1.1 CONSTITUENTS OF THE SOIL
The term soil refers to the outer loose material of the earth crust, it is composed of five major components which include; mineral matter, water, organic matter, air and living organisms. The various components of the soil environment constantly changes and the quantity of these constituents are not the same in all soil but vary with locality. Soil is a natural body known as pedosphere and which performs four important functions; it is a medium for plant growth, it is a means of water storage, supply, and purification, it is a modifier of the atmosphere of Earth, it is a habitat for organisms, all of which in turn modify the soil (Bhagabati et al., 2004). A gram of soil can contain billions of organisms belonging to thousands of species. Soil has a prokaryotic density of roughly 1013 organisms per cubic meter (Donahue et al., 1977). A typical soil has a biomass composition of 70 % microorganisms, 22 % macro fauna and 8 % roots. The living components of an acre of soil may include 900 lb of earthworms, 2400 lb of fungi, 1500 lb of bacteria, 133 lb of protozoa and 890 lb of arthropods and algae (Pimentel, 1995).
Bacteria are a large group of unicellular prokaryotic microorganisms. Bacteria are typically a few micrometers in length and have a wide range of shapes ranging from spheres (cocci), to rods (bacilli) and spirals (spirilla), and of these, Bacilli are the most numerous than the others. The number and type of bacteria present in a particular soil would be greatly influenced by geographical location such as soil temperature, soil pH, soil type, organic matter contents, cultivation, aeration, and moisture content. Bacteria are broadly classified into two groups of Gram positive and Gram negative. The names originated from reactions to Gram stain (Rao and Subba, 1999). Bacteria and Archaea are the smallest organisms in soil apart from viruses and are the most abundant microorganisms in soil. Gram negative bacteria have a thin cell wall consisting of a few layers of peptidoglycan surrounded by a second lipid membrane containing lipopolysaccharides and lipoproteins. These bacteria do not retain crystal violet dye in the Gram staining protocol due to their lipopolysaccharide outer layer. Gram negative aerobic bacteria are found in nearly 7 % of soil resident bacteria population (Boddey et al., 1991).
1.2 ANTIMICROBIAL AGENTS
Antimicrobial agents are natural or synthetic chemical substances which have the capacity of inhibiting or terminating total metabolic cell activity of bacteria. These chemical molecules are classified depending on their targets. They can be referred to as broad spectrum or narrow spectrum depending on its range of action towards their targets. Antibacterial agents can be further subdivided into bactericidal agents which kill bacteria and bacteriostatic agents which slow down or inhibit bacterial growth (Wainwright, 1989). The major classes of antimicrobial agents are Beta-lactams, Aminoglycosides, Tetracyclines, Sulfonamides, Macrolides, Quinolones, and Glycopeptides (Drews, 2000). The British Scientist, Alexander Fleming is credited with being the first to notice that another organism could inhibit bacteria growth in 1928 (Nester et al., 2009). There are a number of bacteria that have potentials to produce antibiotic, example of which is Bacillus species which produce antibiotics like Bacitracin, Pumulin, Gramicidin, which are active against Gram positive bacteria such as Staphylococcus, Corynebacterium, and Streptococcus. Streptomyces species produce antibiotics like Tetracycline, Chloramphenicol, Vancomycin, Gentamycin, which are active against Gram negative bacteria (Waites et al., 2008). Antibacterial antibiotics are commonly classified based on their mechanisms of action, chemical structure or spectrum of activity. Most antibacterial target bacterial functions or growth processes (Calderon and Sabundayo, 2007). Those that target the bacterial cell wall (penicillins and cephalosporins), or the cell membrane (polymyxins), or interfere with essential bacterial enzyme (rifamycin, lipiarmycins, quinolones and sulfonamides) have bactericidal activities. Those that target protein synthesis (macrolides, lincosamides, and tetracyclines) are usually bacteriostatic with the exception of bactericidal aminoglycosides (Finberg et al., 2004).
1.3 TRENDS IN ANTIMICROBIAL RESISTANCE
There is great concern among public health authorities around the globe about the threat of increasing antimicrobial resistance (World Health Organisation, 2001). In response to these concerns, medical experts, professional societies and agencies such as the Centers for Disease Control and Prevention (CDC), have proposed initiatives to curtail the spread of antimicrobial resistance in pathogenic bacteria (Gerard, 2015). Although various strategies to contain the spread of antimicrobial resistance have been proposed, a better understanding to the interplay among antimicrobial use, microbial virulence and microbial adaptation is needed to determine which strategies are likely to be most effective and achievable. The history of resistance among Staphylococcus aureus isolates to various antimicrobial agents illustrates several successive evolutionary stages of resistance. Initially susceptible to penicillin G, this Gram positive coccus quickly developed the ability to produce beta lactamase (i.e penicillinase) that inactivated both the penicillins and the aminopenicillins. This resistance was at first sporadic but it then became more common and it was first observed in the hospital setting but later spread to community (Chambers, 2001). Most attention to emergence of antimicrobial resistant bacteria in hospitals has been focused on Gram positive organisms for which new antimicrobial agents are available for treatment. In contrast, less attention has been focused on emerging multidrug resistant Gram negative organisms, for which there is a current need for new antimicrobials for treatment. Data collected between 1994 and 2002 at one tertiary care center in the United States not only showed the emergence of multidrug resistant Pseudomonas aeruginosa (prevalence, 1 % – 6 %) but also showed the emergence of multidrug resistant Klebsiella species (prevalence, 0.5 % – 17 %) (D’agata, 2004). The most common resistance pattern was co-resistance to Quinolones, third generation Cephalosporins and Aminoglycosides (Manikal et al., 2000).
1.4 JUSTIFICATION FOR THE STUDY
Micro-organisms in soil are important because they affect the structure and fertility of different soil. Soil micro-organisms can be classified as bacteria, actinomycetes, algae, and protozoa (Rao and Subba, 1999). Bacteria and archaea are the most abundant micro-organisms in the soil (Wood and Martin, 1989). Bacteria population is one half of the microbial biomass in soil (Choi and Charles, 2013). Many groups of microorganisms like the Gram negative, Gram positive bacteria and fungi have the ability of synthesizing antimicrobial agents (Sudha et al., 2011). The top cultivable antimicrobial agent producers present in soils are the actinomycetes. The actinomycetes are a group of Gram positive bacteria that exhibit characteristics of both bacteria and fungi. (Thajuddin and Subramanian, 2005). Gram negative enteric pathogens are occasionally acquired from soil rather than from water or via the faecal oral route. Any soil organism may potentially enter water or an aerosol, thus soil is often the origin of water-borne infections. Enteric pathogens may enter soil after contamination of sewage or other human or animal waste and in developing countries, via untreated domestic waste disposal. Soil moisture and adsorption to clay particles may also promote survival of enteric pathogens (Santamaria and Toranzos, 2003). An outbreak of Escherichia coli 0157 disease has been convincingly associated with soil (likely via hands contaminated with mud) from a Scottish scout camp that had been previously grazed by sheep (Mukherjee et al., 2006). Indistinguishable strains of E.coli 0157 also were isolated from a diseased child and from a recently manured garden where the child had played (Ogden et al., 2002). Soil maybe a reservoir for E.coli 0157 in part because of its ability to replicate within the common soil protozoan Acanthamoeba (Barker et al., 1999). In developed countries, antibiotics are used also in farming, making antibiotic resistance a growing problem. Bacteria may also contain plasmids which are small extra chromosomal DNAs that may contain genes for antibiotic resistance (Englhardt and Peter, 1998). Because bacteria are known to swap genes when they come in contact, researchers have speculated that some resistance genes found in the soil may find their way into microbes that cause diseases in humans and animals such as E.coli or Pseudomonas aeruginosa (Williams, 2012). The remarkable increase in antibiotic resistance and the growing problem of transfer of these resistance among bacterial species has led to the search for new sources of antibiotics through the isolation and identification of micro-organisms. Soil is considered one of the most suitable environments for microbial growth and some of these soil micro-organisms have been shown to contain strong antimicrobial substances (Singh et al., 2009). According to World Health Organization, overprescription and the improper use of the antibiotics has led to the generation of antibiotic resistance in many bacterial pathogens. Nowadays the drug resistance strains of pathogens emerge more quickly than the rate of discovery of new drugs and antibiotics. (Sudha et al., 2011). Data collected between 1994 and 2002 at one tertiary care center in the united states not only showed the emergence of multidrug resistant Pseudomonas aeruginosa (prevalence, 1% – 6%) but also showed the emergence of multidrug resistant Klebsiella species (prevalence, 0.5% – 17%) (D’agata, 2004). Because of this, many scientists and pharmaceutical industry have actively involved in isolation and screening of actinomycetes from different untouched habitats for their production of antibiotics (Sudha et al., 2011). Serious infections caused by bacteria have become resistant to commonly used antibiotics and become a major global healthcare problem in the 21st century. Heightened use/misuse of antibiotics in human medicine, agriculture and veterinary is primarily contributing to the phenomenon. There is an alarming increase of antibiotic resistance in bacteria that cause either community infections or hospital acquired infections. Of particular interest are the multidrug resistant pathogens e.g. E. coli, K. pneumoniae, Acinetobacter baumani, methicillin resistant S. aureus penicillin resistant S. pneumoniae, vancomycin resistant Enterococcus and extensively drug resistant Mycobacterium tuberculosis (Alekshun and Levy, 2007). With the growing concern around the world on the increasing rates of antimicrobial resistance to available antimicrobial agents, it becomes imperative to examine the followings; whether soil dwelling Gram negative bacteria isolated in Ekpoma contributes to the pool and source of antimicrobial resistance; and whether the Gram negative bacterial isolates produce substances that inhibit or destroy other microbes. To the best of our knowledge, this study might represent the first of its kind in Ekpoma and it’s environs.
1.5 AIM AND OBJECTIVES OF THE STUDY
This research aims at determining the presence of natural occurring antimicrobial resistant bacteria in soil. The specific objectives are:
- To isolate and characterize soil dwelling Gram negative bacteria
- To determine the antibiotic resistance of isolated soil dwelling Gram negative bacteria
- To determine whether the resistance to antibiotic by isolated bacteria is plasmid or chromosomally mediated
- To determine if the isolated Gram negative bacteria produce antibacterial substances against other bacteria.