as fluorescent-labeled antibodies detected by photomultiplier, 3D digital image is formed. Observation of biofilms that are multilayered and have complex 3D structures requires additional resolution [2]. Images of each layers of biofilm obtained are combined by computer for construction of digital 3D images of whole biofilm.
Biofilm producer microorganisms can be manipulated genetically by tagging of microbial gene of interest by gene cassette encoding GFP as a reporter gene (gfp genes) to monitor gene expression and metabolic physiology activity in biofilms and determine location of microorganism within biofilm [2, 8, 9].
Idea about gene activity in biofilms is given by confocal microscopy applied to 3D localization of nonenzyme reporter systems such as GFP. Growth phase and activity of bacteria embedded in biofilms can be defined by promoter-reporter systems that is designed and fluoresced just in living dividing cells. In situ cellular growth activity of bacteria embedded in biofilms is determined by measuring ribosome-hybridization-signal intensity, due to synthesis rate and content of ribosome correlated with the growth rate (especially in exponential phase). Expression cassette that is active only in growing cells, labeled by GFP and controlled by rRNA promoter, can be constructed to monitor growth phase and activity of bacteria within biofilm [2, 10].
Specific microorganisms present in a heterogeneous biofilm community can be identified by the probes of fluorescent in situ hybridization (FISH) method. GFP that is translated enables procedure not to require fixation or staining. Fluorescent-labeled microorganism within biofilm can also be examined by FISH. DNA probes designed to hybridize 16S rRNA of microorganism integrated to either fluorescent dye such as FITC or Rhodamine or enzyme such as horseradish peroxidase. The advantage of probes conjugated with horseradish peroxidase is not to destroy microorganism within biofilm. The growth rate of microorganism within biofilm can be determined by FISH method, due to the amounts of ribosomes existing in a microorganism that is directly proportional to growth activity of microorganism. Probe must be designed to label conserved region of only a single species (Figure 4) [2, 12].
Fluorescence image of 28 distinct E. coli strains labeled by fluorophore-conjugated oligonucleotides complementary to 16S rRNA of E. coli [11].
Roll plate method is applied for the detection of possible microbial colonization having a potential to develop indwelling device-associated infection on the outer surface of cylindrical materials such as catheters and vascular grafts. Microorganism colonize on external surface of catheter is detected by roll plate method, instead of microorganism colonize on intraluminal site of catheter. Material is touched and rolled on the surface of medium [3].
Congo red agar (CRA) method that is a qualitative assay for detection of biofilm producer microorganism, as a result of color change of colonies inoculated on CRA medium, is described by Freeman et al. The CRA medium is constructed by mixing 0.8 g of Congo red and 36 g of sucrose to 37 g/L of Brain heart infusion (BHI) agar. After incubation period that was 24 h at 37°C, morphology of colonies that undergone to different colors is differentiated as biofilm producers or not. Black colonies with a dry crystalline consistency indicate biofilm producers, whereas colonies retained pink are non-biofilm producers (Figure 5) [13].
CRA method applied on CRA medium. Black crystalline colonies of biofilm producer cell and pinkish-red colonies of biofilm nonproducer cell.
Tube method (TM) that is a qualitative assay for detection of biofilm producer microorganism, as a result of the occurrence of visible film, is described by Christensen et al. [14]. Isolates are inoculated in polystyrene test tube which contained TSB and incubated at 24 h at 37°C. The sessile isolates of which biofilms formed on the walls of polystyrene test tube are stained with safranine for 1 h, after planktonic cells are discharged by rinsing twice with phosphate-buffered saline (PBS). Then, safranine-stained polystyrene test tube is rinsed twice with PBS to discharge stain. After air drying of test tube process, the occurrence of visible film lined the walls, and the bottom of the tube indicates biofilm production (Figure 6) [14].
Tube method. The first two polystyrene test tubes from the left indicate biofilm production. Other test tubes rather than the first two polystyrene test tubes from the left indicate lacking of biofilm production.
Microtiter plate (MtP) assay is a quantitative method to determine biofilm production by microplate reader. Bacterial suspension is prepared in MHB supplemented with 1% glucose and adjusted to 0.5 McFarland (1.108 cfu/ml). This bacterial suspension is 20-fold (1/20) diluted to reach 5.106 cfu/ml. Then 180 μl of Mueller-Hinton Broth (MHB) supplemented with 1% glucose [15] and 20 μl of bacterial suspensions are inoculated into 96-well flat-bottomed sterile polystyrene microplate to obtain 5.105 cfu/ml as a final concentration (tenfold dilution (1/10)). Microplates are incubated at 24 h at 37°C. The sessile isolates of which biofilms formed on the walls of wells of microplate are stained with only 150 μl of safranine for 15 min, after planktonic cells in wells of microplate are discharged by washing twice with phosphate-buffered saline (PBS) (pH 7.2) and wells are dried at 60°C for 1 h [14]. Before staining with safranine, fixation of biofilms can be done by either subjecting to 150 μl of methanol for 20 min or drying at 60°C for 1 h. Then safranine-stained wells of microplates are washed twice with PBS to discharge safranine stain. After air drying process of wells of microplate, dye of biofilms that lined the walls of the microplate is resolubilized by 150 μl of 95% ethanol or 33% glacial acetic acid or methanol. Then microplate is measured spectrophotometrically at 570 nm by a microplate reader [15, 16]. The studies are repeated in triplicates. Uninoculated wells containing sterile MHB supplemented with 1% glucose that are considered to be the negative controls are used as blanks. The blank absorbance values are used to identify whether biofilm formation of isolates exists or not. The wells of isolates of which OD values are higher than blank well are considered to be biofilm producers. Cut off value (ODc) can provide categorization of isolates as biofilm producer or not.
Negative value obtained from this formula and represented as zero indicates lack of biofilm production, whereas positive value indicates biofilm production (Figure 7).
Microtiter plate assay indicating biofilm production.
To interpret results, categorization can be done as no biofilm production (0), weak (+ or 1), moderate (++ or 2), and strong biofilm production (+++ or 3) by the calculation of cutoff value (ODc) shown below [15]:
OD ≤ ODc no biofilm production
ODc< OD ≤ 2 × ODc weak biofilm production
2 × ODc< OD ≤ 4 × ODc moderate biofilm production
4 × ODc< OD strong biofilm production.
PCR techniques is used for not only identification of pathogens by amplifying species-specific nucleic acid sequences but also detection of virulence factors by amplifying target virulence genes such as biofilm genes with the usage of gene-specific primers, even in the uncultured pathogen present in the sample.
Forward and reverse primers of biofilm-associated gene are designed. Firstly, multiple alignments were done in the NCBI to find oligonucleotide sequences specific to the species. Then primer pair of biofilm-associated gene is designed by using Primer3Plus verified by FASTA analysis checking specificity of primers for microbial sequences in the database [17, 18].
Genomic DNA of microorganism is extracted by extraction kits of which protocols can vary according to species and Gram-positivity or Gram-negativity of microorganisms. DNA of microorganism is measured spectrophotometrically by microplate spectrophotometry reader to determine the amount of DNA extracted as microgram per microliter.
Biofilm-associated gene is amplified by PCR such as qualitative real-time PCR, multiplex and conventional PCR that is used to detect whether biofilm-associated gene is present or not in microorganism. If conventional and multiplex PCR protocols are applied to detect biofilm gene, rather than qualitative real-time PCR, PCR product isolated is visualized on an agarose gel containing a DNA-intercalating dye such as ethidium bromide to confirm the presence of amplified gene (Figure 8). Only in qualitative real-time PCR, the amplicon is detected by fluorescence using a pair of specific hybridization probes labeled with fluorescence dye [11].
Image of mecA gene on agarose gel. First sample is DNA size marker (ranging from 250 to 10,000 kb), second sample is ATCC 43300 methicillin-resistant Staphylococcus aureus (MRSA) positive control, third and fourth samples are mecA gene-positive isolates, and fifth sample is ATCC 29213 methicillin-sensitive Staphylococcus aureus (MSSA) as a negative control.
Microtiter plate (MtP) assay is a qualitative assay to detect efficacy of agent against biofilm production by microplate reader.
Bacterial suspension is prepared in MHB supplemented with 1% glucose and adjusted to 0.5 McFarland (1.108 cfu/ml). This bacterial suspension is 20-fold (1/20) diluted to reach 5.106 cfu/ml.
A 180 μl of agent doses and 20 μl of bacterial suspension are dispersed to each well of microplate to obtain 5.105 cfu/ml as a final concentration (tenfold dilution (1/10)). After incubation at 37°C for 24 h, ongoing processes are done according to MtP assay as mentioned previously for the determination of effect of agent against biofilm production [14, 16].
Biofilms remained after eradication by agent are measured by this technique. Biofilms of bacteria that line the walls of wells are formed according to MtP method.
After the content of microplate is discharged, 200 μl of each dose of agents is dispersed to each well of microplate of which the walls are lined with biofilm. A 200 μl of distilled water is added into a well of microplate of which the walls are lined with biofilm as a control. Then the effect of agent against mature biofilm is determined according to MtP assay as mentioned previously. Minimum concentration of agent eradicating mature biofilm that is named as minimum biofilm eradication concentration (MBEC) can be determined by this modified plate assay. MBEC50 and MBEC90 indicate the minimum concentrations of agents inhibiting 50 and 90% of mature biofilm formed.
In summary, biofilm formation process on abiotic surfaces by bacteria is done. Quantification of sessile biofilm-embedded bacteria lined on abiotic surface and sessile biofilm-embedded bacteria remained on abiotic surface after addition of agent on abiotic surface on which mature biofilms formed is determined by plate counting. Bacterial suspension is prepared and adjusted to 0.5 McFarland (1.108 cfu/mL) in Mueller-Hinton Broth (MHB) supplemented with 1% glucose [15]. This bacterial suspension is 200-fold (1/200) diluted to gain 5.105 cfu/mL. Kirschner wire orthopedic implants are placed into each test tube containing 5.105 cfu/mL isolate and incubated at 37°C for 24 h to lead bacteria to produce biofilm on Kirschner wire. After incubation, Kirschner wires on which biofilms are produced are discharged and rinsed with PBS (pH 7.2) and then transferred into each test tubes containing agent concentrations. After incubation at 37°C for 24 h, Kirschner wires are discharged and placed into test tubes containing 1 mL of sterile MHB and sonicated at 42 kHz for 2 min after vortexed for 5 min. Then 100 μl samples of each test tube sonicated and vortexed are inoculated on Mueller-Hinton agar (MHA) and incubated at 37°C for 24 h [19].
The lowest concentration of agent in which bacterial growth is below or equal to control is determined as biofilm minimum inhibitory concentration (bMIC) for biofilm. bMIC50 and bMIC90 indicate the minimum concentrations of agent inhibiting 50 and 90% of biofilm-embedded bacteria. After incubation, the lowest concentration of agent in which colonies of biofilm-embedded bacteria are not grown is determined as biofilm minimum bactericidal concentration (bMBC) of agent for biofilm [19].
Checkerboard assay is used for the determination of combination effects of two different agents. A 250 μl twofold dilutions of each agent from the stock solutions are dispersed to each row and column to obtain final varying concentrations by starting at fourfold of zero MIC for each isolate. So each well contains distinct combination of concentrations of two agents. First wells of rows and columns are left behind for sole treatments of each dose of agents. One well is used for bacterial control (Figure 9). Kirschner wires on which bacterial biofilm is produced are dispersed to each well. This microplate is incubated at 37°C for 24 h. After incubation, Kirschner wires are discharged and sonicated at 42 kHz for 2 min after vortexed for 5 min. The lowest concentration of agent in which bacterial growth that is not observed is determined as biofilm minimum inhibitory concentration (bMIC) of agent for biofilm. Then 100 μl samples of each test tube sonicated and vortexed are inoculated on MHA and incubated at 37°C for 24 h. After incubation, the lowest concentration of agent in which colonies of biofilm-embedded bacteria are not grown is determined as biofilm minimum bactericidal concentration (bMBC) of agent for biofilm. For the determination of whether the synergism is present between agents or not, fractional inhibitory concentrations (FICs) index that are calculated for each agent are summed up according to formula written below:
The schematization of checkerboard assay.
When ∑FIC is equal and lesser than 0.5, between 0.5 and 1, equal to 1, higher than 1 and equal and lesser than 4, and higher than 4, it is interpreted that the effect between agents in combination is synergistic, partial synergistic, additive, indifferent, and antagonistic, respectively [20].
The wells having the highest synergy rates of two agents that constitute the combinations are determined by taking the average and standard deviation of FIC indexes calculated of the wells with the lowest drug combination without bacterial growth in each row and column (Figure 9).
Measurements of biofilm genes repressed or induced by agent are done by quantitative real-time PCR (qPCR). So efficacy of agent against biofilm-associated genes can be detected by qPCR.
Complementary DNA (cDNA) is copied from RNA by enzyme reverse transcriptase. Gene expression in pathogen is monitored by qPCR copying cDNA from RNA of target gene. Amplified cDNA probed for identification. Fluorescent probes such as dye SYBR Green are used to indicate double-stranded DNA, consequently amplification. Accumulation of PCR amplicons labeled fluorescently is monitored through the qPCR processes (Figure 10). Visualization of amplicon on agarose gel is not needed to confirm amplification in qPCR [11].
Quantitative real-time polymerase chain reaction (qPCR) of atl (light blue), 16S RNA (red), mecA (purple), and nuc (light pink) genes of sample; icaA (gray), icaD (plato), atl (pink), mecA (blue), and nuc (green) genes of ATCC 43300 methicillin-resistant Staphylococcus aureus (MRSA); and icaA (orange), 16S RNA (yellow), and nuc (grayish blue) genes of ATCC 29213 methicillin-sensitive Staphylococcus aureus (MSSA). Expressions of all these genes are monitored by qPCR except nuc (light pink) gene sample. Lines below threshold monitored by qPCR indicate the negativity of genes such as icaA and icaD genes samples and icaD (turquoise), atl (plato), and mecA (light pink) genes of ATCC 29213 MSSA. RFI, relative fluorescence intensity.
Total RNA of microorganism is isolated according to protocols of RNA isolation kits. Kit protocols can vary according to the species of microorganism. Total RNA of microorganism is measured spectrophotometrically by microplate spectrophotometry reader to determine the amount of total RNA isolated as microgram per microliter. Then cDNA is synthesized from total RNA with qPCR using primer pair of the biofilm-associated gene, which is designed using Primer3 and verified by FASTA analysis, which controls the specificity of the primers for microbial sequences in the data system, after multiple alignments were done in the NCBI to find oligonucleotide sequences specific to the species [17, 18].
Extracellular polymeric substances (EPS) not only contain polysaccharides but also contain proteins such as extracellular enzymes. These expressed proteins located in the matrix of EPS can be detected and characterized by mass spectrometry (MS) [1]. Large biologic molecules can be also detected and characterized in complex biologic structures such as EPS by MS. Chemicals involved in biofilm process are examined in detail by MS. Electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are the types of MS [2]. In time-of-flight (TOF) mass spectrometer, mass is analyzed by ions desorbed in vacuum chamber. These two technics are combined and called MALDI-TOF.
Sample is ionized and vaporized by laser. Ions generated from sample by laser pass through the column of MALDI-TOF device toward TOF detector by an electric field. Depending on the mass/charge ratio of molecule, measurements are done by TOF. If this ratio is smaller, ions move faster (Figure 11).
MALDI-TOF mass spectrometry device [11].
Bacteria are identified, expression of bacterial proteins such as surface proteins and exoenzymes like β-lactamase in response to antimicrobials can be monitored, and growth of bacteria is measured by applications of MALDI. MS has high sensitivity and requires minimum amount of sample [2].
Biofilm-embedded bacteria can be estimated by biologic assays that is an indirect assay. Biological assays that measure production of microbial product give an opinion about estimation of the number of microorganism within biofilm. Amount of biologic product is correlated with biofilm-embedded microorganism producing the product by standardization of planktonic microorganism. Biologic products produced by planktonic microorganism are similar to biologic products produced by biofilm-embedded bacteria. Standardization curves of each microorganism tested need to be formed. Measurement of total protein at the absorbance is 550 or 950 nm; tryptophan fluorescence, endotoxin [2], ATP production via bioluminescence caused by luciferin and luciferase, urease production to estimate number of attached microorganism, and electron transport via the production of formazan are done by biological assays [3].
Biofilms cause resistance to many antimicrobial agents. The results of biofilm produced on indwelling medical devices are recurrent, untreatable infections and failure of medical device. To overcome chronic and recurrent infections, it is important to detect biofilms of microorganisms, determine antibiofilm activity of agents against biofilm, and determine antibacterial activity of agents against biofilm-embedded microorganism with the appropriate methods by clinical microbiologist and biofilm researcher microbiologist. Identification of genes involved in biofilm formation and measurement of gene expression as a result of antibiofilm and antibacterial activity of agents can be advantageous in biofilm studies.
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She performed research in perioperative autotransfusion and obtained the degree of PhD in 1993 publishing Peri-operative autotransfusion by means of a blood cell separator.\nBlood transfusion had her special interest being the president of the Haemovigilance Chamber TRIP and performing several tasks in local and national blood bank and anticoagulant-blood transfusion guidelines committees. Currently, she is working as an associate professor and up till recently was the dean at the Albert Schweitzer Hospital Dordrecht. She performed (inter)national tasks as vice-president of the Concilium Anaesthesia and related committees. \nShe performed research in several fields, with over 100 publications in (inter)national journals and numerous papers on scientific conferences. \nShe received several awards and is a member of Honour of the Dutch Society of Anaesthesia.",institutionString:null,institution:{name:"Albert Schweitzer Hospital",country:{name:"Gabon"}}},{id:"83089",title:"Prof.",name:"Aaron",middleName:null,surname:"Ojule",slug:"aaron-ojule",fullName:"Aaron Ojule",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Port Harcourt",country:{name:"Nigeria"}}},{id:"295748",title:"Mr.",name:"Abayomi",middleName:null,surname:"Modupe",slug:"abayomi-modupe",fullName:"Abayomi Modupe",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/no_image.jpg",biography:null,institutionString:null,institution:{name:"Landmark University",country:{name:"Nigeria"}}},{id:"94191",title:"Prof.",name:"Abbas",middleName:null,surname:"Moustafa",slug:"abbas-moustafa",fullName:"Abbas Moustafa",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/94191/images/96_n.jpg",biography:"Prof. Moustafa got his doctoral degree in earthquake engineering and structural safety from Indian Institute of Science in 2002. He is currently an associate professor at Department of Civil Engineering, Minia University, Egypt and the chairman of Department of Civil Engineering, High Institute of Engineering and Technology, Giza, Egypt. He is also a consultant engineer and head of structural group at Hamza Associates, Giza, Egypt. Dr. Moustafa was a senior research associate at Vanderbilt University and a JSPS fellow at Kyoto and Nagasaki Universities. He has more than 40 research papers published in international journals and conferences. He acts as an editorial board member and a reviewer for several regional and international journals. His research interest includes earthquake engineering, seismic design, nonlinear dynamics, random vibration, structural reliability, structural health monitoring and uncertainty modeling.",institutionString:null,institution:{name:"Minia University",country:{name:"Egypt"}}},{id:"84562",title:"Dr.",name:"Abbyssinia",middleName:null,surname:"Mushunje",slug:"abbyssinia-mushunje",fullName:"Abbyssinia Mushunje",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Fort Hare",country:{name:"South Africa"}}},{id:"202206",title:"Associate Prof.",name:"Abd Elmoniem",middleName:"Ahmed",surname:"Elzain",slug:"abd-elmoniem-elzain",fullName:"Abd Elmoniem Elzain",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Kassala University",country:{name:"Sudan"}}},{id:"98127",title:"Dr.",name:"Abdallah",middleName:null,surname:"Handoura",slug:"abdallah-handoura",fullName:"Abdallah Handoura",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"École Supérieure des Télécommunications",country:{name:"Morocco"}}},{id:"91404",title:"Prof.",name:"Abdecharif",middleName:null,surname:"Boumaza",slug:"abdecharif-boumaza",fullName:"Abdecharif Boumaza",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Abbès Laghrour University of Khenchela",country:{name:"Algeria"}}},{id:"105795",title:"Prof.",name:"Abdel Ghani",middleName:null,surname:"Aissaoui",slug:"abdel-ghani-aissaoui",fullName:"Abdel Ghani Aissaoui",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/105795/images/system/105795.jpeg",biography:"Abdel Ghani AISSAOUI is a Full Professor of electrical engineering at University of Bechar (ALGERIA). He was born in 1969 in Naama, Algeria. He received his BS degree in 1993, the MS degree in 1997, the PhD degree in 2007 from the Electrical Engineering Institute of Djilali Liabes University of Sidi Bel Abbes (ALGERIA). He is an active member of IRECOM (Interaction Réseaux Electriques - COnvertisseurs Machines) Laboratory and IEEE senior member. He is an editor member for many international journals (IJET, RSE, MER, IJECE, etc.), he serves as a reviewer in international journals (IJAC, ECPS, COMPEL, etc.). He serves as member in technical committee (TPC) and reviewer in international conferences (CHUSER 2011, SHUSER 2012, PECON 2012, SAI 2013, SCSE2013, SDM2014, SEB2014, PEMC2014, PEAM2014, SEB (2014, 2015), ICRERA (2015, 2016, 2017, 2018,-2019), etc.). His current research interest includes power electronics, control of electrical machines, artificial intelligence and Renewable energies.",institutionString:"University of Béchar",institution:{name:"University of Béchar",country:{name:"Algeria"}}},{id:"99749",title:"Dr.",name:"Abdel Hafid",middleName:null,surname:"Essadki",slug:"abdel-hafid-essadki",fullName:"Abdel Hafid Essadki",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"École Nationale Supérieure de Technologie",country:{name:"Algeria"}}},{id:"101208",title:"Prof.",name:"Abdel Karim",middleName:"Mohamad",surname:"El Hemaly",slug:"abdel-karim-el-hemaly",fullName:"Abdel Karim El Hemaly",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/101208/images/733_n.jpg",biography:"OBGYN.net Editorial Advisor Urogynecology.\nAbdel Karim M. A. El-Hemaly, MRCOG, FRCS � Egypt.\n \nAbdel Karim M. A. El-Hemaly\nProfessor OB/GYN & Urogynecology\nFaculty of medicine, Al-Azhar University \nPersonal Information: \nMarried with two children\nWife: Professor Laila A. Moussa MD.\nSons: Mohamad A. M. El-Hemaly Jr. MD. Died March 25-2007\nMostafa A. M. El-Hemaly, Computer Scientist working at Microsoft Seatle, USA. \nQualifications: \n1.\tM.B.-Bch Cairo Univ. June 1963. \n2.\tDiploma Ob./Gyn. Cairo Univ. April 1966. \n3.\tDiploma Surgery Cairo Univ. Oct. 1966. \n4.\tMRCOG London Feb. 1975. \n5.\tF.R.C.S. Glasgow June 1976. \n6.\tPopulation Study Johns Hopkins 1981. \n7.\tGyn. Oncology Johns Hopkins 1983. \n8.\tAdvanced Laparoscopic Surgery, with Prof. Paulson, Alexandria, Virginia USA 1993. \nSocieties & Associations: \n1.\t Member of the Royal College of Ob./Gyn. London. \n2.\tFellow of the Royal College of Surgeons Glasgow UK. \n3.\tMember of the advisory board on urogyn. FIGO. \n4.\tMember of the New York Academy of Sciences. \n5.\tMember of the American Association for the Advancement of Science. \n6.\tFeatured in �Who is Who in the World� from the 16th edition to the 20th edition. \n7.\tFeatured in �Who is Who in Science and Engineering� in the 7th edition. \n8.\tMember of the Egyptian Fertility & Sterility Society. \n9.\tMember of the Egyptian Society of Ob./Gyn. \n10.\tMember of the Egyptian Society of Urogyn. \n\nScientific Publications & Communications:\n1- Abdel Karim M. El Hemaly*, Ibrahim M. Kandil, Asim Kurjak, Ahmad G. Serour, Laila A. S. Mousa, Amr M. Zaied, Khalid Z. El Sheikha. \nImaging the Internal Urethral Sphincter and the Vagina in Normal Women and Women Suffering from Stress Urinary Incontinence and Vaginal Prolapse. Gynaecologia Et Perinatologia, Vol18, No 4; 169-286 October-December 2009.\n2- Abdel Karim M. El Hemaly*, Laila A. S. Mousa Ibrahim M. Kandil, Fatma S. El Sokkary, Ahmad G. Serour, Hossam Hussein.\nFecal Incontinence, A Novel Concept: The Role of the internal Anal sphincter (IAS) in defecation and fecal incontinence. Gynaecologia Et Perinatologia, Vol19, No 2; 79-85 April -June 2010.\n3- Abdel Karim M. El Hemaly*, Laila A. S. Mousa Ibrahim M. Kandil, Fatma S. El Sokkary, Ahmad G. Serour, Hossam Hussein.\nSurgical Treatment of Stress Urinary Incontinence, Fecal Incontinence and Vaginal Prolapse By A Novel Operation \n"Urethro-Ano-Vaginoplasty"\n Gynaecologia Et Perinatologia, Vol19, No 3; 129-188 July-September 2010.\n4- Abdel Karim M. El Hemaly*, Ibrahim M. Kandil, Laila A. S. Mousa and Mohamad A.K.M.El Hemaly.\nUrethro-vaginoplasty, an innovated operation for the treatment of: Stress Urinary Incontinence (SUI), Detursor Overactivity (DO), Mixed Urinary Incontinence and Anterior Vaginal Wall Descent. \nhttp://www.obgyn.net/urogyn/urogyn.asp?page=/urogyn/articles/ urethro-vaginoplasty_01\n\n5- Abdel Karim M. El Hemaly, Ibrahim M Kandil, Mohamed M. Radwan.\n Urethro-raphy a new technique for surgical management of Stress Urinary Incontinence.\nhttp://www.obgyn.net/urogyn/urogyn.asp?page=/urogyn/articles/\nnew-tech-urethro\n\n6- Abdel Karim M. El Hemaly, Ibrahim M Kandil, Mohamad A. Rizk, Nabil Abdel Maksoud H., Mohamad M. Radwan, Khalid Z. El Shieka, Mohamad A. K. M. El Hemaly, and Ahmad T. El Saban.\nUrethro-raphy The New Operation for the treatment of stress urinary incontinence, SUI, detrusor instability, DI, and mixed-type of urinary incontinence; short and long term results. \nhttp://www.obgyn.net/urogyn/urogyn.asp?page=urogyn/articles/\nurethroraphy-09280\n\n7-Abdel Karim M. El Hemaly, Ibrahim M Kandil, and Bahaa E. El Mohamady. Menopause, and Voiding troubles. \nhttp://www.obgyn.net/displayppt.asp?page=/English/pubs/features/presentations/El-Hemaly03/el-hemaly03-ss\n\n8-El Hemaly AKMA, Mousa L.A. Micturition and Urinary\tContinence. Int J Gynecol Obstet 1996; 42: 291-2. \n\n9-Abdel Karim M. El Hemaly.\n Urinary incontinence in gynecology, a review article.\nhttp://www.obgyn.net/urogyn/urogyn.asp?page=/urogyn/articles/abs-urinary_incotinence_gyn_ehemaly \n\n10-El Hemaly AKMA. Nocturnal Enuresis: Pathogenesis and Treatment. \nInt Urogynecol J Pelvic Floor Dysfunct 1998;9: 129-31.\n \n11-El Hemaly AKMA, Mousa L.A.E. Stress Urinary Incontinence, a New Concept. Eur J Obstet Gynecol Reprod Biol 1996; 68: 129-35. \n\n12- El Hemaly AKMA, Kandil I. M. Stress Urinary Incontinence SUI facts and fiction. Is SUI a puzzle?! http://www.obgyn.net/displayppt.asp?page=/English/pubs/features/presentations/El-Hemaly/el-hemaly-ss\n\n13-Abdel Karim El Hemaly, Nabil Abdel Maksoud, Laila A. Mousa, Ibrahim M. Kandil, Asem Anwar, M.A.K El Hemaly and Bahaa E. El Mohamady. \nEvidence based Facts on the Pathogenesis and Management of SUI. http://www.obgyn.net/displayppt.asp?page=/English/pubs/features/presentations/El-Hemaly02/el-hemaly02-ss\n\n14- Abdel Karim M. El Hemaly*, Ibrahim M. Kandil, Mohamad A. Rizk and Mohamad A.K.M.El Hemaly.\n Urethro-plasty, a Novel Operation based on a New Concept, for the Treatment of Stress Urinary Incontinence, S.U.I., Detrusor Instability, D.I., and Mixed-type of Urinary Incontinence.\nhttp://www.obgyn.net/urogyn/urogyn.asp?page=/urogyn/articles/urethro-plasty_01\n\n15-Ibrahim M. Kandil, Abdel Karim M. El Hemaly, Mohamad M. Radwan: Ultrasonic Assessment of the Internal Urethral Sphincter in Stress Urinary Incontinence. The Internet Journal of Gynecology and Obstetrics. 2003. Volume 2 Number 1. \n\n\n16-Abdel Karim M. El Hemaly. Nocturnal Enureses: A Novel Concept on its pathogenesis and Treatment.\nhttp://www.obgyn.net/urogynecolgy/?page=articles/nocturnal_enuresis\n\n17- Abdel Karim M. El Hemaly. Nocturnal Enureses: An Update on the pathogenesis and Treatment.\nhttp://www.obgyn.net/urogynecology/?page=/ENHLIDH/PUBD/FEATURES/\nPresentations/ Nocturnal_Enuresis/nocturnal_enuresis\n\n18-Maternal Mortality in Egypt, a cry for help and attention. The Second International Conference of the African Society of Organization & Gestosis, 1998, 3rd Annual International Conference of Ob/Gyn Department � Sohag Faculty of Medicine University. Feb. 11-13. Luxor, Egypt. \n19-Postmenopausal Osteprosis. The 2nd annual conference of Health Insurance Organization on Family Planning and its role in primary health care. Zagaziz, Egypt, February 26-27, 1997, Center of Complementary Services for Maternity and childhood care. \n20-Laparoscopic Assisted vaginal hysterectomy. 10th International Annual Congress Modern Trends in Reproductive Techniques 23-24 March 1995. Alexandria, Egypt. \n21-Immunological Studies in Pre-eclamptic Toxaemia. Proceedings of 10th Annual Ain Shams Medical Congress. Cairo, Egypt, March 6-10, 1987. \n22-Socio-demographic factorse affecting acceptability of the long-acting contraceptive injections in a rural Egyptian community. Journal of Biosocial Science 29:305, 1987. \n23-Plasma fibronectin levels hypertension during pregnancy. The Journal of the Egypt. Soc. of Ob./Gyn. 13:1, 17-21, Jan. 1987. \n24-Effect of smoking on pregnancy. Journal of Egypt. Soc. of Ob./Gyn. 12:3, 111-121, Sept 1986. \n25-Socio-demographic aspects of nausea and vomiting in early pregnancy. Journal of the Egypt. Soc. of Ob./Gyn. 12:3, 35-42, Sept. 1986. \n26-Effect of intrapartum oxygen inhalation on maternofetal blood gases and pH. Journal of the Egypt. Soc. of Ob./Gyn. 12:3, 57-64, Sept. 1986. \n27-The effect of severe pre-eclampsia on serum transaminases. The Egypt. J. Med. Sci. 7(2): 479-485, 1986. \n28-A study of placental immunoreceptors in pre-eclampsia. The Egypt. J. Med. Sci. 7(2): 211-216, 1986. \n29-Serum human placental lactogen (hpl) in normal, toxaemic and diabetic pregnant women, during pregnancy and its relation to the outcome of pregnancy. Journal of the Egypt. Soc. of Ob./Gyn. 12:2, 11-23, May 1986. \n30-Pregnancy specific B1 Glycoprotein and free estriol in the serum of normal, toxaemic and diabetic pregnant women during pregnancy and after delivery. Journal of the Egypt. Soc. of Ob./Gyn. 12:1, 63-70, Jan. 1986. Also was accepted and presented at Xith World Congress of Gynecology and Obstetrics, Berlin (West), September 15-20, 1985. \n31-Pregnancy and labor in women over the age of forty years. Accepted and presented at Al-Azhar International Medical Conference, Cairo 28-31 Dec. 1985. \n32-Effect of Copper T intra-uterine device on cervico-vaginal flora. Int. J. Gynaecol. Obstet. 23:2, 153-156, April 1985. \n33-Factors affecting the occurrence of post-Caesarean section febrile morbidity. Population Sciences, 6, 139-149, 1985. \n34-Pre-eclamptic toxaemia and its relation to H.L.A. system. Population Sciences, 6, 131-139, 1985. \n35-The menstrual pattern and occurrence of pregnancy one year after discontinuation of Depo-medroxy progesterone acetate as a postpartum contraceptive. Population Sciences, 6, 105-111, 1985. \n36-The menstrual pattern and side effects of Depo-medroxy progesterone acetate as postpartum contraceptive. Population Sciences, 6, 97-105, 1985. \n37-Actinomyces in the vaginas of women with and without intrauterine contraceptive devices. Population Sciences, 6, 77-85, 1985. \n38-Comparative efficacy of ibuprofen and etamsylate in the treatment of I.U.D. menorrhagia. Population Sciences, 6, 63-77, 1985. \n39-Changes in cervical mucus copper and zinc in women using I.U.D.�s. Population Sciences, 6, 35-41, 1985. \n40-Histochemical study of the endometrium of infertile women. Egypt. J. Histol. 8(1) 63-66, 1985. \n41-Genital flora in pre- and post-menopausal women. Egypt. J. Med. Sci. 4(2), 165-172, 1983. \n42-Evaluation of the vaginal rugae and thickness in 8 different groups. Journal of the Egypt. Soc. of Ob./Gyn. 9:2, 101-114, May 1983. \n43-The effect of menopausal status and conjugated oestrogen therapy on serum cholesterol, triglycerides and electrophoretic lipoprotein patterns. Al-Azhar Medical Journal, 12:2, 113-119, April 1983. \n44-Laparoscopic ventrosuspension: A New Technique. Int. J. Gynaecol. Obstet., 20, 129-31, 1982. \n45-The laparoscope: A useful diagnostic tool in general surgery. Al-Azhar Medical Journal, 11:4, 397-401, Oct. 1982. \n46-The value of the laparoscope in the diagnosis of polycystic ovary. Al-Azhar Medical Journal, 11:2, 153-159, April 1982. \n47-An anaesthetic approach to the management of eclampsia. Ain Shams Medical Journal, accepted for publication 1981. \n48-Laparoscopy on patients with previous lower abdominal surgery. Fertility management edited by E. Osman and M. Wahba 1981. \n49-Heart diseases with pregnancy. Population Sciences, 11, 121-130, 1981. \n50-A study of the biosocial factors affecting perinatal mortality in an Egyptian maternity hospital. Population Sciences, 6, 71-90, 1981. \n51-Pregnancy Wastage. Journal of the Egypt. Soc. of Ob./Gyn. 11:3, 57-67, Sept. 1980. \n52-Analysis of maternal deaths in Egyptian maternity hospitals. Population Sciences, 1, 59-65, 1979. \nArticles published on OBGYN.net: \n1- Abdel Karim M. El Hemaly*, Ibrahim M. Kandil, Laila A. S. Mousa and Mohamad A.K.M.El Hemaly.\nUrethro-vaginoplasty, an innovated operation for the treatment of: Stress Urinary Incontinence (SUI), Detursor Overactivity (DO), Mixed Urinary Incontinence and Anterior Vaginal Wall Descent. \nhttp://www.obgyn.net/urogyn/urogyn.asp?page=/urogyn/articles/ urethro-vaginoplasty_01\n\n2- Abdel Karim M. El Hemaly, Ibrahim M Kandil, Mohamed M. Radwan.\n Urethro-raphy a new technique for surgical management of Stress Urinary Incontinence.\nhttp://www.obgyn.net/urogyn/urogyn.asp?page=/urogyn/articles/\nnew-tech-urethro\n\n3- Abdel Karim M. El Hemaly, Ibrahim M Kandil, Mohamad A. Rizk, Nabil Abdel Maksoud H., Mohamad M. Radwan, Khalid Z. El Shieka, Mohamad A. K. M. El Hemaly, and Ahmad T. El Saban.\nUrethro-raphy The New Operation for the treatment of stress urinary incontinence, SUI, detrusor instability, DI, and mixed-type of urinary incontinence; short and long term results. \nhttp://www.obgyn.net/urogyn/urogyn.asp?page=urogyn/articles/\nurethroraphy-09280\n\n4-Abdel Karim M. El Hemaly, Ibrahim M Kandil, and Bahaa E. El Mohamady. Menopause, and Voiding troubles. \nhttp://www.obgyn.net/displayppt.asp?page=/English/pubs/features/presentations/El-Hemaly03/el-hemaly03-ss\n\n5-El Hemaly AKMA, Mousa L.A. Micturition and Urinary\tContinence. Int J Gynecol Obstet 1996; 42: 291-2. \n\n6-Abdel Karim M. El Hemaly.\n Urinary incontinence in gynecology, a review article.\nhttp://www.obgyn.net/urogyn/urogyn.asp?page=/urogyn/articles/abs-urinary_incotinence_gyn_ehemaly \n\n7-El Hemaly AKMA. Nocturnal Enuresis: Pathogenesis and Treatment. \nInt Urogynecol J Pelvic Floor Dysfunct 1998;9: 129-31.\n \n8-El Hemaly AKMA, Mousa L.A.E. Stress Urinary Incontinence, a New Concept. Eur J Obstet Gynecol Reprod Biol 1996; 68: 129-35. \n\n9- El Hemaly AKMA, Kandil I. M. Stress Urinary Incontinence SUI facts and fiction. Is SUI a puzzle?! http://www.obgyn.net/displayppt.asp?page=/English/pubs/features/presentations/El-Hemaly/el-hemaly-ss\n\n10-Abdel Karim El Hemaly, Nabil Abdel Maksoud, Laila A. Mousa, Ibrahim M. Kandil, Asem Anwar, M.A.K El Hemaly and Bahaa E. El Mohamady. \nEvidence based Facts on the Pathogenesis and Management of SUI. http://www.obgyn.net/displayppt.asp?page=/English/pubs/features/presentations/El-Hemaly02/el-hemaly02-ss\n\n11- Abdel Karim M. El Hemaly*, Ibrahim M. Kandil, Mohamad A. Rizk and Mohamad A.K.M.El Hemaly.\n Urethro-plasty, a Novel Operation based on a New Concept, for the Treatment of Stress Urinary Incontinence, S.U.I., Detrusor Instability, D.I., and Mixed-type of Urinary Incontinence.\nhttp://www.obgyn.net/urogyn/urogyn.asp?page=/urogyn/articles/urethro-plasty_01\n\n12-Ibrahim M. Kandil, Abdel Karim M. El Hemaly, Mohamad M. Radwan: Ultrasonic Assessment of the Internal Urethral Sphincter in Stress Urinary Incontinence. The Internet Journal of Gynecology and Obstetrics. 2003. Volume 2 Number 1. \n\n13-Abdel Karim M. El Hemaly. Nocturnal Enureses: A Novel Concept on its pathogenesis and Treatment.\nhttp://www.obgyn.net/urogynecolgy/?page=articles/nocturnal_enuresis\n\n14- Abdel Karim M. El Hemaly. 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