ABSTRACT
A total of 106 actinomycetes isolated from the rhizosphere of plants in abbatoir and refuse dumps in Awka and Onitsha were investigated for the production of antimicrobial substances. Five of them were found to show antimicrobial activity against Gram positive and Gram negative bacteria as well as fungi on solid media. Three of the very active isolates designated MP-75, SP -76 and QP-100 were further investigated in submerged medium in a shake-flask experiment using glucose and soy bean as carbon and nitrogen sources respectively. Isolates MP-75 and SP-76 were found to produce antimicrobial substances. The antimicrobial substance produced by isolates SP-76 showed the highest antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, Klebsiella sp, Staphylococcus aureus and Bacillus sp. The isolates were identified as Streptomyces species based on their characteristic features. Activity of the antimicrobial substance produced by Streptomyces SP-76 was maximum when 4% (w/v) glucose and 2% (w/v) soybean were used in the fermentation process. Influence of surfactants on accumulation of antimicrobial substance by Streptomyces SP-76 in the fermentation broth showed that Tween 80, oleic acid, palmitic acid and stearic acid enhanced antimicrobial activity. The effect of growth promoters on antibiotic production by Streptomyces SP-76 indicated that peptone, casein and yeast extract stimulated antimicrobial activity against the test organisms. The effect of varying pH on antibiotic production by Streptomyces SP-76 showed that there was maximum antimicrobial activity at pH 7. In a time course for antibiotic production, maximum antimicrobial activity was obtained at 120h. Paper chromatography of the culture filtrate of Streptomyces SP-76 indicated that it contains more than one antimicrobial substance. From this, it can be seen that the growth and subsequent production of bioactive metabolites by Streptomyces SP-76 isolated from the soil
CHAPTER ONE
INTRODUCTION
For centuries, preparations derived from living matter were applied to wounds to destroy infection. The fact that a microorganism is capable of destroying one another was not established until the latter half of the 19th century, when Pasteur noted the antagonistic effect of other bacteria on the anthrax organism and pointed out that this action might be put to therapeutic use. Meanwhile, the German Chemist, Paul Ehrlich developed the idea of selective toxicity; that certain chemicals that would be toxic to some organisms like infectious bacteria, would be harmless to other organisms e.g humans (Limbird, 2004).
In 1928, Sir Alexander Fleming, a Scottish biologist, observed that Penicillium notatum, a common mold, had destroyed Staphylococcus bacteria in culture and in 1939, the American microbiologist Rene Dubois demonstrated that a soil bacterium was capable of decomposing the starchlike capsule of the Pneumococcus bacterium, without which the Pneumococcus is harmless and does not cause pneumonia.
Dubois then found in the soil a microbe, Bacillus brevis, from which he obtained a product, tyrothricin, that was highly toxic to a wide range of bacteria (Limbird, 2004). Tyrothricin, a mixture of the two peptides, gramicidin and tyrocidine, was also found to be toxic to red blood and reproductive cells in humans but could be used to good effect when applied as an ointment on body surfaces. Penicillin was finally isolated in 1939, and in 1994 Selman Waksman and Albert Schatz, American microbiologists, isolated streptomycin and a number of other antibiotics from Streptomyces griseus
(Calderon and Sabundayo, 2007).
2
Discovery of new antibiotics produced by Streptomyces still continues. Today, due to the increasing resistance of pathogenic bacteria to our current arsenal of antibiotics, a great need exist for the isolation and discovery of new antibiotics and other drug agents (Jarroff, 1994; Rice, 2003; Wenzel, 2004). Fifty years ago, it was easy to discover new antibiotics by simply screening the fermentation broths of actinomycetes and fungi. Today, it is much more challenging, but there are much better tools to address this problem. Discovering new antibiotics, pharmacophores is a long-term endeavor that requires deft orchestration and support of many innovative sciences (Cuatrecasas, 2006). There are three approaches that can be used to improve our chances of finding new antibiotic substances: new test methods, new organisms, and variation of culture conditions. None of these three options guarantees success alone and the chances are best if the three are combined. There is need for long-term basic microbiological research, which should cover the following areas: methods of isolating and cultivating microorganisms that have not yet been accessed or only with great difficulty, studies on the transportation of antibiotics into the bacterial cell, comparative biochemistry of prokaryotes and eukaryotes, mode of action of antibiotics and pathogenicity factors. In addition to search for new antibiotics, longterm strategies to prevent the development and spreading of resistant bacteria must be developed (Fiedler and Zanher, 1995). Therefore, it is time to define natural product discovery from actinomycetes and other microbes as a major priority for medical sciences and to engage the most creative scientists in academia (Cuatrecasas, 2006) in close collaboration with biotechnology and pharmaceutical companies, which would elevate the science to a new level of achievement so as to be commensurate with past successes and present demonstrated potential.
3
In this study, effort has been made:
– To isolate Streptomyces from the soil, capable of producing antimicrobial substances
– To study the cultural conditions necessary for antibiotic production.
– To determine the time taken for the optimum production of antimicrobial substance.
– To determine the type of antimicrobial substance(s) present in the culture
filtrate.
4
LITERATURE REVIEW
Antibiotics can be defined as agents produced by microorganisms, which can kill or hinder the growth of other microorganisms. They are produced as secondary metabolites (Bibb, 2005). Antibiotics usually are products of bacteria and fungi, though synthetic antibiotics exist.
In terms of monetary value, antibiotics are currently the most important products of microbial biotechnology, apart from such “traditional” products as alcoholic beverages and cheese. Worldwide antibiotic production was estimated at a value of about 8 billion dollars in 1981 and has doubled since that time. Antibiotics are used extensively in human and veterinary medicine, as well as in agriculture, for the purpose of preventing (prophylaxis) or treating microbial infections. They are also used to promote the growth of animals (Gaskins et al., 2002). In some countries, they are used at low doses in animal feeds and are considered to improve the quality of the product, with a lower percentage of fat and higher protein content (Cromwell, 2002).
Peptide antibiotics are quite diverse, amphipathic, either non-ribosomally synthesized (e.g. bacitracins, polymyxins and gramicidins) or ribosomally synthesized (Bdefensins, magainins and thionicins) (Gu et al., 2006). Bacteria and fungi use nonribosomal peptide synthetases to produce broad structural and biologically-active peptides (Sorensen et al., 1996). Naturally occurring antimicrobial peptides are widely distributed among evolutionary divergent organisms including mammals, amphibians, insects, plants and bacteria, and play a significant role in innate immunity
(Zasloff, 1992; Boman, 1995). The mechanism of antimicrobial peptides is elucidated 5
through actions like membrane channel and pore formation, which allows passage of ions and other solutes, inhibition of protein synthesis, DNA damage and interference with cell wall synthesis (Lehrer et al., 1985; Agawa et al., 1991; Gera and Lichenstein, 1991; Debano and Gordee, 1994; Bechinger, 1997; Hancock, 1997;
Lehrer et al., 1998; De Lucca and Walsh, 2000; Selitrennikoff, 2001; Brown and
Hancock, 2006).
Most of the antibiotics in use today are derivatives of natural products of actinomycetes and fungi (Butler and Buss, 2006; Newman and Crag, 2007). Medical chemistry has played a key role in modifying core natural products to optimize the pharmacological properties while minimizing toxicity. Recent studies on various Streptomyces species have shown that many regulatory factors are involved in creating a complex network, which then influences the morphological differentiation and production of secondary metabolites. Such regulatory cascade are consistent with the need of Streptomyces spp. to interact with a variety of environmental changes (Bibb, 1996). If the regulatory mechanism of anabolic biosynthesis can be wholly understood and tightly controlled, this could be a starting point for replacing mutagenesis by UV irradiation or mutagen treatment and the consecutive screening of high-producer strains for improving the productivity of anabolic biosynthesis. Unfortunately, however the molecular processes regulating the events leading to differentiation and simultaneous anabolic biosynthesis are still poorly understood, despite the discovery of numerous useful insights into Streptomyces genetics (Hwang et al., 2002).
6
GOALS OF ANTIBIOTIC RESEARCH
The study and development of antibiotics certainly share some of the same aims as other areas of biotechnology. For example, it is always desirable to try to improve the yield of an antibiotic fermentation and subsequent processing steps. Indeed, in the early stage of the development of penicillin, the crucial achievement was to obtain the compound in amounts sufficient for therapeutic use. Also, for antibiotics that are put mainly to veterinary or agricultural uses, reducing cost through improved yields is of paramount importance. However, much of the research on the antibiotics for human use has a very different focus. When trying to develop a cure for a life-threatening infection, the cost of treatment is not the most important factor (Glazer and Nikaido, 2001).
A very large fraction of antibiotic research is directed towards the development of new agents. They are needed because of the many micro-organisms including most fungi and viruses, for which we do not yet possess truly effective and safe antibiotic agents. Some bacteria, such as Pseudomonas aeruginosa, also are intrinsically resistant to most antibiotics (Kirst and Sides, 1989).
An even more complex problem is the emergence of resistant strains among the organisms that were sensitive to antibiotics before the drugs became widely used. This phenomenon tends to limit the useful life of any new antibiotic, requiring the pharmaceutical industry to come up with new compounds continually. The need is especially acute because of the following unfortunate situation. In any modern hospital, huge amounts of antibiotics are used in the treatment as well as the prevention of infectious diseases. As a result, the hospital environment becomes highly enriched for bacteria that are resistant to those antibiotics. At the same time, 7
the immune and other defence mechanisms of the body are not functioning well in many hospitalized-patients, who are thus especially vulnerable to hospital acquired (nosocomial) infection by these resistant bacteria (Glazer and Nikaido, 2001).
HISTORY OF ANTIBIOTICS
Many cures for infectious diseases prior to the beginning of the twentieth century were based on medicinal folklore. Cures for infection in ancient Chinese medicine using plants with antibiotic– like properties began to be described over 2,500 years ago (Lindbland, 2008). Many other ancient cultures, including the ancient-Egyptians, ancient-Greeks and Madeira Arabs already use molds and plants to treat infections (Forrest, 1982; Wain- Wright, 1989). Cinchona bark was a widely effective plant for treatment of malaria in the 17th Century, the disease caused by protozoan parasites of the genus Plasmodium (Lee, 2002). Scientific endeavors to understand the science behind what caused these diseases, the development of synthetic antibiotic chemotherapy, the isolation of the natural antibiotics marked milestones in antibiotic development (Foster and Raoult, 1974).
Originally known as antibiosis, which means ‘against life’, the term was introduced by the French bacteriologist Vuilleum as a descriptive name of the phenomenon exhibited by these drugs (Calderon and Sabundayo, 2007). Antibiosis was first described in 1877 in bacteria when Louis Pasteur and Robert Koch observed that an airborne bacillus could inhibit the growth of Bacillus anthracis (Landberg, 1949). These drugs were later renamed antibiotics by Selman Waksman,an American microbiologist in 1942 (Waksman, 1947; Calderon and Sabundayo, 2007).
8 Synthetic antibiotic chemotherapy as a science and the story of antibiotic development began in Germany with Paul Ehrlich, a German medical scientist in the late 1880s. He noted that certain dyes would bind to and colour human, animal or bacterial cells, while others did not. He then extended the idea that it might be possible to make certain dyes or chemicals that would act as a magic bullet or selective drug that would bind to and kill bacteria while not harming the human host. After much experimentation, screening hundreds of dyes against various organisms, he discovered a medicinally useful drug, the man-made antibiotic Salvarsan (Limbird 2004; Calderon and Sabundayo, 2007; Bosch and Rosich, 2008). However, the adverse side-effect profile of Salvarsan, coupled with the later discovery of the antibiotic penicillin, superseded its use as an antibiotic. The work of Ehrlich, which marked the birth of the antibiotic revolution, was followed by the discovery of prontosil by Domagk in 1932 (Bosh and Rosich, 2008). Prontosil, the first commercially available antibacterial antibiotic was developed by a research team led by Gerhard Domagk, at the Bayer laboratories of the IG Farben conglomerate in Germany. Prontosil had a relatively broad effect against Gram-positive cocci but not against enterobacteria. The discovery and development of this first sulfonamide drug opened the era of antibiotics.
The discovery of natural antibiotics produced by microorganisms stemmed from earlier work on the observation of antibiosis between micro-organisms. Pasteur observed that “if we could intervene in the antagonism observed between some bacteria, it would offer “perhaps the greatest hopes for therapeutics” (Kingston,
2008). Bacterial antagonism of Penicillium sp. as first described in England by John
Tyndall in 1875 (Kingston, 2008). However, his work went by without much notice
from the scientific community until Alexander Fleming’s discovery of Penicillin in 1928. Even then the therapeutic potential of Penicillin was not pursued. More than ten years later, Ernst Chain and Howard Florey became interested in Fleming’s work, and came up with the purified form of Penicillin. The purified antibiotic displayed antibacterial activity against a wide range of bacteria. It also had low toxicity and could be taken without causing adverse effect. Furthermore, its activity was not inhibited by biological constituents such as pus, unlike the synthetic antibiotic. No one had discovered a compound equaling this activity previous to this. The discovery of penicillin led to renewed interest in the search for antibiotic compounds with similar capabilities. Because of their discovery of penicillin, Ernst Chain, Howard
Florey and Alexander Fleming shared the 1945 Nobel prize in medicine. In 1939, Rene Dubois isolated gramicidin, one of the first commercially manufactured antibiotics in use during world war ll, which proved highly effective in treating wounds and ulcers (van Epps, 2006). Florey credited Dubois for reviving his research in penicillin (van Epps, 2006).
ANTIMICROBIAL PHARMACODYNAMICS.
The assessment of the activity of an antibiotic is crucial to the successful outcome of antimicrobial therapy. Non-microbiological factors such as host defense mechanisms, the location of an infection, the underlying disease as well as the intrinsic pharmacokinetics and pharmacodynamics affect the properties of an antibiotic (Pankey and Sabath, 2004). Fundamentally, antibiotics are classified as either having lethal or bactericidal action against bacteria or are bacteriostatic, preventing bacterial growth. The bactericidal activity of antibiotics may be growth phase dependent and in most but not all cases the action of many bactericidal antibiotics requires ongoing cell activity and cell division for the drugs killing activity (Mascio et al., 2007). These classifications are based on laboratory behaviour; in practice, both of these are capable of ending a bacterial infection (Pelczar et al., 1999; Pankey and Sabath, 2004). ‘In vitro’ characterization of the action of antibiotics to evaluate activity measure the minimum inhibitory concentration and minimum bactericidal
concentration of an antimicrobial and are excellent indicators of antimicrobial potency (Weigand et al., 2008). However, in clinical practice, these measurements alone are insufficient to predict clinical outcome. By combining the pharmacokinetic profile of an antibiotic with the antimicrobial activity, several pharmacological parameters appear to be significant markers of drug efficacy (Spanu et al., 2004). The activity of antibiotics may be concentration-dependent and their characteristic antimicrobial activity increases with progressively higher antibiotic concentrations (Rhee and Gardiner, 2004). They may also be time-dependent, where their antimicrobial activity does not increase with increasing antibiotic concentrations; however, it is critical that a minimum inhibitory serum concentration is maintained for a certain length of time (Rhee and Gardiner, 2004). A laboratory evaluation of the killing kinetics of the antibiotic using killing curves is useful to determine the time-or concentrationdependence of antimicrobial activity (Pankey and Sabath, 2004).
SIDE EFFECTS
Although antibiotics are generally considered safe and well tolerated, they have been associated with a wide range of adverse effects (Slama et al., 2005). Side effects are many, varied and can be very serious depending on the antibiotics used and the microbial organisms targeted. The safety profiles of newer medications may not be as well established as those that have been in use for many years (Slama et al., 2005).
Adverse effects can range from fever and nausea to major allergic reactions including photodermatitis. One of the more common side effects is diarrhea, sometimes caused by the anaerobic bacterium, Clostridium difficille, which results from one antibiotic disrupting the normal balance of the intestinal flora. Such overgrowth of pathogenic bacteria may be alleviated by ingesting probiotics during a course of antibiotics. An antibiotic-induced disruption of the population of the bacteria normally present as constituents of the normal vaginal flora may also occur, and may lead to overgrowth of yeast species of the genus Candida in the vulvo-vaginal area (Pirotta and Garland, 2006). Other side effects can result from interaction with other drugs such as elevated risk of tendon damage from administration of a quinolone antibiotic with a systematic corticosteroid.
ANTIBIOTIC RESISTANCE
Emerging antibiotic resistance has created a major public health dilemma, compounded by a dearth of new antibiotic options. Increasing rates of bacterial resistance among common pathogens are threatening the effectiveness of even the most potent antibiotics. The introduction of new antibiotics has not kept pace with the increasing rate of resistance, leaving clinicians with fewer treatment options.
The emergence of antibiotic resistance is an evolutionary process that is based on selection for organisms that have enhanced ability to survive doses of antibiotics that would have previously been lethal (Cowen, 2008). Antibiotics like penicillin and erythromycin which used to be one-time miracle cures are now less effective because bacteria have become more resistant. Antibiotics themselves act as a selective pressure which allows the growth of resistant bacteria within a population and inhibits susceptible bacteria (Levy, 1994). Antibiotic selection of pre-existing antibiotic resistant mutants within bacterial populations was demonstrated in 1943 by the LuriaDelbruck experiment (Luria and Delbruck, 1943). Survival of bacteria often results from an inheritable resistance (Witte, 2004). Any antibiotic resistance may impose a biological cost and the spread of antibiotic resistant bacteria may be hampered by the reduced fitness associated with the resistance, which proves disadvantageous for survival of the bacteria when antibiotic is not present. Additional mutations, however, may compensate for the fitness cost and aids the survival of these bacteria (Anderson,
2006).
The underlying molecular mechanisms leading to antibiotic resistance can vary.
Intrinsic resistance may naturally occur as a result of the bacteria’s genetic makeup (Alekshun and Levy, 2007). The bacterial chromosome may fail to encode a protein which the antibiotic targets. Acquired resistance results to a mutation in the bacterial chromosome or the acquisition of extra-chromosomal DNA (Alekshun and Levy,
2007).
Antibiotic–producing bacteria have evolved resistance mechanisms which have been shown to be similar to and may have been transferred to antibiotic resistant strains (Marshall et al., 1998; Nikaido, 2009). The spread of antibiotic resistance mechanisms occur through vertical transmission of inherited mutations from previous generations and genetic recombination of DNA by horizontal genetic exchange (Witte, 2004). Antibiotic resistance exchanged between different bacteria by plasmids that carry genes which encode antibiotic resistance, may result in coresistance to multiple antibiotics (Witte, 2004; Baker-Austin et al., 2006). These plasmids can carry different genes with diverse resistance mechanisms to unrelated antibiotics but because they are located on the same plasmid, multiple antibiotic resistance to more than one antibiotic is transferred. (Baker-Austin et al., 2006). Alternatively, cross-resistance to other antibiotics within the bacteria results when the same resistance mechanism responsible for resistance to more than one antibiotic is selected for (Baker-Austin et al., 2006).
CAUSES AND RISK FACTORS
The first rule of antibiotics is try not to use them, and the second rule is try not to use too many of them (Marino, 2007). Inappropriate antibiotic treatment and overuse of antibiotics have been a contributing factor to the emergence of resistant bacteria. The problem is further exacerbated by self-prescribing of antibiotics by individuals without the guidelines of a qualified clinician and the non-therapeutic use of antibiotics as growth promoters in agriculture (Larson, 2007).
Antibiotics are frequently prescribed for indications in which their use is not warranted, an incorrect or sub-optimal antibiotic is prescribed or in some cases for infections likely to resolve without treatment (Slama et al., 2005).
Teaching hospitals and centers that treat critically ill patients are particularly vulnerable to high rates of bacterial resistance. Risk factors associated with increased resistance among patients in the intensive care unit (ICU) include long hospital stay, advanced age, use of invasive devices, immuno suppression, lack of hospital personnel, adherence to infection-control principles and previous antibiotic use (Fish and Ohlinger, 2006). Repeated courses of antimicrobial therapy are common in acutely ill, febrile patients, who frequently have endotracheal tubes, urinary catheters and central venous catheters (Fish and Ohlinger, 2006). In combination with host factors, indwelling devices are routes for transmission and colonization of resistant infections (Davis, 2006). However, two principal drivers of resistance appear to be inadequate: empirical antibiotic therapy and prolonged antibiotic use (Fish and
Ohlinger, 2006).
Lengthy and inappropriate antimicrobial therapy allows microbes to mutate into new forms that help them survive antibiotics and quickly become new, dominant strains (Kallel et al., 2006). In prolonged courses, even effective antibiotics may permit the development of much drug resistance pathogens. In one study, pediatric patients were treated for various respiratory tract infections with either a standard 10-day course of amoxicillin or high-dose, short-course amoxicillin therapy. At the end of 28 days, the high-dose, short-course therapy group had lower rates of penicillin-resistant
Streptococcus pneumoniae and lower risk of resistance to
trimethoprin/sulfamethoxazole (Schraq et al., 2001). The study demonstrated that: bacterial mutants become dominant if pathogens are exposed to an antimicrobial agent for a long period and resistance genes travel together, spreading via conjugation or bacteriophages. These newly emergent resistant strains prey on the weakest patients, leaving hospitals with more severely ill patients, higher health care costs and rising mortality rates (Kallel et al., 2006).
What can be done to slow the relentless progression of resistant pathogens? While the search for new antibiotic options continues, strategies can be employed to slow the development of resistance to the current armamentarium. For example, we must avoid under dosing, which is a common yet often unrecognized factor associated with treatment failure and bacterial resistance (Fish and Ohlinger, 2006). An understanding of pharmacokinetic and pharmacodynamic principles can optimize antibiotic use, such as by increasing the time above the minimum inhibitory concentration and by maximizing the peak level or area under the contraction curve
(Craig, 1998).
Resistant containment depends on very early empirical and aggressive treatment for potentially resistant pathogens, followed by de-escalation and narrowing of the antimicrobial spectrum after identifying the pathogen. Empirical diagnosis of infection seems unlikely. De-escalation is a crucial infection management technique and an effective strategy that balances the need to provide early adequate antibiotic therapy to high-risk patients and the objective of avoiding antibiotic overuse (Kollef,
2001).
Other strategies include avoiding prolonged antibiotic use, prescribing drugs that have more than one mechanism of action or target, combining agents (where appropriate) to improve killing and decreasing the duration of therapy (Peterson, 2005; Fish and Ohlinger, 2006). Patients with ventilator-associated pneumonia (VAP) who received antimicrobial treatment for 8 days had no greater mortality or recurrent infections than did those who received 15 days of antibiotics. They did, however, have more antibiotic free-days (Chastre et al., 2003). Finally, adherence to infection control principles by hospital personnel, which will often require further training and education, will create an improved best-practice environment for infection control.
Only a continued commitment to these challenges and vigilance with respect to the
use of antibiotics will allow advancement to the next era – one of renewed successes against infectious diseases.
In addition to the above mentioned strategies, the first generation of drugs that might treat infectious diseases without incurring the cost of resistance is being developed. For example, Balaban (1998) described an agent that inhibits density-dependent expression of virulence factors, while Maurelli (1998) expressed hope in the production of enterotoxin inhibitors. Vaccines against virulence determinants are becoming serious possibilities (Li, 1997). These drugs differ from their predecessor by neutralizing or penalizing the pathogen rather than genetically killing the microbe.
New technologies are promising to identify new generation drug targets and more rapidly assess the efficacy of new–generation drugs. High-throughput geneexpression analysis (Lennon, 2000), such as DNA micro arrays, would rapidly diagnose the effects of drugs that do not kill microorganisms but that alter the expression of virulence genes. Furthermore, genomic and protein function analysis are producing targets to inhibit and to manipulate pathogens (Schmid, 1998; Loferer, 2000).
Several correlated reductions in resistance rates and drug use (Seppala, 1997) can be seen as anecdotal evidence that resistance will reduce with the ‘wise’ application of old drug, or the introduction of new drugs that act in a similar manner to current drugs
(Heinemann, 1999).
Phage therapy: This is an approach that has been extensively researched and utilized as a therapeutic agent for over 60 years, especially in the Soviet Union. It is an alternative that might help with the problem of resistance. Phage therapy was widely used in the United States until the discovery of antibiotics, in the early 1940’s. Bacteriophages or phages’ are viruses that invade bacterial cells and in the case of lytic phages, disrupt bacterial metabolism and cause the bacterium to lyse. Phage therapy is the therapeutic use of lytic bacteriophages to treat pathogenic bacterial infections (Chanishvili et al., 2001; Mulczyk and Gorski, 2003; Jikia et al., 2005).