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CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
1.0 Introduction
Pseudomonas aeruginosa is an important opportunistic pathogen of humans. It is a Gram-negative, aerobic rod, belonging to the family Pseudomonadaceae.
Members of this family are widespread in nature, inhabiting soil, sewage, water and surfaces of plants and animals including humans. P. aeruginosa is found in faeces, skin especially hands, external ear, axilla and perineum and occasionally in other sites in healthy persons (Forbes et al., 2007; Cheesbrough, 1994; Rosebury, 1962).
Pseudomonas aeruginosa has become an important cause of infections, especially in persons with compromised host defence mechanisms such as loss of the integrity of skin and mucous membrane or underlying immune deficiency. It causes urinary tract infections (UTI), respiratory tract infections, dermatitis, skin and soft tissue infections, eye and ear infections, bacteremia and a variety of systemic infections, particularly in patients with severe burns and in cancer and AIDS patients. P. aeruginosa, together with Escherichia coli and Staphylococcus aureus are nosocomial
(hospital-acquired) pathogens responsible for serious infections particularly in immunocompromised patients. In U.S.A, P. aeruginosa ranks second among the causes of nosocomial pneumonia, third among the causes of UTI and is the fourth most common cause of surgical site infections (Insler and Gore, 1986; Qarah, et al.,
2003; Davies et al., 1983). Out of 6704 strains isolated from hospitalized burns patients, the overall frequency of P. aeruginosa was 73.1% and of S. aureus 10.3%,
while 16.6% consisted of other organisms (Rastegar Lari et al., 2005). High mortality rates have been associated with P. aeruginosa infections, particularly when it involves immunocompromised patients (Boffi et al., 2000). Virulent strains of E. coli can cause gastroenteritis, urinary tract infections, and neonatal meningitis. In rarer cases, virulent strains are also responsible for haemolytic-uremic syndrome (HUS), peritonitis, mastitis, septicemia and pneumonia (Yagi, et al., 1997). S. aureus causes various infections which include wound infections, bacteremia, endocarditis, toxic shock syndrome and food poisoning. S. aureus infections generally involve intensive suppuration and destruction (necrosis) of tissue. Salmonella typhi and paratyphi cause typhoid fever (enteric fever), a life-threatening infection characterized by prolonged fever and multisystem involvement, including lymph nodes, liver and spleen (Forbes et al., 2007). Strains of these pathogens have increasingly shown resistance to the mainstream antibiotics used in hospitals. The circulation of such resistant and virulent pathogens in a hospital setting poses a potential danger which cannot be overlooked, moreso as Pseudomonas infection can be complicated and life-threatening. P. aeruginosa is responsible for acute fulminant infections such as pneumonia, sepsis, burn wound infections and meningitis which are associated with extremely high mortality. The nosocomial infection of Pseudomonas is escalated by:
• Its resistance to many chemical disinfectants in which it has been found to
grow in some cases. As a result, it can thrive in certain hospital environments
such as respirators, sinks and toilets. Many nosocomial infections have been
traced to soaps, disinfectants and other aqueous solutions that have become
contaminated with the organism (Arino, et al., 1998).
• modern medical practices including the extensive use of antimicrobial drugs
and invasive devices such as mechanical ventilators and catheters.
• Its ability to grow in moist, nutrient poor environments such as the water in
the humidifier of a mechanical ventilator (Bruun, et al., 1976).
• Its resistance to many antibiotics (Nester, et al., 2001).
P. aeruginosa shows a particular propensity for the development of resistance and is therefore a particularly dangerous and dreaded pathogen. The emergence of resistance in P. aeruginosa limits future therapeutic choices and is associated with increased rates of mortality and morbidity and higher costs. There are a limited number of antimicrobial agents with reliable activity against P. aeruginosa, including antipseudomonal penicillins and cephalosporins, carbapenems, and fluoroquinolones, particularly ciprofloxacin. For each of these agents, emergence of resistance during therapy has been described and has been recognized as a cause of treatment failure
(Cometta, et al., 1994; Fink et al., 1994; Milatovic and Braveny, 1987; CMPT, 1997;
Ozumba, 2003). The excessive morbidity and mortality associated with ineffective empirical therapy in P. aeruginosa infections has been reported (Boffi et al., 2000).
Pseudomonas aeruginosa is naturally resistant to many antibiotics due to the permeability barrier afforded by its outer membrane lipopolysaccharide (LPS). Also, its tendency to colonize surfaces in a biofilm form makes the cells impervious to therapeutic concentrations of antibiotics. Because its natural habitat is the soil, living in association with bacilli, actinomycetes and molds, it has developed resistance to a variety of their naturally occurring antibiotics (Wood, et al., 1998; Qarah et al., 2003). In addition, Pseudomonas maintains antibiotic resistance plasmids, both resistant factors (R factors) and resistant transfer factors (RTFs), and is able to transfer these genes by means of the bacterial processes of transduction and conjugation (Forbes, et al., 2007). Scientists have characterized several fundamentally different mechanisms of antibiotic resistance, and
hundreds of variants. Bacteria also share resistance-encoding genes in a number of ways. Bacteria can share genes without mating and exchanging DNA one-to-one.
They can even adopt loose genes from other species, or become infected by viruses that can move genes from one bacterium to others. Bacteria can also acquire multiple different genes for resistance, making them resistant to multiple families of antibiotic drugs. Such multiple drug resistant (MDR) strains present the greatest clinical challenge and are becoming more common (http://www.accelr8.com/index.php).
Resistance rates can evolve rapidly in P. aeruginos (Fridkin and Gaynes, 1999;
Ramirez-Ronda et al., 1999). P. aeruginosa isolates from leg ulcers showed a marked increase in resistance to ciprofloxacin from 9% in 1992 to 24% in 1996, while oxacillin resistance in S. aureus increased from 24% to 50% (Colsky et al., 1998). It was found that emergence of resistance to at least one antibiotic occurred in 10.2% of the patients during antipseudomonal therapy. Resistance emerged during treatment with each class of antibiotic (Carmeli, et al., 1999). By 1993, multidrug-resistant Sal. typhi were isolated in Viet Nam. They were resistant to the usual firstline antibiotics: chloramphenicol, ampicillin and co-trimoxazole but fully susceptible to fluoroquinolones and third generation cephalosporins. Thus fluoroquinolones became the drug of choice for multi-drug-resistant typhoid fever (Wain, et al., 1997).
The spreading of resistant bacteria, especially in hospitals, raises concern for the effectiveness of antimicrobial therapy, and calls for renewed interest and strategies on treatment and prevention. Proposed solutions are outlined by the Centres for
Disease Control (CDC) as a multipronged approach that includes: prevention (such as vaccination); improved monitoring; and the development of new treatments. It is this last solution that would encompass the development of new antimicrobials from plants (Fauci, 1998).
2
Historically, plants have provided a source of inspiration for novel drug compounds, and plant-derived medicines have made large contributions to human health and well-being. Their role is two-fold in the development of new drugs: they may become the base for the development of conventional drugs, a natural blueprint for the development of new drugs or a phytomedicine to be used for the treatment of disease. It is estimated today that plant materials are present in, or have provided the models for about 50% of conventional drugs. Currently, 121 prescription drugs sold world-wide come from plant-derived sources (Robbers et al., 1996; Taylor, 2005).
Globally, there is a renewed interest in drugs derived from natural sources. This is as a result of the general belief that they are superior, have minimal adverse side effects and are less expensive. Plant-derived medicines are relatively safer than their synthetic alternatives, and offer profound therapeutic benefits and more affordable treatment. They are commonly found in the localities where they grow and therefore easily available. Moreover, scientists have realized that using medicinal plants identified by native herbalists makes research more efficient and less expensive. A large number of medicinal plants are used in Nigeria to treat illnesses in form of decoctions, tinctures and poultice (Gills, 1992; Iwu, 1993). Formerly, traditional medicine was viewed with a great deal of criticism, especially by practitioners of orthodox medicine. The claims of native doctors that a single plant could cure a wide range of diseases fed the skepticism of ‘doubting Thomases’. The lack of standardization, and the vital link between traditional medicine and spiritism further discouraged many people of higher socio-economic status from seeking herbal healing. The herbal healers were patronized mostly by the rural dwellers, the poor, and people with sicknesses for which no standard drug could be effective. However, with the emergence antibiotic resistance, medical doctors now work in conjunction
6 with herbalists, sending patients to them for herbal treatment when antibiotic therapy has failed. This is an indication that plant-based antimicrobials may provide a solution to the problem of drug resistance. In Igbo ethnomedicine, local plants are used to treat infectious diseases. Bryophyllum pinnatum leaf juice, Picralima nitida stem bark, ocimum gratissimum (nchu anwu) extracts, Ageratum conyzoides and Cassia alata extracts and a host of other medicinal plants have been used successfully to treat bacterial, viral and fungal infections (Obaseiki-Ebor, 1985; Fakeye et al., 2002;
Ofokansi et al., 2003; Durodola, 1977). These phytomedicines are usually dispensed by native doctors referred to in Igbo language as ‘dibias’. Africa has a large forest reserve, most of which have not been analyzed for their phytochemical composition and pharmacological properties. Moreover, the native doctors engaged in herbal medicine are very conservative and most of them are dying without divulging their wealth of knowledge. There is need for research workers to partner with them in a bid to discover more plants with antimicrobial activity. Plant chemicals may well help us in our ongoing struggle with constantly evolving pathogens, including bacteria, viruses and fungi, which are mutating against our mainstream drugs and becoming resistant to them (Taylor, 2005).
Pseudomonas aeruginosa causes a number of human diseases which include:-
Endocarditis: P. aeruginosa infects heart valves of those who abuse intravenous (IV) drugs and prosthetic heart valves. Left-sided endocarditis typically presents with symptoms of congestive heart failure (Qarah et al, 2003).
Skin and soft tissue Infections: Pseudomonas aeruginosa causes surgical wound sepsis and is often a cause of concern in the surgical ward. The death rate in post operative wound sepsis has increased with the emergence of multiple-drug-resistant strains which defy antibiotic therapy. Infection of traumatic wounds with P.
= aeruginosa has sometimes led to amputation of the leg due to gas gangrene (Edmonds et al., 1972; Kalayi, 2000).
Eye Infections: P. aeruginosa is one of the common causes of bacterial keratitis and neonatal ophthalmia. Eye infection can be rapidly destructive and lead to loss of the entire eye ((McLaughln-Borlace et al., 1998; Hazlett et al., 1981).
Ear Infections: P. aeruginosa is the predominant bacterial pathogen in some cases of otitis externa (Swimmer’s ear) and chronic otitis media. Patients present with pain, pruritis, and ear discharge (Van Asperon et al., 1995).
Central Nervous System (CNS) Infections: P. aeruginosa can cause meningitis and brain abscess. The organism invades the CNS from the inner ear or paranasal sinus, or is inoculated directly by means of head trauma, surgery or invasive diagnostic procedures (Qarah et al, 2003).
Urinary Tract Infections (UTI): P. aeruginosa infections of the urinary tract usually are hospital-acquired as a secondary condition and related to catheterization, instrumentation and surgery. P. aeruginosa invade the bloodstream from the urinary tract, accounting for 40% of Pseudomonas bacteremias (Olayinka et al, 2004).
Gastrointestinal Tract (GIT) Infections: P. aeruginosa can cause GIT infection in very young children and adults with hematologic malignancies and chemotherapy- induced neutropenia. The spectrum of disease can range from perirectal infections to severe necrotizing enterocolitis, with significant morbidity and mortality (Qarah et al,
2003).
Respiratory Tract Infections: P. aeruginosa causes primary pneumonia in patients with immunosuppression (such as AIDS and cancer patients) and patients with chronic lung disease. In cystic fibrosis (CF), chronic infection of the lower respiratory tract leads to pulmonary failure in most CF patients resulting in the high rate of
? morbidity and mortality associated with the disease (Schwarzman and Boring III,
1971; Simpson et al., 1988).
Bacteremia: P. aeruginosa causes bacteremia in patients with hematological malignancies, immunodeficiency relating to AIDS, neutropenia, diabetes mellitus and severe burns. The mortality rate of Pseudomonas bacteremia remains greater than
10% (Horan et al., 1988).
The large number of diseases caused by P. aeruginosa especially in the hospital setting is a cause for serious concern. Its resistance to antimicrobial agents is an increasing clinical problem and a recognized public health threat. The need to search for alternative means of therapy for P. aeruginosa-related diseases is the major aim of the present research.
+
1.1 AIMS AND OBJECTIVES
The aim of the study was to evaluate the in vitro activities of some medicinal plant extracts on antibiotic-resistant Pseudomonas aeruginosa and some other pathogens and investigate the toxicity of active constituents on mammalian cells with a view of recommending any effective extract for use in the development of alternative drugs in cases of resistant infections. The study has the following objectives:
1. Identifying resistant P. aeruginosa isolates from clinical specimens.
2. Exposing the P. aeruginosa isolates to different medicinal plants to find out
effective ones.
3. Testing the toxicity of the effective medicinal plants.
4. Analyzing the phytochemical composition of the effective medicinal plants.
1.2.0 LITERATURE REVIEW
1.2.1 General Description of Pseudomonas
1.2.1.1 The Genus Pseudomonas
The genus Pseudomonas comprises a relatively large and important group of
Gram-negative bacteria, belonging to the family Pseudomonadaceae. The family includes Xanthomonas which together with Pseudomonas comprise the informal group of bacteria known as Pseudomonads. Members of the genus Pseudomonas are found abundantly as free-living organisms in soils, fresh water and marine environments and in many other natural habitats. They may also be found in association with plants and animals as normal flora or as agents of disease (Starr et al., 1981; Van Asperon et al., 1995; Insler and Gore 1986; Cowan and Steel 1993).
Morphologically, Pseudomonas may be described as Gram-negative, straight or slightly curved rods, 0.5 – 0.8µm x 1 – 3µm. They are typically motile by means of one or more polar flagella. They are catalase-positive, indole-negative, and usually oxidase-positive. No organic growth factors are required. They may produce a diffusible or insoluble pigment which may be used in diagnosis (Holt et al., 1994;
Singleton, 1999; Cheesbrough 1994).
1.2.1.2 Cultural Characteristics of Pseudomonas aeruginosa
On solid media, Pseudomonas aeruginosa may produce three colony types: the small, rough colony produced by natural isolates from soil or water, the large, smooth colony with a fried egg appearance, elevated, with flat edges and the mucoid
colonies yielded by isolates from respiratory and urinary tract secretions. P. aeruginosa grows well on Nutrient agar, MacConkey agar, and other media containing bile salts. In the laboratory, colonies of P. aeruginosa are identified using several cultural characteristics and biochemical tests: On Blood agar, they produce beta-haemolytic colonies that have a typical flat “fringy” morphology. On
MacConkey agar, P. aeruginosa forms non-lactose-fermenting colonies. On Nutrient agar, most P. aeruginosa strains produce a soluble (diffusible), blue-green pigment, pyocyanin, which is a diagnostic feature. Apyocyanogenic strains are identified on the basis of motility by polar monotrichous flagella, growth at 42oC, oxidative utilization of glucose and acid production from glucose but not lactose or sucrose. They also give a positive oxidase reaction and produce a fruity distinctive grape-like smell due to 2-aminoacetophenone production (Pollack, 2000; Starr et al., 1981; Cheesbrough,
1994; Cowan and Steel, 1993).
1.2.1.3 Nutrition and Growth of Pseudomonas
Pseudomonas species have simple minimal nutritional requirements and can grow in media without organic growth factors (Holt et al., 1994). Strains of
Pseudomonas can be extremely nutritionally versatile. They can grow well in mineral salts media supplemented with any of a large number of carbon sources. The optimum temperature for the growth of P. aeruginosa is 37oC and it is able to grow at temperatures as high as 42oC (Cheesbrough, 1994). However it fails to grow at 4oC–
5oC and this can separate P. aeruginosa from P. fluorescens which does not usually grow at 37oC but can grow at 5oC (Cowan and Steel, 1993). Pseudomonas strains grow in media with neutral pH and most strains fail to grow under acidic conditions of pH 4.5 (Holt et al., 1994).
1.2.1.4 Pigments of Pseudomonas
P. aeruginosa produces two types of soluble pigments, pyocyanin and pyoverdin. Pyocyanin is a non-fluorescent blue-green pigment that is diagnostic for only this species. Pyocyanin refers to “blue pus” which is characteristic of suppurative infections caused by P. aeruginosa. However, about 4% of P. aeruginosa strains do not produce pyocyanin (Cheesbrough, 1994). Pyoverdin is a fluorescent yellow-green pigment produced by P. aeruginosa, P. fluorescens and P. putida. The colonies fluoresce green in ultra violet (UV) light. Pyoverdin is produced under conditions of low iron availability. It is known to play a role as siderophore in the iron intake systems of the bacteria and hence its production is enhanced under conditions of iron deficiency. Production of the pigments is readily demonstrated by culturing the bacteria in King’s Medium B, which contains no added iron (Appendix 1). P. mendocina is related to P. stutzeri. The colonies are flat and smooth and have a yellowish colour due to the presence of carotenoid pigments (Starr et al., 1981;
Cheesbrough, 1994).
1.2.1.5 Genome Structure of Pseudomonas
The genome structure of P. aeruginosa, P. putida and Pseudomonas syringae has been sequenced. Pseudomonas syringae pathovar tomato DC 3000, the tomato pathogen, has genome 6.5 megabases in size that is comprised of a circular chromosome and two plasmids. Two hundred and ninety eight (298) virulence genes were found including those that encode for 31 confirmed and 19 predicted proteins dealing with secretion systems. Over 12% of the genes were found to deal with
regulation. This may be due to the bacterium’s need for rapid adaptation to the diverse environments encountered during epiphytic growth and pathogenesis. Many similarities were found among the genomes of P. syringae pathovar tomato DC 3000,
P. putida and P. aeruginosa but 1,159 genes were found to be unique to DC3000
(Buell et al., 2003).
Plasmids: Thirteen (13) compatibility groups of plasmids have been identified in
Pseudomonas. Group P–1 contains “wide host-range plasmids” which are capable of transfer to strains of practically any gram-negative species. Group P-2 contains over half the transmissible plasmids identified in the species. P–2 plasmids carry a variety of markers determining resistance to various antibiotics and simple chemicals e.g mercury and borate. In addition, they confer resistance to tellurite and tellurate, which makes these agents useful for selective isolation. Group P–9 contains typically catabolic plasmids and they contribute significantly to the nutritional diversity of some Pseudomonas species. In addition, some plasmids of Pseudomonas determine resistance to physical agents, bacteriophages and bacteriocins.
1.2.1.6 Ecology of Pseudomonas
Pseudomonads are ubiquitous organisms with world-wide distribution and found in a variety of habitats. They predominate in soils and water under aerobic, mesophilic and neutral conditions. Pseudomonas occur regularly on the surfaces of plants and animals. P. aeruginosa is sometimes present as part of the normal flora of humans but the prevalence of colonization of healthy individuals outside the hospital is relatively low ranging from 0 to 24 percent depending on the anatomical locale
(Kominos et al., 1972; Rosebury, 1962; Qarah et al., 2003). The ubiquity of the
Pseudomonads seems to be a consequence of their minimal nutritional requirements,
the range of carbon compounds they can utilize, and the diversity of their metabolism, which includes autotrophy, lithotrophy and anaerobic mode of respiration. The nutritional versatility of Pseudomonas makes them important in the balance of nature and economy of humans. They occupy a prominent position in nature for their active participation in the carbon cycle. Pseudomonas species possess unique catabolic pathways and their ability to degrade many natural and artificially synthesized compounds offers a solution to the problems of environmental pollution (Sagardia et al., 1975; Arino et al., 1998; Lessie and Phibbs, 1984).
Pseudomonads flourish in the soil under conditions which favour the growth of aerobic Actinomycetes of the genus Streptomyces and other species of bacteria.
The latter specialize in aerobic decomposition of organic compounds and could provide the Pseudomonads with monomeric carbon sources which they require. The notorious resistance of Pseudomonas to Streptomycete antibiotics may be a consequence of their natural association in the same ecological niches. In nature,
Pseudomonas species exist both as saprophytes and as parasites, depending on whether they are growing on dead or living organic matter. Pseudomonads that are phytopathogenic are specific for their host plants. The young pathological lesions form special ecological niches in which the pseudomonads exist as homogeneous populations. This makes their isolation in pure culture a relatively straight-forward process. The animal pathogens are far less host specific than the phytopathogens.
They can be found in both human and animal hosts. Most of them are considered to be opportunistic pathogens. The ability of P. aeruginosa to thrive in moist, nutrient poor environments has led to its continued existence in hospital settings and its success as a nosocomial pathogen (Bruun et al., 1976).
2
1.2.1.7 Pseudomonas Species of Medical Importance
The Pseudomonads are better known as pathogens of plants rather than animals. However some species are known to be pathogenic for humans and animals:
P. aeruginosa is historically the most significant of these pathogenic species and is considered the epitome of opportunistic pathogens (Wilson and Ahean 1977; Wilson et al., 1981). P. aeruginosa is a leading cause of nosocomial infections. According to the US Centers for Disease Control and Prevention (CDC), the overall incidence of P. aeruginosa infections in US hospitals averages about 0.4 percent and the bacterium is the fourth most commonly isolated nosocomial pathogen, accounting for 10.1 percent of all hospital-acquired infections (Qarah et al., 2003; Nester, et al., 2001). It can be acquired nosocomially in the intensive care unit (ICU) setting and is associated with the use of invasive devices, instrumentation and surgery (Brunn et al., 1976). P. aeruginosa infections can involve any part of the body. Isolates have been recovered from CF sputum, acute pneumonia sputum, blood, urine, skin wounds and burns wounds (Qarah et al., 2003). When conditions are favourable, P. aeruginosa can infect wounds, burnt areas, urinary and respiratory tracts and blood and may commonly be isolated from such sites (Olayinka et al., 2004; Schwarzmann and
Boring III, 1971; Woods et al., 1986). They may also be involved in pneumonia, endocarditis, meningitis and other pathological conditions in humans. In animals, P. aeruginosa is associated with pneumonia, enteritis, vaginitis, mastitis and endometritis
P. fluorescens and P. putida
6
These fluorescent species were described a few years after the description of
P. aeruginosa. It is becoming evident that P. fluorescens and P. putida may also be serious opportunistic pathogens. Clinical sources from which strains of these species have been isolated include respiratory tract specimens, pleural fluid, urine, cerebrospinal fluid, faeces and blood. However, the clinical importance of these two species is still being debated. This is mainly due to their inability to grow at body temperature and indeed they are rarely pathogenic to humans. Bacteria in the P. fluorescens and P. putida complex have been isolated from lizards, insects and mammals (Qarah et al., 2003).
Non-fluorescent Pseudomonas Pathogens
The non-fluorescent Pseudomonas species which have been encountered in clinical laboratories include P. stutzeri, P. mendocina and P. alcaligenes.
P. stutzeri is a common soil inhabitant, also found in water and plant materials. It has been isolated from clinical materials and can grow at body temperature. However, its pathogenic status has not been clearly demonstrated. P. mendocina has rarely been isolated from materials of clinical origin and has not been incriminated as a cause of infection in humans. However, strains of P. mendocina have been found to produce alginate, a property of P. aeruginosa strains isolated from cystic fibrosis (CF) patients, which protects them from phagocytosis by alveolar macrophages. P. alcaligenes has occasionally been associated with infection in humans and is considered as a rare opportunistic pathogen (Schwarzmann and Boring III, 1971).
1.2.2 Pathogenesis of P. aeruginosa Infections
1.2.2.1 Sources of Infection
=
The exact source and mode of transmission of P. aeruginosa are often unclear because of the ubiquitous presence of the pathogen in the environment. Infection may arise from any of a great number of sources:
• Hospital environment: In the hospital, P. aeruginosa is found in sinks, toilets,
washbasins and drains, water fountains and ice machines (Edmonds et al.,
1972; Bruun et al., 1976).
• Invasive medical devices: Nosocomial infections most often result from
medical devices that breach the first-line barriers of the normal host defence.
These include urinary catheters, indwelling catheters used to deliver
intravenous fluids, tracheostomes and mechanical ventilators (Crouch et al,
1996).
• Health care workers: Health personnel may be involved in infection. A
member of a surgical team who is a carrier can inoculate the pathogen directly
into a surgical site.
• Autoinfection: Patients’ own normal flora can be a potential source of
infective agents. P. aeruginosa is frequently isolated from noses, throats,
sputum, surgical wounds, urine and stool of patients. Nearly all invasive
procedures can transmit normal flora to otherwise sterile body sites.
Autoinfection by strains already acquired on carrier sites is significant (Bruun
et al., 1976).
• Soaps, disinfectants and aqueous solutions: Many nosocomial infections have
been traced to soaps and solutions of disinfectants and antiseptics that have
become contaminated with P. aeruginosa. Water, saline solutions and
dextrose solutions used in infant feeding are sometimes contaminated
(Thomas et al., 1975).
?
• Pharmaceuticals: Pseudomonas have been found in pharmaceutical
preparations such as eye drops, cosmetics and preparations containing
medicinal plant materials.
• Foods have been implicated as sources of infection in hospitals.
Pseudomonas species have been isolated from many natural and
manufactured foods e.g. vegetables used in hospital kitchen. Pyocin types of
P. aeruginosa isolated from clinical specimens were frequently identical with
those recovered from the vegetables, thus implicating vegetables as an
important source and vehicle by which the organism colonizes the intestinal
tract (Kominos et al., 1972).
• Visitors to patients can transport the bacteria as contaminants in foods,
flowers and presents.
• Cross infections are attributed to several factors such as concentration of
debilitated patients, high frequency of tracheostomied and catheterized
patients and use of different kinds of special treatments and equipment
(Bruun, et al, 1976).
P. aeruginosa can remain viable for long periods of time in many different habitats.
This makes it difficult to control the organism in a hospital setting and prevention of contamination is practically impossible.
1.2.2.2 Virulence Factors of P. aeruginosa
Pseudomonas aeruginosa elaborates several extra-cellular products which are reported to contribute to its virulence. They include the following:
• Two protein toxins, exoenzyme S and exotoxin A, which are adenosine
diphosphate ribosyltransferases (Sokol et al., 1981; Morihara et al., 1981).
+
• Lipopolysaccharide (an endotoxin).
• Several proteases including elastase and alkaline protease (Mull and Callahan,
1963; Cryz and Iglewski, 1980).
• Two soluble protein hemolysins, phospholipase C, and a lecithinase (Berka et
al., 1981; Berk, 1963).
• Cytotoxin, a soluble protein, (mw 25,000).
• Alginate, produced by mucoid strains of P. aeruginosa. Alginate is a viscous,
high molecular weight exopolysaccharide, a linear non-repeating polymer of
D-mannuronic acid and L-guluronic acid (Evans and Linker, 1973).
• Pigments of Pseudomonas are probably virulence factors.
Studies have shown that there is no significant difference between strains isolated from environmental sources and those from bacteremic patients in the production of these virulence factors. This suggests that pathogenic potential may be a general property of P. aeruginosa from all sources (Nicas and Iglewski, 1986).
1.2.2.3 Mechanism of Infection
Pseudomonas aeruginosa is the epitome of an opportunistic pathogen. It rarely infects uncompromised tissues. In most cases of infection, there is usually loss of integrity of skin or mucous membrane or underlying immune deficiency (Qarah et al.,
2003).`Since the tissue invasiveness of the organism is limited, P. aeruginosa uses accidental portals of entry such as burns, wounds, catheterization and surgical procedures to gain access to its host (Pollack, 2000). The pathogenesis of
Pseudomonas infections is multifactorial, due to the large number of virulence determinants possessed by the bacterium. Most of the infections are both invasive and toxinogenic. The disease process begins with the alteration or circumvention of
normal host defences. The ultimate Pseudomonas infection may be seen as composed of three distinct stages:
1. Bacterial attachment and colonization
2. Local invasion
3. Disseminated systemic disease
Each stage is mediated by particular virulence factors which are responsible for the characteristic syndromes that accompany the disease.
Attachment and Colonization
Pseudomonas cells attach to the host’s surfaces by means of one or more of several adhesins:
• Fimbriae (N-methyl-phenylalanine pili) adhere to epithelial cells of the upper
respiratory tract. These adhesins appear to bind to specific galactose, mannose
or sialic acid (N-acetylneuraminic acid) receptors on tracheal epithelial cells.
This colonization may be aided by production of a protease enzyme (elastase)
that degrades fibronectin in order to expose the underlying fimbrial receptors
on the epithelial cell surface. Tissue injury may play a role in colonization of
the respiratory tract. This has been called opportunistic adherence and may be
an important step in infections of the respiratory tract as well as Pseudomonas
keratitis and urinary tract infections (UTI).
• Alginate produced by mucoid strains, forms an additional or alternative
adhesin which attaches to the tracheobronchial mucin (N-acetylglucosamine).
• Surface-bound exoenzyme S, which could likely serve as an adhesin for
glycolipids on respiratory surfaces (Sokol et al., 1981).
Alginate slime further forms the matrix of the Pseudomonas biofilm which anchors the cells to their environment and in medical situations, protects the bacteria from host
defences such as lymphocytes, phagocytes, ciliary action of the respiratory tract, antibodies and complement. It also makes them less susceptible to antibiotics
(Mclaughln-Borlace et al., 1998; Schwarzmann and Boring 111, 1971).
Local Invasion
Pseudomonas aeruginosa invades tissues by its ability to resist phagocytosis and the host immune defences. The bacterial capsule (glycocalyx) effectively protects the cells from opsonization by antibodies, complement deposition and phagocytic engulfment. P. aeruginosa produces two proteases which break down physical barriers and contribute to bacterial invasion:
• Elastase cleaves collagen, IgG, IgA and complement. It disrupts the
respiratory epithelium and interfers with ciliary function. It has been reported
to cause a necrotizing vasculitis in tissues of patients with infections of P.
aeruginosa (Mull and Callahan, 1963).
• Alkaline protease interfers with fibrin formation and lyses fibrin.
These two proteases together destroy the ground substance of the cornea and
other supporting structures composed of fibrin and elastin. They also cause the
inactivation of gamma interferon and Tumor Necrosis factor (TNF).
Three soluble proteins also play a role in invasiveness:
• Cytotoxin, a pore-forming protein, and
• Two hemolysins: Phospholipase C, and a lecithinase, which act synergistically
to break down lipids and lecithin.
These three proteins contribute to invasion through their cytotoxic effects on
eukaryotic cells.
• Pigments of Pseudomonas appear to play a role in invasiveness. The blue-
green pigment, pyocyanin, impairs the normal function of human nasal cilia,
and disrupts the respiratory epithelium (Berka et al., 1981; Berk, 1963; Evans
and Linker, 1973).
Disseminated Systemic Disease and Toxinogenesis
Blood stream invasion and dissemination of Pseudomonas from local sites of infection is probably mediated by the same cell-associated and extracellular products responsible for the localized disease. In addition, lipopolysaccharide (endotoxin) mediates the usual pathological aspects of Gram-negative septicemia e.g fever, hypotension, intravascular coagulation etc. The two extracellular protein toxins, exoenzyme S and exotoxin A, produced by P. aeruginosa also play a role (Pollack et al., 1977).
Exoenzyme S is produced by bacteria growing in burned tissue and may be demonstrated in the blood before the bacteria are detected. It may act to impair the function of phagocytic cells in the bloodstream and internal organs to prepare for invasion of P. aeruginosa (Sokol et al.,1981).
Exotoxin A is produced by most isolates of P. aeruginosa, under conditions of iron limitation that characterize many animal tissues. The target of this toxin is one of the elongation factors in translation during protein synthesis. Exotoxin A has necrotizing activity at the site of bacterial colonization and is thought to contribute to the colonization process. Toxinogenic strains cause a more virulent form of pneumonia than nontoxinogenic strains. Exotoxin A causes ocular damage and lens cataract of mice to eyes which previously had a non-penetrating wound on the corneal surface (Hazlett et al., 1981). Purified exotoxin A is highly lethal for animals including primates. Exotoxin A has adenosine-5-diphosphate ribosyl (ADPR) transferase activity similar to that shown in diphtheria toxin. This enables it to inhibit
eukaryotic protein synthesis (Morihara et al., 1981). Both exotoxin A and exoenzyme
S have ADPR transferase activity but exoenzyme S differs from exotoxin A in being heat-stable. The production of exotoxin A and exoenzyme S has been shown to increase the mortality rate of patients separately, and more if the infecting strain produces S and A together (Sokol et al, 1981).
1.2.3 Diseases Caused by P. aeruginosa
Pseudomonas aeruginosa causes a number of human diseases which include:
Endocarditis
P. aeruginosa infects heart valves of those who abuse intravenous (IV) drugs and prosthetic heart valves. Right-sided or left-sided valve infection may occur. Non- specific symptoms include fever and malaise while more specific symptoms depend on which cardiac valve is involved. Left-sided endocarditis typically presents with symptoms of congestive heart failure (Qarah et al, 2003).
Skin and soft tissue Infections
Pseudomonas aeruginosa causes surgical wound sepsis and is often a cause of concern in the surgical ward. The death rate in post operative wound sepsis has increased with the emergence of multiple-drug-resistant strains which defy antibiotic therapy. Infection of traumatic wounds with P. aeruginosa has sometimes led to amputation of the leg due to gas gangrene. Pseudomonas has emerged as an important source of burn wound sepsis (Edmonds et al., 1972; Kalayi, 2000). A burn wound is considered to have sepsis when there is bacterial proliferation of 100,000 organisms per gram of tissue, with involvement of subjacent unburned tissue. Pseudomonas does not grow on dry skin but flourishes on moist skin. It causes the green nail syndrome, a suppurative inflammation around a finger nail that can develop in individuals whose
hands are frequently submerged in water. Secondary wound infections occur in patients with decubiti (pressure sore), eczema, and tenia pedis. These infections may have characteristic blue-green exudates with a fruity odor. Pseudomonas is a common cause of hot tub or swimming pool folliculitis. Pseudomonas bacteremia produces distinctive skin lesions known as ecthyma gangrenosum. It is also implicated in unmanageable forms of acne vulgaris (Colsky et al., 1998).
Eye Infections
P. aeruginosa is one of the common causes of bacterial keratitis and scleral abscess (McLaughln-Borlace et al., 1998). It is also the etiologic agent of neonatal ophthalmia. If the defences of the ocular environment are compromised in any way the bacterium can proliferate rapidly and through the production of enzymes such as elastase, alkaline protease and exotoxin A, cause a rapidly destructive infection that can lead to loss of the entire eye (Hazlett et al., 1981). Other predisposing conditions for corneal involvement are exposure to intensive care unit (ICU) environment and
AIDS. Corneal lesions can progress to endophthalmitis and orbital cellulitis.
Symptoms are pain, redness, swelling and impaired vision. Investigators have demonstrated that pseudomonads are not constituents of the normal eye flora but rather are transient opportunists that often enter the outer eye from contaminated ocular products or environmental sources (Wilson and Ahearn, 1977; Wilson et al.,
1981; Insler and Gore, 1986). Eye infection usually follows a breach in the integrity of the corneal surface either by mechanical or chemical trauma and use of contact lenses (Galentine et al., 1984).
Ear Infections
P. aeruginosa often inhabits the external auditory canal in association with injury, maceration, inflammation or simply wet and humid conditions. It is the
2 predominant bacterial pathogen in some cases of otitis externa (Swimmer’s ear). It is also a common cause of chronic otitis media. Patients present with pain, pruritis, and ear discharge (Van Asperon et al., 1995).
Central Nervous System (CNS) Infections
P. aeruginosa can cause meningitis and brain abscess. The organism invades the CNS from the inner ear or paranasal sinus, or is inoculated directly by means of head trauma, surgery or invasive diagnostic procedures. In some cases, it may be due to hematogenous spread from infective endocarditis, pneumonia or UTI. Patients present with fever, headache and confusion (Qarah et al, 2003).
Bone and Joint Infections
The most common sites of infection are the vertebral column, pelvis and the sternoclavicular joint. P. aeruginosa has a particular tropism for fibro-cartilagenous joints of the axial skeleton. Vertebral osteomyelitis may involve the cervical spine and patients present with neck or back pain lasting weeks to months. P. aeruginosa is also implicated on osteochondritis after puncture wounds of the foot. Infection may be blood-borne, as in individuals who abuse intravenous drugs or inpatients with pelvic infections or urinary tract infections. Alternatively, the infection may be contiguous, related to penetrating trauma, surgery or overlying soft tissue infections (Qarah et al,
2003).
Urinary Tract Infections (UTIs)
P. aeruginosa infections of the urinary tract usually are hospital-acquired as a secondary condition and related to catheterization, instrumentation and surgery. As in the case of E. coli UTI, the infection can occur via an ascending or descending route or through bacteremia. Conversely, P. aeruginosa invade the bloodstream from the
6 urinary tract, accounting for 40% of Pseudomonas bacteremias. P. aeruginosa UTI presents with all the symptoms of other forms of UTI (Olayinka et al, 2004).
Gastrointestinal Tract (GIT) Infections
P. aeruginosa infection can affect any portion of the gastrointestinal tract
(GIT). It affects very young children and adults with hematologic malignancies and chemotherapy-induced neutropenia. The spectrum of disease can range from perirectal infections to severe necrotizing enterocolitis, with significant morbidity and mortality. Epidemics of pseudomonal diarrhoea can occur in nurseries. The infants may present with irritability, vomiting, diarrhoea and dehydration. Pseudomonas infection can cause enteritis, with patients presenting with prostration, headache, fever and diarrhoea (Shanghai fever). The GI tract is an important portal of entry in
Pseudomonas septicemia. Pseudomonas lymphitis occurs in patients with neutropenia resulting from acute leukemia (Qarah et al, 2003).
Respiratory Tract Infections
P. aeruginosa causes primary pneumonia in patients with immunosuppression and chronic lung disease. Bacteremic pneumonia is common in neutropenic cancer patients undergoing chemotherapy and in AIDS patients. Symptoms of pneumonia include fever, chills, severe dyspnea, cyanosis, productive cough, confusion and other signs of a systemic inflammatory response. In cystic fibrosis, chronic infection of the lower respiratory tract by mucoid strains is common and is difficult if not impossible to treat. This leads to pulmonary failure in most CF patients (Di Sant Agnese and
Davis, 1979). The persistence of mucoid P. aeruginosa strains in the lungs may be related to the strains increased resistance to phagocytosis compared with that of non-
= mucoid strains. The strains are difficult to eradicate in these infections and is responsible for much of the morbidity and mortality associated with the disease
(Schwarzman and Boring III, 1971; Simpson et al., 1988).
Bacteremia
P. aeruginosa causes bacteremia primarily in immunocompromised patients.
Predisposing conditions include hematological malignancies, immunodeficiency relating to AIDS, neutropenia, diabetes mellitus and severe burns. Bacteremia may be acquired via medical devices in hospitals and nursing homes. Pseudomonas accounts for 25% of all hospital acquired Gram-negative bacteremias and the mortality rate remains greater than 10% (Horan et al., 1988).
1.2.4 Susceptibility of P. aeruginosa to Antibiotic Therapy
All infections caused by Pseudomonas aeruginosa are treatable and potentially curable. Antibiotics form part of the therapy of P. aeruginosa infections.
However, there is a limited number of antimicrobial agents with reliable activity against P. aeruginosa. These include antipseudomonal penicillins and cephalosporins, carbapenems, and fluoroquinolones, particularly ciprofloxacin. Aminoglycosides are frequently used as part of combination regimens for treatment of serious
Pseudomonas infections but are generally not recommended as single drugs (Cometta, et al., 1994; Fink et al., 1994).
1.2.4.1 Effects of Antipseudomonal Penicillins
Carboxypenicillins
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These beta-lactams can destroy P. aeruginosa and indole-positive Proteus species. Examples of carboxypenicillins are carbenicillin and ticarcillin.
Carbenicillin was the first penicillin with activity against P. aeruginosa. It is given parenterally. Ticarcillin is four times more active against P. aeruginosa than is carbenicillin. It is given by intramuscular or slow intravenous (I.V) injection or rapid
I.V infusion. Both carbenicillin and ticarcillin are disodium salts and each 1g delivers about 5.4 millimoles (mmol) of sodium. This source of sodium should be borne in mind when treating patients with impaired cardiac or renal function.
Carboxypenicillins inactivate aminoglycosides and so are not administered in the same syringe or I.V. infusion system (Garber and Friedman, 1970).
Ureidopenicillins
Ureidopenicillins include azlocillin, piperacillin and sodium piperacillin.
Azlocillin is highly effective against P. aeruginosa infections. Piperacillin has the same or slightly greater activity as azlocillin against P. aeruginosa, but more effective against the common Gram-negative organisms. Sodium piperacillin has been found effective for topical use in experimental models of burn sepsis. These drugs have a major advantage over the carboxypenicillins because as monosodium salts they deliver only about 2 millimoles (mmol) of sodium per gram and are thus safer where sodium overload should be avoided. Ureidopenicillins are given parenterally and eliminated in the urine. For Pseudomonas septicaemia, a ureidopenicillin plus an aminoglycoside provides a synergistic effect but co-administration in the same fluid results in inactivation of the aminoglycoside (Laurence et al., 1999).
The Cephalosporins
This class of β-lactams include cephalexin, cefuroxime, ceftazidime and ceftriaxone. Ceftazidime is effective in treatment of chronic and recurrent respiratory
+ infections caused by P. aeruginosa. It is effective and safe in the treatment of serious infections of the lower respiratory tract (Davies et al, 1983). Ceftazidime is the antibiotic of choice in Pseudomonas meningitis, because of its high penetration into the subarachnoid space and the high susceptibility of Pseudomonas to this drug.
However, its use in aerosolized form for CF patients and in monotherapy for
Pseudomonas bacteremia is still controversial (Laurence et al., 1999).
Carbapenems
This class of β-lactam antibacterials include imipenem and meropenem.
Imipenem has the widest spectrum of all currently available antimicrobials, being bactericidal against most Gram-positive and Gram-negative aerobic and anaerobic pathogenic bacteria. Imipenem is used to treat pseudomonal septicaemia, particularly of renal origin, urinary tract infections, intra-abdominal infection and nosocomial pneumonia. 1 – 2 g/d is given by I.V. infusion in 3 – 4 doses. Meropenem is similar to imipenem in antibacterial therapy and in addition, penetrates into the cerebrospinal fluid (CSF) (Cometta, et al., 1994).
Monobactams
Aztreonam is the first member of this class of β - lactams antibiotics. It is active against Gram–negative organisms including P. aeruginosa, Haemophilus influenza, Neisseria meningitidis and N. gonorrhoeae. It is used to treat septicaemia, urinary tract infections and gonorrhoea (Laurence et al., 1999).
1.2.4.2 Effects of Aminoglycosides
Aminoglycosides are bactericidal. They are generally active against aerobic Gram- negative organisms and Staphylococci. Aminoglycosides include gentamicin, tobramycin, amikacin and a number of others. Gentamicin remains the drug of choice
for Gram - negative bacillary infections including those caused by P. aeruginosa. It is often used singly or in combined therapy in septicaemia and endocarditis. 2 – 5 mg/kg body weight is the highest dose for serious infections given singly or in 3 equally divided doses (Hatch and Schiller, 1998). Tobramycin is preferred to gentamicin for infections caused by P. aeruginosa. It is more active against most strains of P. aeruginosa and is less nephrotoxic (Van der Auwera and Schuyleneer, 1983).
Amikacin has the widest antibacterial spectrum among the aminoglycosides but is best reserved for infections caused by gentamicin-resistant organisms. Neomycin and
Framycetin are used topically for skin, eye and ear infections since they are too toxic for systemic use. Aminoglycosides are water-soluble and do not readily cross cell membranes. They are poorly absorbed from the intestine and so are administered intravenously or intramuscularly for systemic use. They distribute mainly to the extracellular fluid. Transfer into the cerebrospinal fluid is poor even when the meninges are inflamed. Their t½ is 2-5hrs and they are eliminated unchanged in the urine. Nephrotoxicity and ototoxicity are major limitations to the use of aminoglycosides (Schentag et al, 1981).
1.2.4.3 Effects of Sulphonamides and Sulphonamide Combinations
These drugs include co-trimoxazole and sulphadiazine. Co-trimoxazole
(sulphonamide-trimethoprim), commonly known as septrin, is used to treat urinary tract infections, acute exacerbations of chronic bronchitis and otitis media in children.
Many pathogenic strains of bacteria, including Pseudomonas have developed resistance to septrin. It is only used when there is evidence that the infecting bacteria is susceptible (Loudon, 1987). Sulphadiazine (in form of Silver sulphadiazine), is
used for treatment of infected burns, leg ulcers and pressure sores because of its wide antibacterial spectrum which includes pseudomonads (Laurence et al., 1999).
1.2.4.4 Effects of Quinolones (4-quinolones, Fluoro-quinolones)
Quinolones are active against Gram-negative organisms including P. aeruginosa, E. coli, Salmonella species, Shigella species, Neisseria species,
Haemophilus influenza and Legionella pneumophila. The first widely used quinolone was nalidixic acid which was effective for urinary tract infections but had little systemic activity. It was subsequently found that fluorination of the quinolone structure produced compounds that were up to 60 times more active than nalidixic acid and with a wide spectrum. They act by inhibiting bacterial (not human) DNA gyrase so preventing supercoiling of DNA, a process necessary for compacting chromosomes into the bacterial cell. They are bactericidal. Members of this group of antibiotics include ciprofloxacin, norfloxacin, ofloxacin and nalidixic acid.
Ciprofloxacin is active against a wide range of bacteria, particularly the Gram- negative organisms listed above, and less active against Gram-positive bacteria such as Streptococcus pneumonia and Enterococcus faecalis. Ciprofloxacin is indicated for infections of the urinary, gastrointestinal and respiratory tracts, tissue infection, gonorrhoea and septicaemia. It is preferably used for infections caused by organisms that are resistant to other drugs. Norfloxacin is used for acute or chronic recurrent urinary tract infections. Ofloxacin is indicated for urinary and respiratory tract infections and gonorrhoea. Nalidixic acid is now used principally for prevention of urinary tract infections (Wain, et al., 1997; Kesah, et al., 1999).
Quinolones are well absorbed from the gut and widely distributed in body tissue. In humans, the bioavailability of fluoroquinolones exceeds 90%, evidencing a good overall absorption (Bressole et al., 1994). In the case of ciprofloxacin, an
“absorption window” along the gastrointestinal tract has been suggested (Harder et al.,1990). Although quinolones are generally well tolerated, central nervous system toxicity may occur, including headache, confusion, hallucinations, anxiety, nervousness, nightmares and even seizures (Domagala, 1994).
1.2.4.5 Effects of Polypeptide Antibiotics
These are mostly used for topical application and include colistin, polymixin B and gramicidin. Colistin is effective against Gram-negative organisms, particularly P. aeruginosa. It is used for bowel sterilization in neutropenic patients. It is also topically applied to skin, including external ear infections. Polymixin B is active against Gram-negative organisms, especially P. aeruginosa. Its principal use is topical application for skin, eye, and external ear infections. Gramicidin is used in various topical applications as eye and ear drops, in combination with neomycin and framycetin (Laurence et al., 1999).
1.2.4.6 Effects of Antibiotic Combinations
For most Pseudomonas infections, treatment with a single antimicrobial is sufficient. Where, two or more antibiotics are recommended, the reason may be to avoid the development of drug resistance, especially in chronic infections like tuberculosis; to broaden the spectrum of antibacterial activity (i) in a known mixed infection, example, peritonitis following gut perforation or (ii) where treatment must be given before a diagnosis has been reached, example, when sepsis is suspected in
patients with neutropenia. In both cases, full doses of each drug are required; to obtain potentiation i.e an effect which is not obtained when each drug is used singly.
Aminoglycosides are usually combined with β - lactams or cephalosporins.
For instance, in the treatment of pulmonary infection in cystic fibrosis patients, piperacilin combined with tobramycin group III, elicited a better clinical response than when piperacillin is used alone (HoogKamp-Korstanje and Van der laag, 1983).
Den Hollander et al., (1997) also demonstrated synergistic activity between tobramycin and ceftazidime against a P. aeruginosa strain resistant to both antibiotics. Synergism was demonstrated at declining antibiotic concentrations below the MIC resulting in pronounced killing of the resistant P. aeruginosa strain.
1.2.5 Resistance of P. aeruginosa to Therapeutic and Chemical Agents
Pseudomonas aeruginosa has a particular propensity for the development of resistance and strains of the organism have increasingly shown resistance to the mainstream antibiotics used in hospitals. Pseudomonas infections can be complicated and life-threatening and infections with resistant strains are associated with extremely high mortality. Resistance to antimicrobial agents is an increasing clinical problem and is a recognized public health threat. The emergence of resistance in P. aeruginosa limits future therapeutic choices. There are a limited number of antimicrobial agents with reliable activity against P. aeruginosa, including antipseudomonal penicillins and cephalosporins, carbapenems, and fluoroquinolones, particularly ciprofloxacin.
Aminoglycosides are frequently used as part of combination regimens for treatment of serious Pseudomonas infections but are generally not recommended as single drugs.
For each of these agents, emergence of resistance during therapy has been described and has been recognized as a cause of treatment failure (Cometta, et al., 1994; Fink et
al., 1994; Milatovic and Braveny, 1987; CMPT, 1997; Ozumba, 2003). However,
“pan-resistant” strains (resistant to all available antibiotics) are still fortunately rare.
Therefore, effective drugs do exist for almost all infections. But finding an effective drug out of the 90-100 available antibiotics requires laboratory testing of antibiotic susceptibility and resistance. Today's methods rely on bacterial cultures, and cultures typically require two to three days to provide enough bacteria for analysis
(http://www.accelr8.com/index.php). Physicians in Intensive Care Units (ICU) urgently need rapid bacterial identification and antibiotic susceptibility testing that produce accurate results within a few hours after the patient presents with symptoms.
1.2.5.1 Resistance to Chemical Agents
Many Pseudomonas aeruginosa strains are resistant to soaps, germicides, disinfectants and detergents. Many nosocomial infections have been traced to soaps, disinfectants and other aqueous solutions that have become contaminated with the organism (Arino, et al., 1998). Some researchers have actually reported some strains of P. aeruginosa growing in solutions of these antimicrobial agents This metabolic feat has enabled P. aeruginosa to remain a steadfast inhabitant of many niches in the hospitals (Thomas et al, 1975).
1.2.5.2 Resistance to Antibiotics
History of Antibiotic Resistance: Powerful antibiotics first became commercially available in the 1940s and have saved untold millions of lives. Many antibiotics originated from natural sources – one type of organism producing chemical weapons against others. Many different types of organisms, including bacteria themselves, produce toxins against bacteria. In response, bacteria evolve defences which include
2 resistance to the antibiotics produced by other organisms. Perhaps the earliest practical example is penicillin, the first marketed modern antibiotic. Fleming discovered penicillin in 1928, produced by the Penicillium chrysogenum mold
(formerly identified as P. notatum). A decade later, Florey, Chain, Heatley, and
Abraham (Oxford) began studies on Fleming's “mould juice.” They demonstrated the therapeutic potential in a mouse infection study in 1940, in one of medicine's greatest experiments. Human studies began in 1941. Production efforts grew to the point where adequate supplies became available by 1944. At the end of the world war, penicillin became widely available and won widespread acceptance. Soon after, resistance to penicillin was observed in bacteria. The researchers themselves used penicillin-resistant bacteria in their own studies. In his 1945 Nobel Prize lecture,
Fleming himself warned of the danger of resistance – “It is not difficult to make microbes resistant to penicillin in the laboratory by exposing them to concentrations not sufficient to kill them, and the same thing has occasionally happened in the body…….” It took little time for Staphylococcus aureus to develop resistance to penicillin. In 1947 physicians observed the first case of clinical resistance. With few exceptions, each introduction of a new antibiotic has been followed within a few years by the first cases of resistance. Resistance then spreads. In addition, more variations of resistance mechanism against the same drug may also continue to emerge over time, compounding the problem (http://www.accelr8.com/index.php).
According to World Health Organization (WHO) annual report titled
“Overcoming Antimicrobial Resistance”, almost all major infectious diseases are slowly but surely becoming resistant to existing medicines. P. aeruginosa is the most notorious of all the resistant pathogens with many multi-resistant strains. Strains of the organism are especially resistant to the older and more commonly used antibiotics
6
(WHO, 1988; O’Brien, 1992; http://www.accelr8.com/index.php). By 1993, multiple- drug-resistant Sal. typhi were isolated in Viet Nam. They were resistant to the usual firstline antibiotics: chloramphenicol, ampicillin and co-trimoxazole but fully susceptible to fluoroquinolones (Wain, et al., 1997).
Development of Resistance to beta-lactams: Pathogenic bacteria that produce beta- lactamase enzymes are naturally resistant to penicillins. β- lactamases open the β- lactam ring in the penicillin molecule and terminate their activity. Virtually all strains of P. aeruginosa produce the Sabbath and Abraham’s enzyme (a β- lactamase) which inactivates penicillins making them resistant to these antibiotics. Members of the genus Staphylococcus also produce beta-lactamases which confer penicillin-resistance on them (Laurence et al., 1999). Various molecular alterations in the beta-lactam structure have been developed to protect the beta-lactam ring against enzymic hydrolysis. This development has resulted in the production of more effective antibiotics in this class. This strategy has been applied to develop penicillins and cephalosporins that are more resistant to beta-lactamases produced by Gram-negative bacilli (Forbes, et al., 2007).
Development of Resistance to Cephalosporins: Cephalosporins, (a group of β- lactam antibiotics) were first obtained from the fungus Cephalosporium. They are closely related to Penicillin in molecular structure and have a wide range of activity and low toxicity. By addition of various side chains on the cephalosporin molecule, many semi-synthetic forms have been introduced with a variety of pharmacokinetic and antibacterial activities. Cephalosporins resist attack by β - lactamases but bacteria develop resistance to them by other means. E. coli is inherently susceptible to cephems, although it usually produces a low level of an uninducible chromosomal
AmpC-type cephalosporinase. Recently however, with increasing use of broad
= spectrum cephalosporins, resistant E. coli isolates are commonly encountered in various clinical settings (Yagi, et al., 1997).
Development of Resistance to Aminoglycosides: Streptomycin was first obtained from Streptomyces griseus in 1944, and used as an effective therapy for tuberculosis.
Other aminoglycosides have since been developed and used effectively as broad spectrum antibiotics. However, bacterial resistance to aminoglycosides has since emerged and is an increasing problem, notably by acquisition of plasmids. Resistance to streptomycin, gentamicin, tobramycin and amikacin has been reported by several workers (Udo, et al., 1998; Oduyebo, et al., 1997; Ozumba, 2003; Kesah, et al.,
1999).
Development of Resistance to Fluoroquinolones: The fluoroquinolones have been very useful in treating resistant infections caused by both Gram-negative and Gram- positive bacteria. Kesah et al., (1999) found 0% resistance to ciprofloxacin and ofloxacin. With the passage of time however, different pathogenic strains developed resistance to the quinolones. Salmonella typhi remained fully susceptible to fluoroquinolones and third generation cephalosporins until 1993 when nalidixic acid- resistant Sal. typhi emerged (Wain, et al., 1997). By 2003, the emergence of P. aeruginosa resistance to quinolones was observed, and has been reported to be on the increase (Ozumba, 2003; Olayinka et al., 2004; Eldere, 2003).
Factors responsible for development of antibiotic resistance: Antimicrobial resistance is a natural phenomenon, amplified manifold due to human misuse and neglect of antimicrobial drugs. The situation of antimicrobial resistance appears to be due to a combination of factors which include socio-economic status, overuse of antibiotics in food production, inappropriate use of antimicrobial drugs, heavy burden of bacterial infectious diseases, huge population without even the rudiments of
? primary health care, rapid spread through crowding, poor sanitation and sexual contact, and self-prescription and easy accessibility of drugs in local pharmacies or open-air markets which is common in most developing countries (Gustafsson and
Wide, 1981).
The social causes that encourage the development of resistance are paradoxical. In poorer countries, underuse of drugs encourages resistance. Patients are unable to afford the full course of the drugs or can only afford to buy counterfeit drugs. This exerts a selective pressure that favours the survival of the more resistant microbes. In wealthy countries, resistance is emerging because of overuse of drugs.
Unnecessary demands for drugs by patients are often met by health services prone to over – prescription. Similarly, overuse of antimicrobials in food production is also contributing to increased drug resistance. Currently, 50% of all antibiotic production is used to treat sick animals, promote livestock and poultry growth or rid cultivated foods of destructive organisms. Globalization, increased travel and trade ensure that resistant strains are quickly spread from their point of origin to other places. A World
Health Organization (WHO) report shows that Africa, the Middle East, Latin America and Asia, containing three quarters of the world`s population, have the highest rates of resistance to the older antimicrobial drugs. Antibiotic resistance causes tens of thousands of deaths each year (WHO, 1988). In hospitals, inappropriate use of antimicrobial agents tends to create a selective pressure that promotes the emergence of resistant strains and predisposes patients to colonization with such organisms. In many instances, poorly planned or haphazard use of medicine has caused the world to lose these drugs as quickly as scientists have discovered them. Those admitted to hospital wards are especially vulnerable. Globally, 60% of hospital-acquired infections are caused by drug resistant microbes. The increasing use of invasive
2+ devices, immunosuppressive drugs and technological advances in medicine has escalated the spreading of resistant stains (Weinstein, 1991).
Antibiotic-Resistance Patterns:
The multiple antibiotic resistance (MAR) of P. aeruginosa has been reported by many research workers. Udo, et al., (1998), reported P aeruginosa and E. coli as some of the faecal flora showing multiple antibiotic resistance (MAR) to the older and commonly used antibiotics, such as ampicillin, chloramphenicol, tetracycline, cotrimoxazole, and streptomycin. Percentage of MAR strains was in the order: P. aeruginosa (23.0%) > E. coli (5.8%). Olayinka et al., (2004) in a study conducted at
Ahmadu Bello University Teaching Hospital (ABUTH), reported that all 13
P.aeruginosa isolated from urine samples were resistant to the cheap easily available antibiotics- rifampicin, ampicillin/cloxacillin, erythromycin, chloramphenicol, and ampicillin but were uniformly susceptible to ciprofloxacin. Oduyebo, et al., (1997) reported the prevalence of multiple antibiotic resistances in strains of P. aeruginosa isolated at the Lagos University Teaching Hospital (LUTH) from 1994 to 1996. The percentage resistance of the isolates to various antibiotics were in the order: amoxycillin-clavulanic acid (92%) > cefotaxime (39.7%)> piperacillin (21.6%) > imipenem (12.5%), while percentage susceptibility were in the order: amikacin
(95.5%) > ciprofloxacin (90%) > gentamicin (60%) > ceftazidime (54.2%). Colsky et al., (1998), analysed the susceptibilities of P. aeruginosa and S. aureus isolated from skin wounds in hospitalized dermatology patients. Thirty-six (36%) of P. aeruginosa isolated from leg ulcers were resistant to ciprofloxacin, while 50% of S. aureus from leg ulcers and 25% from superficial wounds were resistant to oxacillin.
2
Kesah, et al., (1999), at Lagos University Teaching Hospital (LUTH), studied the resistance to commonly used antimicrobials, of nosocomial bacterial pathogens isolated from paediatric patients. Resistances of 125 P. aeruginosa isolates were
100% in cotrimoxazole, tetracycline, streptomycin, amoxycillin-clavulanate, ampicillin, ceftriaxone and cefuroxime. Their resistances to other antibiotics were in the order: nitrofurantoin (62%) > nalidixic acid (56%) > gentamicin (32%) > colistin
(20%)> amikacin (6.4%). Percentage resistances of 175 S. aureus strains were in the order: penicillin and ampicillin (98.9%) > chloramphenicol (75.4%) > tetracycline
(72.6%) > streptomycin (68.0%) > erythromycin (50.3%) > gentamicin (49.7%) > cotrimoxazole (47.4%) > cloxacillin (26.3%) > amoxicillin-clavulanic acid (21.1%) > ciprofloxacin and vancomycin (0.00%). Percentage resistances of 82 E. coli strains were in the order: ampicillin (100%) > tetracycline (90.2%) > cotrimoxazole (82.9%)
> streptomycin (69.5%) > amoxicillin-clavulanic acid (57,3%) > gentamicin (42.7%)
> cefuroxime (20.7%) > nitrofurantoin (9.8%) > nalidixic acid (6.1%) > colistin, ofloxacin and ciprofloxacin (0.00%). In a study of antibiotic susceptibility pattern of
229 P. aeruginosa isolated between 1998 and 2000 at University of Nigeria Teaching
Hospital (UNTH) Enugu, percentage resistances were in the order: ceftazidime
(11.5%) > colistin (16.3%) > ofloxacin (37.5%) > ciprofloxacin (37.9%) (Ozumba,
2003). In another study, Olayinka et al., (2004), investigated the prevalence of multi- drug resistance of P. aeruginosa isolated in Surgical Unit in Ahmadu Bello University
Teaching Hospital (ABUTH). Of all the 92 isolates, 19.6% were resistant to three or more of the antibiotics tested, which include ceftazidime, amikacin, gentamicin, imipenem, ciprofloxacin and perfloxacin. The most prevalent resistance pattern was ceftazidme+gentamicin+perfloxacin+ofloxacin (27.8%).
2
Rastegar Lari, et al., (2005), in a study conducted in Tohid Burn Center in Iran from 1995-1999, found high rates of resistance in P. aeruginosa and S. aureus. The frequency of P. aeruginosa resistance to ciprofloxacin, amikacin and gentamcin was over 85% while, the rate of S. aureus resistance to cloxacillin and cephalexin was
90%.: In a study involving seven hundred and sixteen (716) P. aeruginosa isolates collected from 40 different hospitals in Belgium and the Grand Duchy of Luxembourg in 1999, resistance rates varied significantly between hospitals. Of the fluoroquinolones, ofloxacin showed highest resistance (37.5%), levofloxacin showed
27.5% resistance and ciprofloxacin 24%. Of the aminoglycosides, gentamicin was the most resistant antibiotic (23.5% resistance), followed by tobramycin (19.5%), isepamicin (12%), and amikacin (10.5%). Of the ß-lactam antibiotics, aztreonam was the most resistant (55.5), followed by ticarcillin/clavulanic acid (37%), cefepime
(29.5%), ceftazidime (28.5%), piperacillin/tazobactam (17.5%), piperacillin (24%), meropenem (9.5%). These results reveal that the resistance of P. aeruginosa to penicillins, cephalosporins, fluoroquinolones and aminoglycosides varies between hospitals, but is increasing (Eldere, 2003).
Resistance rates can evolve rapidly in P. aeruginosa. Susceptibility to ciprofloxacin, for example, has gradually evolved from 87% in 1991, to 82–84% in the period 1993–1995, 70% in 1998–1999, to 71% in 2003. For amikacin, susceptibility was 96% in 1994–1995, 91% in 1997, 74% in 1998–1999 and 85% in
2003. For cefepime, it was found that 73% of P. aeruginosa isolates were still susceptible in 1997, compared with 64% in 1998–1999 and 50% in 2003. For meropenem, susceptibility was 86% in 1998–1999 and 81% in 2003. The result supports the need for correct, high dosing and combination therapy to minimize the risk of resistance development in cases of P. aeruginosa infection (Eldere, 2003).
2
1.2.5.3 Resistance Determinants
Natural (inherent) drug resistance The inherent resistance in Pseudomonas results from natural attributes which offer a barrier to antibiotic penetration into the bacterial cell or reduce the intracellular concentration of antimicrobials. These natural attributes include
• Possession of outer membrane lipopolysaccharide (LPS) with a low
permeability, through which antimicrobial agents diffuse very slowly (Masuda
et al., 1995).
• The alginate slime produced by mucoid strains which prevents the diffusion of
drugs into the cell. Hatch and Schiller, 1998 demonstrated that a 0.2%
suspension of P. aeruginosa alignate completely blocked the diffusion of
gentamicin and tobramycin, while a preparation of P. aeruginosa alginate
lyase degraded the diffusion barrier.
• The propensity to construct protective biofilms in medical situations The P.
aeruginosa biofilm has been attributed to the production of alginate
(Mclaughln-Borlace et al., 1998).
• Possession of drug efflux systems which decrease the intracellular
concentration of the antimicrobial agents (Nikaido, 1994).
Resistance genes, R-factors and RTFs
Genes encoding various resistance determinants are found in many bacterial species.
These genes may be chromosomal or plasmid-borne. Strains of P. aeruginosa are often carriers of plasmids containing genes that confer the capacity for antibiotic resistance to the cell. These plasmids are referred to as Resistance Factors (R-factors) and Resistance Transfer Factors (RTFs). Whether chromosomal or plasmid-borne,
2 these genes control the formation of enzymes that modify drugs and render them inactive against the microbe (Biddlecom et al., 1976).
1.2.5.4 Resistance Mechanisms
Mutations against antibiotics
Mutations are alterations in the base sequence of genes leading to alterations in the sequence of amino acids. Some mutations confer various properties to microorganisms, including the ability to resist chemicals and antimicrobials.
Mutations in the parC, parE and gyrA genes which confer fluoroquinolone-resistance to some species of bacteria have been reported (Gonzalez et al., 1998). In clinical isolates of P. aeruginosa mutations in genes GyrA and ParC play a substantial role in conferring of fluoroquinolone-resistance. These findings suggest that DNA gyrase may be a primary target of fluoroquinolones and simultaneous alterations in GyrA and
ParC may be associated with development of higher-level fluoroquinolone -resistance in clinical isolates of P. aeruginosa (Nakano et al., 1997). In P. aeruginosa strain
P2540, mutations designated nalB, nfxB and nfxC result in the over-expression of multidrug efflux systems that confer resistance to a number of antibiotics (Nikaido,
1994; Hirai et al., 1987).
Drug-Modifying Enzymes
Some chromosomal and plasmid–borne genes control the formation of enzymes which modify the structure of drugs. Drug-modifying enzymes include the following:
Beta-lactam-Modifying enzymes:
Some bacterial species produce -lactamases that destroy penicillins and some cephalosporins. Virtually all strains of P. aeruginosa produce a chromosomally-
2 determined, inducible -lactamase, the Sabath and Abraham’s (SA) or ld enzyme. In vitro studies indicate SA enzyme to be active against ampicillins, penicillin G and cephaloridine (Sabath, et al., 1965; Livermore, 1983). However, Carbenicillin has been found to be SA stable and inhibits SA enzyme (Garber and Friedman, 1970). In
E. coli, hyperproduction of the chromosomal AmpC enzyme results in moderate to high-level cephem resistance (Livermore, 1995; Yagi, et al., 1997).
Aminoglycoside-modifying enzymes:
The presence of modifying enzymes is the most frequent cause of bacterial resistance to aminoglycosides. The gentamicin resistance-determinant gene aacC3, encoding aminoglycoside-(3)- N- acetyltransferase 111 has been found so far only in
P. aeruginosa (Biddlecom et al., 1976, Miller et al. 1980). This gene has been cloned and could be expressed in P. putida K t 2440 and E. coli (Vliegenthart et al., 1991;
Van Boxtel and De Klundert 1998).
Efflux systems
The innate resistance of P. aeruginosa to a variety of antimicrobials was previously attributed to a highly impermeable outer membrane (Masuda et al., 1995), but is now recognized to result from the synergy between broadly specific drug efflux pumps and low outer membrane permeability. Active efflux systems that decrease the intracellular concentrations of antimicrobial agents have been assumed to be an especially effective mechanism of drug resistance in P. aeruginosa (Nikaido, 1994).
In quinolone resistance by P.aeruginosa strains, mutations designated nalB, nfxB and nfxC has resulted in the over-expression of multidrug efflux systems (Hirai et al., 1987). Three distinct multidrug efflux operons have been identified: Mex AB-
OprM, over expressed by nalB mutants (Poole et al., 1996), which confers resistance to quinolones, -lactams, tetracyclines, chloramphnicol and trimetoprim (Kohler et
22 al.,1996; Li et al., 1995). MexCD-oprJ operon, over-expressed by nfxB-type mutants, confers resistance to quinolones (Hirai et al., 1987), erythromycin, Zwitteronic cephems and trimethoprim (Kohler et al., 1996). MexEF-oprN efflux operon endows resistance to quinolones, chloramphenicol and trimethoprim and is over- expressed by nfx-C type mutants.
Transmission of resistance genes
Transmission of genes from one organism to another is very common among the bacteria. Bacteria transmit genes to others through the processes of conjugation and transduction. Plasmids carrying resistance markers including R-factors and RTFs can be transferred to other bacteria of the same or unrelated species. The transfer in nature of resistance markers to other Gram-negative species of medical importance is a matter of some concern. However, in comparison with clinical strains of other species, the proportion of P. aeruginosa strains carrying transmissible resistance plasmids is fairly low (Forbes, et al., 2007).
1.2.6 Mortality Rates in Pseudomonas-related Diseases
Antibiotic resistance causes tens of thousands of deaths each year. In the United
States alone, about 14,000 patients died in one year as a result of infection caused by drug resistant pathogens picked up in hospitals (WHO, 1988; O’Brien, 1992; http://www.accelr8.com/index.php). The emergence of resistance in P. aeruginosa is associated with increased rates of mortality and morbidity and higher costs. The excess morbidity and mortality associated with ineffective empirical therapy in P. aeruginosa infections has been reported (Boffi et al., 2000). P. aeruginosa is responsible for acute fulminant infections such as pneumonia, sepsis, burn wound infections and meningitis which are associated with extremely high mortality. In
26 cystic fibrosis, (CF) it is the characteristic pathogen responsible for most cases of morbidity and mortality. P. aeruginosa accounts for 11% of all isolates recovered from fatal nosocomial infections (Qarah et al., 2003; Boffi et al., 2000; Hoiby, 1993).
1.2.7 New Trends in the Control of Resistant Pseudomonas Infections
Since infection by antibiotic resistant strains of P. aeruginosa became a serious problem in immunocompromised patients, a major area of antibiotic research has been to develop new agents against this pathogen. With regard to this, attention has increasingly been turned to vaccination and medicinal plants.
1.2.7.1 Use of Vaccines in Prevention of Pseudomonas Infections
Most cases of mortality in P. aeruginosa infections are due to resistant strains of the organism (Lindberg, et al., 1965). As the problem of resistance has become more serious with the passage of time, some research workers have approached the problem by studying active immunization as a means of preventing Pseudomonas infections.
Markley and Smallman, (1968), reported that mice given thermal injury could be protected against a local Pseudomonas challenge by both specific and non-specific
Pseudomonas vaccines, prepared either from bacterial cells or from the medium in which they were grown. Pyoimmunogen, a polycomponent vaccine against P. aeruginosa infections has been found to afford a high level of protection with low toxicity in mice. The vaccine, obtained from strains belonging to O-serotypes most frequently occurring in clinical practice, has been used for producing protective antigens in both laboratory and semi-industrial conditions (Stanislavskii, et al., 1982).
Grishina et al., (1982), immunized 20 patients having burn wounds with pyoimmunogen. The immunization improved healing and reduced the period of
2= hospitalization. A toxoid, prepared by treating Exotoxin A of P. aeruginosa with formalin and lysine, induced high titers of antibody to exotoxin A in the sera of mice and rabbits. Antiserum to the toxoid neutralized mouse lethality, cytotoxicity and
ADP-ribosyl transferase activity of untreated exotoxin A (Pollack et al., 1982). An intranasal vaccine against respiratory infections of P.aeruginosa and Aerobacter levanicum has been tried on mice. The vaccine, composed of liposomes containing bacterial polysaccharide and IL-2, increased production of secretory IgA titres and specific pulmonary plasma cells (Abraham and Shah, 1992). Grutman, et al., (2001) also reported the protective action of immune sera from rabbits that were immunized with liposomal form of complex surface antigen preparations with added immunomodulators against homo- and heterological strains of bacteria including
E.coli KI, S. typhimurium M, P. aeruginosa 02 and Proteus mirabilis 708.
More recently, immunization with DNA vaccines has been the focus of many research efforts, and the immunoprophylactic potential of many DNA vaccines has been assessed (Soission et al., 1992). In studies of genetic immunization against
Bacillus anthracis and P. aeruginosa, high titred antisera were demonstrated against several Pseudomonas gene products (Naval Medical Research Center Immunology,
2005). Reports on the safety of DNA vaccines, indicate that plasmid DNA vaccines are safe with no adverse effects in experimental animals. The major risk of DNA vaccines is that of deleterious integration into the host cell genome. This has been studied and shown to pose a non-significant safely concern (Martin et al., 1999).
1.2.7.2 Use of Medicinal Plants in the Treatment of Diseases
Plants have been used as sources of drugs for many centuries, in all parts of the world.
In developing countries, about 90% of people still depend on traditional medicine
2? based on different species of plants for their primary health care (Taylor, 2005; Iwu,
1993; Mbam, 2007; Ugwuozor, 2006). The medicinal activities of plants have been attributed to the presence of a variety of secondary metabolites. Scientists involved in medicinal plant research have found the presence of secondary metabolites such as alkaloids, flavonoids, glycosides, cardiac glycosides, saponins, tannins and essential oils (Muller et al., 1979; De Pascual Teresa et al., 1980; Adesina and Sofowora, 1979;
Ofokansi et al., 2003; Vera, 1993). Levan et al., (1979), and Ibrahim et al., (1997), proposed that the presence of tannins, alkaloids, flavonoids and saponins suggest possible antimicrobial activity by plants.
The use of plants for drug development has its undeniable benefits. In research, using medicinal plants makes research less expensive and more efficient, as laboratory synthesis of new medicines is becoming increasingly costly (Taylor, 2005).
In therapy, plant drugs are considered to be more superior since they have a wide range of activities, with little or none of the adverse effects associated with synthetic antimicrobials. They are effective and yet gentle and many have tropisms to specific organs or systems in the body. Plant drugs usually have multiple effects on the body.
Their actions often go beyond the symptomatic treatment of disease. An example of this is Hydrates Canadensis. This plant not only has antimicrobial activity, but also increases blood supply to the spleen, promoting optimal activity of the spleen to release mediating compounds (Murray, 1995). Plant-derived drugs are new and it will take a long time for pathogens to develop resistance to them. They are easily available wherever they grow naturally and therefore more affordable. Economically, harnessing these forest resources for commercial purposes will be a good source of income. In addition, the herbal products market offers many opportunities for cultivating medicinal plant crops so as to meet the demands of the growing market.
6+
Bioprospecting: Recently, and on a global scale, attention has been shifted to plants and other natural products as sources of medicine. Many researchers are now involved in conducting follow-up research to verify the authenticity of information by indigenous peoples concerning the medicinal uses of plants. In the past, native doctors were very reluctant to part with information concerning their medicinal plants, but now, many bioprospectors are working side by side with herbal healers to gain from their knowledge of medicinal plants. Such a transfer of knowledge will help to build up a library of information on medicinal plants which would have been lost by the death of an experienced herbalist (Taylor, 2005). Besides, out of about 500,000 species of plants, only about 2% have been subjected to a complete ethnobotanical and biochemical analysis and less than 10% have been tested for biological activity
(Asuzu 2001; Okafor, 2002). This calls for continued effort on the part of researchers, bioprospectors, and centers engaged in biodiversity programmes.
The development of plant-based drugs begins with the extraction of plant components in different solvents, followed by detailed biological assays. Next, acute and chronic toxicity studies are conducted in animals. If the substance has an acceptable safety index, phytochemical analysis is carried out, with the isolation and identification of the active constituents. Finally, there is formulation of dosage forms, followed by several phases of clinical studies designed to establish efficacy and pharmacokinetic profile of the new drug. Formulation and trial production of the dosage forms are structured to mimic the traditional use of the herb (Iwu, 1993).
Some Plants with Antibacterial Activities
Traditionally, plants have been used to treat infectious diseases of bacterial etiology, and in literature, innumerable reports from researchers bear witness to the
6 antibacterial activities of these plants in vitro and in vivo. A brief review of some plants and their antibacterial activities will suffice.
• Bryophyllum pinnatum leaf juice exhibited wide spectrum activity against B.
subtilis, S. aureus, Strept. pyogenes, E. coli, Proteus species, Klebsiella
species, Shigella species, Salmonella species, Serratia marcenscens and P.
aeruginosa (Obaseiki-Ebor, 1985).
• Picralima nitida stem bark: the methanolic extract exhibited activity against a
wide range of Gram-positive bacteria and fungi (Fakeye et al., 2002).
• Salacia pyriformis ethanolic extract exhibited activity against Proteus
mirabilis and E. coli at concentration of 100 mg/ml (Poh, 2000).
• Terminalia avicennoides and Ocimum gratissimum (nchu anwu) extracts were
active against multiple antibiotic-resistant Shigella species isolated from
patients with bacillary dysentery, at crude concentrations of 3, 000 µg/ml
(Iwalokun et al., 2001).
• T. avicennoides, in another study, exhibited very high activity against Vibrio
cholerae (Akinsinde and Olukoya, 1995).
• Ocimum gratissimum (nchu anwu): Ofokansi et al., (2003), demonstrated the
activity of its leaf extract against E. coli and B. subtilis.
• Jatropha podagrica was found to be active against Gram-positive organisms
only (Odebiyi, 1999).
• Cymbopogon citrates (lemon grass) volatile oil exhibited activity against
Gram-negative and Gram-positive bacteria (Onawunmi et al., 1984).
• Synclisia scarbida exhibited activity against two strains of S. aureus, MIC 5
and 10 mg/ml (Sokomba et al., 1986).
6
• Spilanthes filicaulis inhibited the growth of E. coli, and Klebsiella pneumoniae
but not B. subtilis and S. aureus at concentration of 120 mg/ml (Ekor et al.,
2005).
• Cassia alata aqueous leaf extracts showed broad spectrum activity against
Gram-negative and Gram-positive bacteria including S. aureus, B. subtilis,
E. coli and P. aeruginosa, comparable with the activity of streptomycin
sulphate standard I mg/ml (Akinde, et al., 1999).
• Anacardium occidentale and Angeiossus schimperi exhibited high activity
against E. coli and P. aeruginosa (Kudi et al., 1999).
• Acalypha wilkesiana had broad spectrum activity against B. subitlis, E. coli,
Kleb. pneumoniae, Proteus vulgaris, Serratia marcensens and S. aureus
(Adesino, et al., 1980).
• Acalypha torta exhibited antibacterial and antifungal properties (Akinyanju et
al., 1986).
• Ageratum conyzoides: Extracts of the whole plant inhibited in vitro
development of S. aureus, P. aeruginosa, B. subtilis and E. coli (Durodola,
1977). This, in addition to its analgesic, antipyretic, anti-inflammatory, stytic,
and wound healing properties makes A. conyzoides an excellent remedy
(Database entry for A. conyzoides, 2005).
The antimicrobial activity demonstrated in medicinal plants is a confirmation of their effectiveness in the treatment of infectious diseases by herbalists. However, there is the need to provide information on their effectiveness against drug-resistant organisms as well as their toxicity to mammalian cells. Hence there is need to study medicinal plants in details to find out if they can provide a solution to the problem of
6 antibiotic resistance which is becoming life-threatening and creating very serious problems in the hospitals.
CHAPTER TWO
2.0 MATERIALS AND METHODS
2.1.0 Collection of Pseudomonas aeruginosa Isolated from Clinical Specimens in Hospitals and Diagnostic Laboratories
Bacterial isolates identified as Pseudomonas aeruginosa were collected from the diagnostic laboratories of National Orthopaedic Hospital, Enugu, Federal Medical
Centre, Abakaliki, Ebonyi State and Departments of Microbiology and Veterinary
Microbiology, University of Nigeria, Nsukka, between 2005 and 2007. They were isolated from sputum, urine, wound and oral swab. The isolates were transferred to
Microbiology Department, University of Nigeria, Nsukka for confirmation of their identity using standard biochemical tests.
2.2.0 Confirmation of the Identity of the Clinical Specimens from the Hospitals and Diagnostic Laboratories using Standard Biochemical Tests for P. aeruginosa
Loopfuls of each isolate were streaked on Nutrient agar (Fluka), MacConkey agar
(Antec Diagnostics, UK) and Blood agar plates and incubated at 37oC for 18-24 hours. After incubation, P. aeruginosa was identified based on colony characteristics and biochemical reactions.
6
2.2.1 Colony Characteristics
Discrete colonies on the plates were observed for colony morphology and characteristics of P. aeruginosa. Non-lactose fermenting (pale) colonies on
MacConkey agar; beta-haemolytic colonies with flat, fringy morphology on Blood agar; cream, discrete, smooth and raised colonies on Nutrient agar; the production of blue-green, non-fluorescent and diffusible pigment on Nutrient agar, which is diagnostic of P. aeruginosa, and a fruity, grape-like odor of aminoacetophenone were used as criteria for identifying P. aeruginosa.
2.2.2 Biochemical Reactions
Discreet colonies with the characteristics of P. aeruginosa were picked from the growth medium and further purified by subculturing on nutrient agar plates. They were stored on Nutrient agar slants in a refrigerator at 4oC. The isolates were subjected to biochemical tests for further identification. The Gram reaction, oxidase reaction, oxidation-fermentation tests and growth at 5oC, 37oC, and 42oC were carried out according to standard techniques (Cowan and Steel, 1993; Cheesbrough, 1994).
2.2.2.1 Gram Reaction
The Gram stain was carried out according to standard techniques (Appendix 7), to differentiate Gram-positive organisms such as S. aureus which retain the primary stain (purple), from Gram-negative organisms such as Pseudomonas and Escherichia coli which stain pink.
2.2.2.2 Oxidase Reaction
6
This test was carried out to differentiate Pseudomonas species (oxidase- positive) from members of the Enterobacteriaceae (oxidase- negative). A spot oxidase test was done using the Kovac’s method.
A few drops of oxidase reagent (1 % solution of tetramethyl-p-phenylenediamine dihydrochloride) were placed on a piece of Whatman No. 2 filter paper in a Petri dish.
A sterilized wire loop was used to collect a portion of a discrete colony from a 24 hour culture of the test organism. This was smeared over the reagent and observed for colour change (a positive reaction is indicated by the appearance of a dark purple colour on the paper within 10 seconds).
2.2.2.3 Sugar Fermentation Test
This test was carried out to differentiate Pseudomonas, which utilizes substrates oxidatively, from enterobacteria, which utilize substrates fermentatively.
Method
Ten milliliters (10ml) of Peptone water containing the indicator methyl red (Appendix
6) were poured into each of several Bijou bottles inside which Durham tubes were inserted. 0.1 gram of glucose, sucrose and lactose sugars were weighed out and added to the Peptone water in the different bottles, mixed and autoclaved at 121oC for 10 minutes. After cooling, two loopfuls of test organism were inoculated into each bottle and incubated at 37oC for 18 hours. After incubation, the medium was observed for colour change and gas production. (Colour change of the solution from red to yellow shows acid production, indicating that the organism utilizes the sugar oxidatively, while gas production indicates that the organism utilizes the sugar fermentatively, and absence of gas with no colour change indicates non- utilization of the sugar).
2.2.3 Collection of Other Organisms for the Study
62
A reference culture, ATCC 10145 of P. aeruginosa as well as Escherichia coli (n=2),
Staphylococcus aureus (n=2) and Salmonella typhi (n=1) were included in the study.
These were obtained from Departments of Microbiology and Veterinary
Microbiology, University of Nigeria, Nsukka. The test organisms were stored on
Nutrient agar slants in a refrigerator at 4oC and subcultured every two weeks during the study (Laboratory media used, their composition and preparation are shown in
Appendices 2-6).
2.3.0 Antibiotic Sensitivity Testing on All the Test Organisms
The sensitivity of all the P. aeruginosa and other test organisms to antibiotics were tested. The tests were carried out on Mueller-Hinton agar (Oxoid Ltd.,
Basingstoke, Hampshire, England) and antibiotic multodiscs (Optun Nig.) were used.
The Gram-negative organisms were screened against ciprofloxacin 10g, ofloxacin
10g, perfloxacin 10g, streptomycin 30g, co-trimoxazole (septrin) 30g, ampicillin
30µg, ceporex 10µg, gentamicin 10µg, amoxicillin–clavulanic acid (augmentin) 30µg and nalidixic acid 30µg. The Gram-positive organisms were screened against ofloxacin 5µg, erythromycin 10µg, ciprofloxacin 5µg, clindamycin 10µg, gentamicin
10µg, cephaplexin 30µg, co-trimoxaxole 50µg, ampicillin-cloxacillin (ampiclox)
30µg, floxapen 30µg and amoxicillin–clavulanic acid (augmentin) 30µg. The Kirby-
Bauer disc diffusion method was employed. Twenty-five milliliters (25 ml) of sterile molten Mueller-Hinton agar were poured into sterile Petri dishes and allowed to solidify. The test organisms were sub-cultured on Nutrient Agar plates and incubated at 37oC for 18-24 hours. The growth from each Nutrient Agar plate was transferred into test tubes containing 5ml of 0.9% sterile saline and the volume adjusted in a spectrophotometer to obtain a turbidity which matches that of 0.5 McFarland standard
66
(containing 1.5 × 108 cfu/ml) (Appendix 9). This cell suspension was used to swab the entire surface of the Mueller-Hinton agar as follows: A sterile cotton swab was immersed in the bacterial suspension. Excess fluid was expressed by rotating the swab against the inside wall of the test tube. This was then used to swab the surface of the Mueller-Hinton agar plates while rotating the plate anticlockwise until the entire surface was swabbed. Within 15 minutes of inoculation, multodiscs containing the antibiotics were placed on the plates inoculated with the test organisms, in replicates, and allowed to stand for 1 hour for proper diffusion. The plates were then incubated aerobically at an inverted position at 37oC for 24 hours. After incubation, diameters of the zones of inhibition (IZDs) around each antibiotic disc were measured. IZDs (in millimeters) were recorded by calculating the mean of IZD in replicate plates. The results were interpreted according to CLSI Standards (Appendix
10) (Nelson, 1991; Bauer et al., 1966; CLSI, 2006; Forbes, et al., 2007).
2.4.0 Collection and Identification of the Medicinal Plant Materials used
The plants used in this study were selected after consultation with local herbalists, based on their uses in traditional medicine. They were collected from
Nsukka in Enugu State and Iboko in Izzi Local Government Area of Ebonyi State.
The three plants tested were Alchornea cordifolia leaves, Spondias mombin leaves and Terminalia schimperiana root bark and leaves. The plants were identified by
Plant Taxonomist, Professor J. C. Okafor and a Laboratory Technologist, Mr. A. O.
Ozioko of Department of Botany, Faculty of Biological Sciences, University of
Nigeria, Nsukka. Voucher specimens were deposited at Department of Botany
Herbarium, University of Nigeria, Nsukka and given numbers 97G, 303i, 241D and
578 respectively (Table 1).
6=
Table 1: Medicinal plants used for the study
S/N Botanical Name Common/Local Name Part Place of
(Family) collected collection
1 Alchornea cordifolia cn: Christmas bush Leaves Nsukka
(Euphorbiaceae) Igbo: Ububo
2 Spondias mombin cn: Hog plum Leaves Iboko
(Anacardiaceae) Igbo: Ijikara, gogori
3 Terminalia schimperiana Igbo: Oshioku Root bark Iboko
(Syn: T. glaucescens) Leaves
KEY: cn = common name
6?
2.4.1 Alchornea cordifolia (Schum et Thon.) Muell. Arg. (Common name:
Christmas bush; Igbo: Ububo or Abaraugba; Hausa: bambami; Yoruba: ewe-epa) is a multistemmed shrub or small tree, up to 5m tall and 30cm girth. It is widespread in tropical secondary forests in West, East and South African countries (Figure 1). The leaves are commonly used for wrapping fermenting ‘Ugba’ (Sliced oilbean seeds used to prepare ‘African salad’). In Igbo ethnomedicine, the herbalists use extracts of this plant for the treatment of dysentery. A slurry of the fruit is administered for cough. A decoction of the leaves alone is used as an eye lotion. The leaves and bark when powdered are drunk in water or eaten in food for piles. The juice of the leaves and fruits is rubbed in ringworm and other skin infections as a curative agent (Iwu, 1993).
=+
Figure 1: Alchornea cordifolia (Abaraugba) leaves
=
2.4.2 Spondias mombin Linn. (Common name: Hog plum; Igbo: Ijikara, gogori;
Peruvian: Ubos) is a tropical deciduous tree, found in moist forests. It is erect, 20m tall, and 60-75cm in diameter. The trunk is slightly buttressed with thick, fissured, corky grayish bark. The leaves are 20-45cm long and hairy underneath (Figure 2).
The flowers are small, white fragrant flowers occuring in panicles. The fruits are plum-like, green at first and golden yellow on ripening, with juicy pulp, very acidic and sour-tasting. S. mombin is commonly used as a hedge plant in Igboland. The fruit pulp is used to make jams, juices and ice creams. It is used by herbalists in native medicine as broad spectrum antiseptic, antibacterial, antiviral, antiparasitic and anthelminthic agent, and for menstrual regulation and treatment of menstrual pain and cramps. It is used to treat vaginal and yeast infections, stress, anxiety and as a nervine.
In Nigeria, it is used for tonsillitis, sore throat, thrush and wounds. In Africa, it is used to stop bleeding, chronic diarrhoea, constipation, coughs, fever, gonorrhoea, stomach ailments, tapeworm, yaws and postpartum hemorrhage (bleeding after childbirth) (Corthout et al., 1987; Ajao et al., 1985; Abo et al., 1999; Database entry for Ubos, 2005; Personal communication-Ugwuozor, 2006).
=
Figure 2: Spondias mombin (Ijikara) leaves
=
2.4.3 Terminalia schimperiana Hochst is synonymous with T. glaucescens Planch. ex Benth. (Igbo: Oshioku). Terminalia is a genus of large trees comprising around
100 species distributed in tropical regions of the world. There are 11 species found in
West Africa. Species of Terminalia are most widely used for medicinal purposes and some like T. catappa, have edible fruits. Trees of this genus are known as a source of secondary metabolites-cyclic triterpenes and their glycoside derivatives, flavonoids, tannins and other aromatic compounds (McGraw et al., 2001, Adjanahoun et al.,
1991).
T. schimperiana is native to tropical Africa. In Nigeria, it is found in the woodland savanna. It is a broad leaved, small tree that can reach up to 714m. It is deciduous to semi-evergreen, depending on the climate. The leaves are alternate, simple, elliptic to obvate, entire, 9-15cm long and 3-8cm broad, green above with pale undersides. The flowers are tiny and form spikes at the base of the leaves. The fruit is a samara with a single wing, 6-9cm long which turns brown with age (African Plants Database;
Arbonnier, 2004) (Figures 3 and 4).
The botanical classification of T. schimperiana is shown below:
Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Myrtales
Family: Combretaceae
Genus: Terminalia
Species: T. schimperiana (Wikipedia, 2007).
In parts of West Africa, T. schimperiana is used as a medicinal plant (Sofonara,
1982). The stem bark is applied to wounds. Herbalists use the root bark to treat burn
= wounds in Izzi Local Government Area of Ebonyi State where it is known as
‘Oshioku’ (Personal communication- Mbam, V. 2007). When applied to wounds, it produces a burning sensation, similar to what is experienced when iodine is applied to wounds. The twigs may be chewed to promote oral hygiene. The leaves are used to treat bronchitis and dysentery.
=
Figure 3: Terminalia schimperiana (Oshioku) leaves
=2
Figure 4: Terminalia schimperiana (Oshioku) tree
=6
2.5.0 Extraction of the Active Antimicrobial Substances from the Plant Materials
2.5.1 Drying and Pulverization of Plant Parts
The plant parts used for the tests were dried for two weeks at room temperature. They were each pulverized in a mill into dry powder, packaged in clean black polythene bags and kept for further studies. Extraction of the components of each plant was done using the solvents Ethanol (96%, Analar) and sterile distilled water.
2.5.2 Ethanolic Extraction
Fifty grams (50 g) of each pulverized material was introduced into a 1000 ml conical flask and 250 ml (or equivalent w/v concentrations for higher volumes) of
96% Ethanol solvent was added to the powder. The mixture was stirred with a glass rod and macerated for 48 hours at room temperature. After 48 hours, the mixture was filtered with Whatman No. 1 filter papers inserted in a funnel to separate the filtrate
(extract) from the residue (marc). The marc was rinsed with half the volume of solvent previously used and filtered again. The extract was concentrated using a rotary evaporator under low pressure at 40oC and then dried at room temperature. The extract was stored in labelled, sterile amber bottles and kept in a refrigerator at 4oC.
2.5.3 Aqueous Extraction (at 4oC)
Fifty grams (50 g) of each pulverized material was extracted in 250 ml of distilled water (solvent) employing the same procedure as in section 2.5.2. However, the aqueous mixtures were macerated at 4oC in a refrigerator to prevent deterioration of the extract with time. The aqueous extract was air dried in open trays at room
== temperature and the extracts stored in labelled, sterile amber bottles and kept in a refrigerator at 4oC.
2.6.0 Preliminary Antimicrobial Screening of Crude Extracts
2.6.1 Serial Dilution of Crude Extract
Different concentrations of each of the extracts were prepared using a suitable solvent. The solubility of the extracts was first determined by introducing 0.02 g of extract in 1ml of a solvent. The solvents- Dimethylsulfoxide (DMSO), Tween 80 and
Propylene glycol and Distilled water- in which the extracts dissolved were used.
Solvents found to have activity against the test organisms were diluted out with sterile nutrient broth or distilled water (Appendix 11). Five dilutions of each crude extract were prepared as follows: 0.02 g of crude extract was weighed with a microgram balance (Mettler H8) and introduced into a sterile Bijou bottle containing 1ml of the solvent (or equivalent w/v concentrations for higher volumes of solvent). This was thoroughly mixed using a vortex mixer to give a 20 mg/ml concentration. Serial dilution was carried out by using a sterile pipette to transfer 2 ml of this dilution into another bijou bottle containing an equal amount of solvent to obtain a 10 mg/ml dilution. This procedure was used to prepare two-fold dilutions of 5 mg/ml, 2.5 mg/ml and 1.25 mg/ml of the crude extracts.
2.6.2 Sensitivity Test of Organisms with Plant Extracts using Agar-well
Diffusion Method
The test organisms were sub-cultured on nutrient agar plates and incubated at
37oC for 18-24 hours. The growth from each plate was transferred into test tubes containing 5 ml of 0.9% sterile saline and the volume adjusted in a spectrophotometer to obtain a turbidity which matches that of 0.5 McFarland Standard (containing 1.5 ×
=?
108 cfu/ml). A sterile cotton swab was immersed in the bacterial suspension. Excess fluid was expressed by rotating the swab against the inside wall of the test tube. This was then used to swab the surface of the Mueller-Hinton agar plates while rotating the plate anticlockwise until the entire surface has been swabbed.
The sensitivities of the test organisms to the crude extracts were tested using the agar-well diffusion method (Baron and Finegold, 1990). A sterile cork borer with a diameter of 8mm was used to bore wells into the seeded Mueller-Hinton agar plates.
A drop of molten agar was placed in each well to seal the bottom. Using a sterile pipette, 0.1ml of each of the dilutions (20 mg/ml, 10 mg/ml, 5 mg/ml, 2.5 mg/ml and
1.25 mg/ml) of each extract was introduced into a labeled well. The same quantity of gentamicin (standard antibiotic) and solvent were used as positive and negative controls respectively. Gentamicin was used in five dilutions (10 µg/ml, 5 µg/ml, 2.5
µg/ml, 1.25 µg/ml and 0.625 µg/ml (Appendix 12). Replicate plates were prepared for each organism. The plates were allowed to stand on the bench for 1 hour at room temperature for proper diffusion to take place and subsequently incubated at 37oC for
24 hours. After incubation, the plates were examined and inhibition zone diameters
(IZD) measured with a ruler. The IZD (mm) was recorded by calculating the mean of
IZDs for each set of replicate plates.
2.7.0 Bioactivity-guided Fractionation
After the preliminary antimicrobial screening of the crude extracts, the extract that exhibited significant activity against the test organisms was selected for fractionation.
?+
2.7.1 Separation of the crude ethanolic extract
The selected extract was separated into five soluble fractions to find out the fraction that contained the active ingredients. A hundred gram (100 g) of any crude extract that showed activity was mixed with twice its mass of silica gel in a small quantity of alcohol that was enough to make a homogeneous mixture. This mixture was homogenized with a mortar and pestle and then placed in a large, transparent bottle with a cover. The extract was separated with five solvents viz n-hexane, chloroform, ethylacetate, acetone and ethanol to yield fractions. The solvents were used in order of increasing polarity.
First n-hexane was poured into the bottle and shaken vigorously to mix thoroughly with the extract. The n-hexane was then decanted from the bottle. Fresh n- hexane was poured into the bottle again and the process was repeated. This was done five times until all constituents extractable by n-hexane were exhausted and the eluate became colourless. The extract was then removed from the bottle, dried and replaced in the bottle. The process was repeated with chloroform until all constituents extractable by chloroform were exhausted. This procedure was repeated with ethylacetate, acetone and finally ethanol to obtain n-hexane, chloroform, ethylacetate, acetone and ethanol-soluble fractions.
2.7.2 Concentration of the Soluble Fractions
Each soluble fraction was poured into a glass plate and air dried at room temperature until all the solvent evaporated. The five fractions were weighed to determine the yield and then stored in sterile amber bottles at 4oC for further tests.
?
2.7.3 Antimicrobial screening of the soluble fractions against the test organisms
Activities of the five fractions against the test organisms were carried out according to standard methods previously described in sections 2.6.1 to 2.6.2. The soluble fraction that gave the highest activity against the test organisms was further separated using Thin Layer Chromatography.
2.7.4 Thin Layer Chromatography (TLC) and Antimicrobial Screening of TLC
Fractions
Preparatory Thin Layer Chromatography was first employed to determine the suitable solvent system to be used (Appendix 13). For the main TLC procedure, silica-coated glass chromatography plates with 20cm by 20cm dimensions were used.
The thickness of the silica layer was 0.5mm. The starting line or origin was marked
2cm from the lower edge of the silica layer. Another line, the solvent front, was marked 15 cm from the starting line. This marks the distance the solvent is allowed to travel. The fraction was dissolved in ethanol and a 1ml syringe with needle was used to apply a thick band of the solution along the starting line. This band was allowed to dry. The TLC tank containing the solvent system was covered with a glass plate and allowed to stand for 10 minutes for chamber saturation. The plate was then placed in the tank for fractionation, with the silica layer dipping in the solvent up to a height of
1 cm. As the solvent system (mobile phase) moves up the adsorbent medium (silica gel), the components of the fraction were separated according to their different rates of movement in the solvent. Those that are less strongly adsorbed to the stationary phase move faster than those more strongly adsorbed. At the end of the TLC, the plate was brought out from the jar and allowed to dry.
?
To detect the colour of the separate bands, an iodine tank was prepared. This was a glass tank containing a few crystals of iodine and covered with a glass plate.
The rim was smeared with vaseline to prevent escape of iodine fumes. The iodine crystals vaporize through sublimation to fill the tank with iodine vapour. The TLC plates were then placed inside the tank which was again covered with the glass plate and enclosed with a black polythene bag to shut out light. After 3 minutes the plates were removed and examined. The separate bands on the TLC plates were carefully scrapped into separate clear glass bottles and ethanol was added. Each mixture was stirred with a glass rod and the silica gel was allowed to settle. The clear liquid containing the fraction was decanted into a watch glass to allow the ethanol to evaporate. The TLC fractions were screened against the test organisms using the method previously described in sections 2.6.1 to 2.6.2.
2.8.0 Determination of Minimum Inhibitory Concentration (MIC) values of the
Extracts with Activity
The MIC values of the active crude extracts and fractions were determined from the results of the antimicrobial screening. Microsoft Excel was used to plot the graph of mean IZD2 against log drug concentration. A trend line was fitted into the scatter diagram to obtain intercept on the log drug conc axis (C), after which the MIC values (antilog of C) were obtained (Appendices 14 to 28).
2.9.0 Acute Toxicity Studies and Determination of Median Lethal Dose (LD50)
The crude extract and soluble fraction that showed highest activity were used to conduct toxicity studies on mice. This was carried out according to the method described by Locke (1983).
?
2.9.1 Acute Toxicity Studies with the Active Crude Extract
Albino mice of both sexes were obtained from the Animal house of Faculty of
Pharmaceutical Sciences, University of Nigeria, Nsukka. Eighteen (18) mice were used. They were housed three animals per cage in six plastic cages labeled A, B, C,
D, E and F and allowed free access to food and water. The mice were weighed in a balance to obtain their body mass. They were grouped into six. The first group was placed in Cage A and labeled A1, A2, and A3. The second group was placed in cage
B and labeled B1, B2 and B3 while the third group was placed in cage C and labeled
C1, C2, C3 etc. The mice were dosed by intraperitoneal injection with graded doses of the crude extract.
Group A received 10 mg/kg body mass
Group B received 100 mg/kg body mass
Group C received 200 mg/kg body mass
Group D received 400 mg/kg body mass
Group E received 800 mg/kg body mass
Group F received 1000 mg/kg body mass
The mice were observed for 24 hr for signs of acute toxicity such as changes in behaviour, convulsion, excitement, weakness and death. The death pattern (if any) was recorded. LD50 was calculated as square root of A x B: where A = largest dose that causes no death; B = smallest dose that causes 100% death.
2.9.2 Acute Toxicity Studies with Active Soluble Fraction
Eighteen (18) albino mice were used to repeat the toxicity test with soluble fraction. This time, the mice were dosed orally with the aid of a gavage. Graded doses of 10 mg/kg, 100 mg/kg, 1000 mg/kg, 5000 mg/kg, 7000 mg/kg and 10,000
? mg/kg were administered. The mice were observed for 24hr for signs of acute toxicity. LD50 was calculated as square root of A x B: where A = largest dose that causes no death; B = smallest dose that causes 100% death
2.10 Phytochemical Analyses of Crude Extracts and Fractions
The extracts and fractions that exhibited activity against the test organisms were subjected to phytochemical analysis. The tests were carried out in the
Department of Pharmacognosy, University of Nigeria Nsukka, based on procedures outlined by Harbourne (1973) and Trease and Evans (1989).
Test for carbohydrate
Molisch Test
A small quantity (0.1 g) of each extract was boiled with 2 ml of distilled water and filtered. To the filtrate, few drops of naphthol solution in ethanol (Molisch’s reagent) were added. Concentrated sulphuric acid was then gently poured down the side of the test tube to form a lower layer. A purple interfacial ring indicates the presence of carbohydrate.
Test for Alkaloids
Twenty milliliters (20 ml) of 3% sulphuric acid in 50% ethanol was added to
2g of the extract and heated in a boiling water bath for 10 minutes, cooled and filtered. 2 ml of the filtrate was tested with a few drops of Mayer’s reagent
(potassium mercuric iodide solution), Dragendorff’s reagent (bismuth potassium iodide solution), Wagner’s reagent (iodine in potassium iodide solution), and picric acid solution (1%).
The remaining filtrate was placed in 100 ml separatory funnel and made alkaline with dilute ammonia solution. The aqueous alkaline solution was separated
? and extracted with two 5 ml portions of dilute sulphuric acid. The extract was tested with a few drops of Mayer’s, Wagner’s, Dragendorff’s reagents and picric acid solution. Alkaloids give a milky precipitate with few drops of Mayer’s reagent; reddish brown precipitate with few drops of Wagner’s reagent; yellowish precipitate with few drops of picric acid and brick red precipitate with few drops of
Dragendorff’s reagent.
Test for reducing sugar
Five milliliters (5 ml) of a mixture of equal parts of Fehling’s solution I and II were added to 5 ml of aqueous extract and heated on a water bath for 5 minutes. A brick red precipitate shows the presence of reducing sugar.
Test for Glycosides
Five milliliters (5 ml) of dilute sulphuric acid was added to 0.1 g of the extract in a test tube and boiled for 15 minutes on a water bath, then cooled and neutralized with 20% potassium hydroxide solution. 10 ml of a mixture of equal parts of
Fehling’s solution I and II was added and boiled for 5 minutes. A more dense brick red precipitate indicates the presence of glycoside.
Test for Saponins
Twenty milliliters (20 ml) of distilled water was added to 0.25 g of the extract and boiled on a hot water bath for 2 minutes. The mixture was filtered while hot and allowed to cool and filtrate was used for the following tests:
(a) Frothing Test
Five milliliters (5 ml) of filtrate was diluted with 15 ml of distilled water and shaken vigorously. A stable froth (foam) upon standing indicates the presence of saponins.
?2
(b) Emulsion Test
To the frothing solution was added 2 drops of olive oil and the contents shaken vigorously. The formation of emulsion indicates the presence of saponins.
(c) Fehling’s Test
To 5 ml of the filtrate was added 5 ml of Fehling’s solution (equal parts of 1 and II) and the contents were heated in a water bath. A reddish precipitate which turns brick red on further heating with sulphuric acid indicates the presence of saponins.
Test for Tannins
One gram (1 g) of the powdered material was boiled with 20 ml of water, filtered and used for the following test.
(a) Ferric chloride Test
To 3 ml of the filtrate, few drops of ferric chloride were added. A greenish black precipitate indicates the presence of tannins.
(b) Lead Acetate Test
To a little of the filtrate was added lead acetate solution. A reddish colour indicates the presence of tannins.
Test for Flavonoids
Ten milliliters (10 ml) of ethylacetate was added to 0.2 g of the extract and heated in a water bath for 3 minutes. The mixture was cooled, filtered and the filtrate was used for the following tests.
(a) Ammonium Test
Four milliliters (4 ml) of filtrate was shaken with 1ml of dilute ammonia solution. The layers were allowed to separate and the yellow colour in the ammoniacal layer indicates the presence of flavonoids.
?6
(b) 1% Aluminium Chloride solution Test
Another 4 ml portion of the filtrate was shaken with 1 ml of 1% aluminium chloride solution. The layers were allowed to separate. A yellow colour in the aluminium chloride layer indicates the presence of flavonoids.
Test for Resins
(a) Precipitation Test
A small quantity (0.2 g) of the extract was extracted with 15 ml of 96% ethanol. The alcoholic extract was then poured into 20ml of distilled water in a beaker. A precipitate occurring indicates the presence of resins.
(b) Colour Test
A small quantity (0.2 g) of the extract was extracted with chloroform and the extract was concentrated to dryness. The residue was redissolved in 3ml of acetone and another 3ml concentrated hydrochloric acid was added. This mixture was heated in a water bath for 30 minutes. A pink colour which changes to magenta red indicates the presence of resins.
Test for Fats and Oil
A small quantity (0.1 g) of the extract was pressed between filter paper and the paper was observed. A control was also prepared by placing 2 drops of olive oil on filter paper. Translucency of the filter paper indicates the presence of fats and oil.
Test for Steroids and Terpenoids
Nine milliliters (9 ml) of ethanol was added to 1 g of the extract and refluxed for a few minutes and filtered. The filtrate was concentrated to 2.5 ml on a boiling water bath. 5ml of hot distilled water was added to the concentrated solution, the mixture was allowed to stand for 1 hour and the waxy matter was filtered off. The filtrate was extracted with 2.5 ml of chloroform using separating funnel. To 0.5 ml of
?= the chloroform extract in a test tube was carefully added 1ml of concentrated sulphuric acid to form a lower layer. A reddish brown interface shows the presence of steroids. Another 0.5 ml of the chloroform extract was evaporated to dryness in a water bath and heated with 3 ml of concentrated sulphuric acid for 10 minutes in a water bath. A grey colour indicates the presence of terpenoids.
Test for Acidic Compounds
A small quantity of the extract (0.1 g) was placed in a clear dry test tube and sufficient water added. This was warmed in a hot water bath and then cooled. A piece of water-wetted litmus paper was dipped into the filtrate and the colour change on the litmus paper was observed. Acidic compounds turn blue litmus paper red.
Ultraviolet Spectroscopy of TLC Fractions (F1, F7, and F9)
Ultraviolet Spectroscopy of TLC Fractions (F1, F7, and F9) was carried out at the Department of Pharmacognosy, University of Nigeria, Nsukka.
2.11 Statistical Analysis
The values obtained in the experiments were expressed statistically as mean
+SEM. They were analyzed for statistically significant differences using Analysis of
Variance and Independent Samples t-test. P<0.05 was chosen for significance.
Analysis of Variance was used to determine whether there were significant differences in the antimicrobial activities of the different plants and of gentamicin against the test organisms.
Independent Samples t-test, employing Levene’s Test for Equality of
Variances, was used to determine whether there were significant differences in the susceptibilities of gentamicin-sensitive and gentamicin-resistant test organisms to the plant extracts and fractions.
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CHAPTER THREE
3.0 RESULTS
3.1.1 Results of Confirmatory Tests for Pseudomonas aeruginosa
Out of the twenty-three (23) isolates collected from different hospitals and laboratories, only fourteen (14) gave typical colony characteristics and biochemical reactions of P. aeruginosa. On MacConkey agar, they produced non-lactose fermenting (pale) colonies. On Blood agar, they produced beta-haemolytic colonies with flat, fringy morphology. On Nutrient agar, they were discrete, cream and raised colonies. They all produced a blue-green, non-fluorescent and diffusible pigment on nutrient agar, which is diagnostic of P. aeruginosa. They produced a fruity, grape-like odour of aminoacetophenone and Gram staining yielded Gram-negative straight rods.
Oxidase test produced a positive spot oxidase reaction. From the results of sugar fermentation test, Pseudomonas isolates were oxidative, producing acid and no gas from glucose, but none from sucrose or lactose (Appendix 8).
3.1.2 Test organisms used for the study
Fourteen P. aeruginosa isolates, a reference culture, ATCC 10145 of P. aeruginosa as well as Escherichia coli (n=2), Staphylococcus aureus (n=2) and
Salmonella typhi (n=1) were used for the study (Table 2).
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Table 2: Test organisms used for the study
Test organism Site of Clinical sample Source P. aeruginosa 1 Sputum FMC P. aeruginosa 2 Urine FMC P. aeruginosa 3 Urine Vet. Microbiology UNN P. aeruginosa 4 Wound NOH P. aeruginosa 5 Wound NOH P. aeruginosa 6 Wound NOH P. aeruginosa 7 Wound NOH P. aeruginosa 8 Wound NOH P. aeruginosa 9 Wound NOH P. aeruginosa 10 Sputum Microbiology UNN P. aeruginosa 11 Oral swab Vet. Microbiology UNN P. aeruginosa 12 Oral swab Vet. Microbiology UNN P. aeruginosa 13 Urine Vet. Microbiology UNN P. aeruginosa 14 Urine Vet. Microbiology UNN P. aeruginosa 15* Microbiology UNN E. coli 1 ~ Microbiology UNN E. coli 2 ~ Vet. Microbiology UNN S. aureus 1 ~ Microbiology UNN S. aureus 2 ~ Vet. Microbiology UNN Sal. typhi ~ Microbiology UNN KEY: FMC- Federal Medical Center Abakaliki, NOH- National Orthopaedic Hospital, Enugu, UNN- University of Nigeria, Nsukka, *ATCC Typed Sample, ~ unspecified.
3.2.0 Results of Antibiotic Sensitivity Tests on all the Test Organisms
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The results of the antibiotic tests revealed that out of the 15 isolates of P. aeruginosa studied, five were resistant to all 10 antibiotics tested. Two of the remaining ten isolates were resistant to 9 antibiotics, one was resistant to 6 antibiotics, while five were resistant to 5 antibiotics and two were resistant to 4 antibiotics. E. coli isolates 1 and 2 were resistant to 8 and 5 antibiotics respectively, while Sal. typhi was resistant to 6 antibiotics. S. aureus isolates 1 and 2 were both resistsant to 7 antibiotics
(Table 3). Percentage resistance (%R) of P. aeruginosa isolates to the ten antibiotics were as follows: Ciprofloxacin (33.3%), perfloxacin (46.6%), ofloxacin (53.3%), ceporex and gentamicin (60%), cotrimoxazole and streptomycin (80%), ampicillin, amoxicillin-clavulanic acid (augmentin) and nalidixic acid (100%) (Table 4). The interpretive chart for antibiotic test is shown in Appendix 10.
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Table 3: Susceptibility of Pseudomonas aeruginosa, Escherichia coli, Salmonella typhi and Staphylococcus aureus to the antibiotics, measured as inhibition zone diameters (mm) Test isolate PN AU NA S GN CEP SXT OFX PEF CPX
P. aeruginosa 1 0 0 0 0 28 22 34 15 28 39 P. aeruginosa 2 0 0 0 0 16 32 34 30 27 32 P. aeruginosa 3 0 0 0 0 0 0 0 0 0 0 P. aeruginosa 4 0 0 0 0 0 0 0 0 0 18 P. aeruginosa 5 0 0 0 0 0 0 0 0 0 24 P. aeruginosa 6 0 0 0 0 22 24 14 24 28 36 P. aeruginosa 7 0 0 0 21.5 12 0 13 24 30 34 P. aeruginosa 8 0 0 0 0 14 24 20 24 30 24 P. aeruginosa 9 0 0 0 0 0 0 0 0 0 0 P. aeruginosa 10 0 0 0 0 0 0 0 0 0 22 P. aeruginosa 11 0 0 0 0 0 0 0 0 0 0 P. aeruginosa 12 0 0 0 0 0 0 0 0 0 0 P. aeruginosa 13 0 0 0 0 18 28 13 26 28 39 P. aeruginosa 14 0 0 0 20 16 0 12 18 29 28 P. aeruginosa15* 0 0 0 21 15 21 15 21 29 34 E. coli 1 0 0 0 19 19 0 0 0 0 0 E. coli 2 0 20.5 0 30 24 0 0 14 21 29 Sal. typhi 0 18 0 22 16 0 0 0 0 28 AMPX SXT CD CP FX GN AU E OFX CPX S. aureus 1 0 0 0 0 0 12 0 26 28 32 S. aureus 2 0 0 0 0 0 0 0 24 25 29
KEY: PN- Ampicillin, AU-Amoxicillin-clavulanic acid (Augmentin), NA- Nalidixic acid, S- Streptomycin, GN- Gentamicin, CEP-Ceporex, SXT- Co-trimoxazole (Septrin), OFX-Ofloxacin, PEF-Perfloxacin, CPX-Ciprofloxacin, AMPX- Ampicillin/cloxacilin, CD-Clindamycin, CP-Cephaplexin, FX-Floxapen, E-Erythromycin, * ATCC Typed Sample
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Table 4: Percentage resistance (%R) of the fifteen isolates of P. aeruginosa studied to different antibiotics tested Antibiotic %Resistance
Ciprofloxacin (CPX) 33.30
Perfloxacin (PEF) 46.60
Ofloxacin (OFX) 53.30
Ceporex (CEP) 60.00
Gentamicin (GN) 60.00
Septrin (SXT) 80.00
Streptomycin (S) 80.00
Ampicillin (PN) 100.00
Augmentin (AU) 100.00
Nalidixic acid (NA) 100.00
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3.3.0 Results of Antimicrobial Screening with Plant Extracts and Gentamicin against the Test Organisms
3.3.1 Sensitivity of the Test Organisms to Gentamicin
Seven (7) P. aeruginosa isolates tested were resistant to gentamicin standard up to a concentration of 10 µg/ml. Eight (8) P. aeruginosa isolates were susceptible to it even at the low concentration of 2.5 µg/ml, with inhibition zone diameters (IZDs) ranging from 15.3 mm at 2.5 mg/ml to 23.0 mm at 10 µg/ml. Seven P. aeruginosa isolates were resistant to 9-10 antibiotics as well as to the gentamicin control. Eight other P. aeruginosa isolates were resistant to 4-6 antibiotics but sensitive to the gentamicin control. E. coli 1 and 2 were susceptible to gentamicin up to a concentration of 2.5 µg/ml, with inhibition zone diameters (IZDs) ranging from 15.3 mm to 21.0 mm. S. aureus 2 was susceptible to gentamicin only at 10 µg/ml with inhibition zone diameter of 15.2 mm while Sal. typhi was susceptible to gentamicin at concentration of 10 µg/ml and 5 µg/ml, with inhibition zone diameters of 17.5 mm and 15.2 mm respectively (Table 5).
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Table 5: Inhibition zone diameters (mm) exhibited by gentamicin on the test organisms Test organism Concentration of gentamicin in µg/ml 10 5 2.5 1.25 0.625 P. aeruginosa 1 22.0±0.50 20.0±0.50 17.7±0.34 0 0 P. aeruginosa 2 23.0 ±0.23 22.5±0.05 19.8±0.44 0 0 P. aeruginosa 3 0 0 0 0 0 P. aeruginosa 4 0 0 0 0 0 P. aeruginosa 5 0 0 0 0 0 P. aeruginosa 6 22.0 ±0.50 19.0±0.00 17.0±0.23 0 0 P. aeruginosa 7 22.2 ±0.17 19.0±0.58 18.0±0.00 0 0 P. aeruginosa 8 21.0 ±0.50 20.5±0.50 16.0±0.58 0 0 P. aeruginosa 9 0 0 0 0 0 P. aeruginosa 10 0 0 0 0 0 P. aeruginosa 11 0 0 0 0 0 P. aeruginosa 12 0 0 0 0 0 P. aeruginosa 13 18.0±0.50 11.3±0.67 10.0±0.00 0 0 P. aeruginosa 14 14.3±0.34 12.2±0.17 10.0±0.23 0 0 P. aeruginosa 15* 21.8±0.44 21.0±0.58 18.0±0.50 0 0 E. coli 1 20.0±0.50 18.0±0.23 15.3±0.67 0 0 E. coli 2 21.0±0.50 19.0±0.00 17.2±0.17 0 0 S. aureus 1 1 3 . 8 ± 0 . 1 7 12.0±0.05 10.5±0.58 0 0 S. aureus 2 15.2±0.17 13.5±0.05 11.3±0.34 10.0±0.23 0 Sal. typhi 17.5±0.05 15.0±0.58 13.5±0.05 0 0 n = 3- Values represent means ± standard error of the mean, *ATCC Typed Sample
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3.3.2 Effects of Crude Extracts of Terminalia schimperiana Root Bark on the
Test Organisms
The ethanolic extract of T. schimperiana root bark exhibited activity against the P. aeruginosa isolates even at the low concentration of 2.5 mg/ml. The inhibition zone diameters (IZDs) ranged from 10.5 mm at 2.5 mg/ml to 18.0 mm at 20 mg/ml. It inhibited the growth of S. aureus 1 and 2 with inhibition zone diameters ranging from
11.7 mm at 2.5 mg/ml to 14.5 mm at 20 mg/ml. It also inhibited the growth of Sal. typhi with inhibition zone diameters ranging from 11.5 mm at 2.5 mg/ml to 15.0 mm at 20 mg/ml. The extract did not have any effect at the concentration of 1.25 mg/ml. It also did not have any effect on E. coli at any of the concentrations tested (Table 6).
The aqueous extract, out of all the crude extracts, was found to have the highest activity against P. aeruginosa isolates even at the low concentration of 1.25 mg/ml. The inhibition zone diameters ranged from 16.5 mm at 1.25 mg/ml to 22.5 mm at concentration of 20 mg/ml. It is also interesting to note that seven (7) P. aeruginosa isolates which were resistant to 10 µg/ml of gentamicin standard were susceptible to T. schimperiana extracts up to 5.0 mg/ml. P. aeruginosa 3 was susceptible even at the lowest concentration of 1.25 mg/ml (Table 7).
+6
Table 6: Inhibition zone diameters (mm) exhibited by ethanolic extract of T. schimperiana root bark on the test organisms Test organism Concentration of extract in mg/ml 20 10 5 2.5 1.25 P. aeruginosa 1 15.5±0.23 12.0±0.56 11.0±0.58 0 0 P. aeruginosa 2 14.5±0.50 12.0±0.50 0 0 0 P. aeruginosa 3 16.5±0.50 14.5±0.23 13.0±0.00 11.0±0.00 0 P. aeruginosa 4 15.5±0.58 13.5±0.23 10.5±0.50 0 0 P. aeruginosa 5 15.0±0.00 15.0±1.00 13.5±0.50 0 0 P. aeruginosa 6 14.5±0.23 11.5±0.50 0 0 0 P. aeruginosa 7 14.5±0.50 11.7±0.34 0 0 0 P. aeruginosa 8 15.5±0.50 14.0±0.00 12.5±0.50 10.5±0.50 0 P. aeruginosa 9 13.0±0.00 0 0 0 0 P. aeruginosa 10 14.5±0.87 12.0±0.67 10.0±0.58 0 0 P. aeruginosa 11 14.5±0.23 12.5±0.23 0 0 0 P. aeruginosa 12 18.0±0.00 16.5±0.50 14.5±0.50 11.0±0.00 0 P. aeruginosa 13 17.2±0.17 16.3±0.34 15.0±0.00 12.0±0.00 0 P. aeruginosa 14 15.0±0.58 13.5±0.50 11.5±0.87 0 0 P. aeruginosa 15* 16.5±0.50 15.0 + 0.00 13.5±0.50 0 0 E. coli 1 0 0 0 0 0 E. coli 2 0 0 0 0 0 S. aureus 1 13.0±0.23 11.7±0.34 10.0±0.00 0 0 S. aureus 2 14.5±0.87 13.5±0.50 12.5±0.50 11.7±0.17 0 Sal. typhi 15.0±0.00 13.3±0.67 12.5±0.50 11.5±0.50 0 n = 3, *ATCC Typed Sample
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Table 7: Inhibition zone diameters (mm) exhibited by aqueous extract of T. schimperiana root bark on the test organisms Test organism Concentration of extract in mg/ml
20 10 5 2.5 1.25
P. aeruginosa 1 17.0±0.58 15.0±0.50 13.5±0.50 0 0
P. aeruginosa 2 22.5±0.50 22.5±0.50 21.0±1.00 18.5±0.89 16.5±0.50
P. aeruginosa 3 18.2±0.17 15.5±0.50 14.0±0.00 12.5±0.50 12.0±0.00
P. aeruginosa 4 15.8±0.44 13.7±0.34 12.0±0.00 0 0
P. aeruginosa 5 16.0±0.56 14.0±0.00 12.5±0.23 0 0
P. aeruginosa 6 17.5±0.50 15.5±0.23 14.0±0.00 12.0±0.00 0
P. aeruginosa 7 22.5±0.87 20.5±0.50 19.5±0.50 17.5±0.23 15.7±0.34
P. aeruginosa 8 21.2±0.17 19.5±0.23 18.0±0.00 16.0±0.00 14.5±0.50
P. aeruginosa 9 15.5±0.50 14.5±0.50 12.5±0.50 0 0
P. aeruginosa 10 15.0±1.00 12.7±0.93 10.5±0.50 0 0
P. aeruginosa 11 15.0±0.00 13.0±0.50 10.3±0.34 0 0
P. aeruginosa 12 14.0±0.23 12.7±0.17 10.5±0.50 0 0
P. aeruginosa 13 15.8±0.44 14.5±0.50 12.5±0.50 10.0±0.50 0
P. aeruginosa 14 15.5±0.23 14.5±0.50 13.0±0.00 11.7±0.17 0
P. aeruginosa 15* 17.5±0.50 15.0±0.58 13.5±0.50 0 0 n = 3, *ATCC Typed Sample
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3.3.3 Effects of Crude Ethanolic Extract of Terminalia schimperiana Leaves on the Test Organisms
The ethanolic extract of T. schimperiana leaves exhibited activity against the
P. aeruginosa isolates even at the lowest concentration of 1.25 mg/ml. The inhibition zone diameters ranged from 10.0 mm at 1.25 mg/ml to 16.0 mm at 20 mg/ml. It also inhibited the growth of S. aureus 1 and 2 and Sal. typhi with inhibition zone diameters ranging from of 10.3 mm at 2.5 mg/ml to 16.0 mm at 20 mg/ml. P. aeruginosa 3 and
4 which showed resistance to gentamicin were susceptible to the extract even at the lowest concentration of 1.25 mg/ml. It did not however significantly inhibit the growth of E. coli 1 and 2 (Table 8).
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Table 8: Inhibition zone diameter (mm) exhibited by ethanolic extract of T. schimperiana leaves on the test organisms Test organism Concentration of extract in mg/ml 20 10 5 2.5 1.25 P. aeruginosa 1 14.2±0.17 12.0±0.23 10.0±0.00 0 0 P. aeruginosa 2 13.5±0.50 11.5±0.50 10.7±0.34 0 0 P. aeruginosa 3 16.0±0.50 14.5±0.50 13.5±0.23 12.0±0.00 10.0±0.58 P. aeruginosa 4 14.0±1.00 12.5±0.50 12.5±0.50 11.0±0.58 10.0±0.00 P. aeruginosa 5 13.5±0.87 12.5±1.00 11.5±0.50 10.5±0.23 0 P. aeruginosa 6 12.7±0.93 11.5±0.87 10.5±0.50 10.0±1.00 0 P. aeruginosa 7 12.7±0.17 11.3±0.34 10.0±0.00 0 0 P. aeruginosa 8 13.0±1.00 12.0±0.58 10.5±0.50 0 0 P. aeruginosa 9 14.0±0.23 12.0±0.00 10.5±0.23 0 0 P. aeruginosa 10 13.8±0.44 12.0±0.50 10.5±0.50 0 0 P. aeruginosa 11 13.0±0.23 11.0±0.23 10.7±0.17 0 0 P. aeruginosa 12 15.2±0.17 13.7±0.88 11.5±0.50 10.0±0.58 0 P. aeruginosa 13 14.0± 1.00 12.3±0.67 10.5±0.50 10.0±0.00 0 P. aeruginosa 14 14.0±0.00 12.5±0.50 10.5±0.23 0 0 P. aeruginosa 15* 14.0±0.56 12.7±0.17 10.3±0.88 0 0 E. coli 1 11.0±0.00 10.0±0.00 0 0 0 E. coli 2 0 0 0 0 0 S. aureus 1 14.5±0.50 12.5±0.50 11.0±1.00 0 0 S. aureus 2 15.0±0.23 13.5±0.50 11.5±0.50 0 0 Sal. typhi 15.7±0.17 13.0±0.00 12.3±0.34 10.3±0.67 0 n = 3, *ATCC Typed Sample
3.3.4 Effects of Crude Extracts of A. cordifolia Leaves on the Test Organisms
Compared to other extracts, A. cordifolia leave extracts was not very effective against the test organisms even at the high concentration of 40 mg/ml. The ethanolic extract produced inhibition zone diameters ranging from 10.2 mm at 5 mg/ml to 17.0 mm at the concentration of 40 mg/ml against P. aeruginosa isolates.
The aqueous extract did not inhibit the growth of P. aeruginosa isolates. Ethanolic extract inhibited the growth of E. coli 1 and 2 with inhibition zone diameters ranging from 10.0 mm at 10 mg/ml to 12.0 mm at the concentration of 40 mg/ml but the aqueous extract did not inhibit their growth. Ethanolic extract exhibited a high activity against Sal. typhi with inhibition zone diameters ranging from 10.0 mm at 2.5 mg/ml to 17.3 mm at concentration of 40 mg/ml. This was more active than the aqueous extract with inhibition zone diameters ranging from 11.0 mm at 5 mg/ml to 14.3 mm at the concentration of 20 mg/ml. None of the extracts inhibited the growth of S. aureus (Table 9).
Table 9: Inhibition zone diameters (mm) exhibited by ethanolic and aqueous extracts of A. cordifolia leaves on the test organisms Test organism Concentration of extract in mg/ml Ethanolic Extract Aqueous extract 40 20 10 5 2.5 20 10 5 2.5 1.25 P. aeruginosa 1 12.5±0.50 11.5±0.23 10.5±0.23 9.8±0.17 0 0 0 0 0 0 P. aeruginosa 2 12.5± 0.50 11.5±0.23 10.0±0.00 0 0 0 0 0 0 0 P. aeruginosa 3 15.0±0.50 12.7±0.34 10.5±0.23 0 0 0 0 0 0 0 P. aeruginosa 4 13.0±0.50 12.5±0.23 11.2±0.17 10.2±0.00 0 0 0 0 0 0 P. aeruginosa 5 13.0±0.58 10.5±0.50 0 0 0 0 0 0 0 0 P. aeruginosa 6 15.8±0.44 14.5±0.23 11.5±0.23 0 0 0 0 0 0 0 P. aeruginosa 7 12.0±0.58 10.0±0.00 0 0 0 0 0 0 0 0 P. aeruginosa 8 12.5±0.50 11.0±0.00 0 0 0 0 0 0 0 0 P. aeruginosa 9 13.8±0.44 11.7±0.34 10.0±0.00 0 0 0 0 0 0 0 P. aeruginosa 10 12.0±0.00 10.0±0.00 0 0 0 0 0 0 0 0 P. aeruginosa 11 17.0±0.58 15.5±0.50 14.0±0.50 12.0±0.00 0 0 0 0 0 0 P. aeruginosa 12 14.0±0.50 12.3±0.34 10.0±0.00 0 0 0 0 0 0 0 P. aeruginosa 13 16.0±0.23 14.2±0.17 11.8±0.17 0 0 0 0 0 0 0 P. aeruginosa 14 14.0±0.50 12.0±0.23 11.0±0.00 10.0±0.00 0 0 0 0 0 0 P. aeruginosa 15* 14.0±0.50 12.5±0.23 12.0±0.00 11.0±0.00 0 0 0 0 0 0 E. coli. 1 12.0±0.00 11.0±0.00 10.5±0.50 0 0 0 0 0 0 0 E. coli. 2 12.0±0.23 10.8±0.17 10.0±0.00 0 0 0 0 0 0 0 S. aureus 1 0 0 0 0 0 0 0 0 0 0 S. aureus 2 0 0 0 0 0 0 0 0 0 0 Sal. typhi 17.0 ±0.34 16.0±0.23 14.0±0.23 12.0±0.00 10.0 14.3 12.8 11.0 0 0 ±0.23 ±0.34 ±0.17 ±0.00 n = 3, *ATCC Typed Sample
3.3.5 Effects of Crude Extracts of S. mombin Leaves on the Test Organisms
Ethanolic and aqueous extracts of S. mombin leaves did not inhibit the growth of P. aeruginosa at concentrations of 1.25 mg/ml to 20 mg/ml. Similarly, they did not inhibit the growth of E. coli. S. aureus 1 and 2 were not susceptible to ethanolic extract but were susceptible to aqueous extract with inhibition zone diameters ranging from 10.0 mm at 1.25 mg/ml to 17.3 mm at 20 mg/ml. Both extracts were active against Sal. typhi with inhibition zone diameters ranging from 11.2 mm at concentration of 5.0 mg/ml to 14.0 at 20 mg/ml (Tables 10 – 11).
Table 10: Inhibition zone diameters (mm) exhibited by ethanolic extract of S. mombin leaves on the test organisms Test Organism Concen tration of Extra ct in mg/m l 20 10 5 2.5 1.25 P. aeruginosa 0 0 0 0 0
E. coli 1 0 0 0 0 0
E. coli 2 0 0 0 0 0
S. aureus 1 0 0 0 0 0
S. aureus 2 0 0 0 0 0
Sal. typhi 12.5±0.50 1 2 . 0 ± 0.00 10.5±0.23 0 0 n=3
Table 11: Inhibition zone diameters (mm) exhibited by aqueous extract of S. mombin leaves on the test organisms Test Organism Concen tration of Ext ract in mg/m l 20 10 5 2.5 1.25 P.aeruginosa 0 0 0 0 0
E. coli 1 0 0 0 0 0
E. coli 2 0 0 0 0 0
S. aureus 1 17.3±0.50 15.3±0.34 14.0±0.23 12.0±0.00 10.0±0.00
S. aureus 2 16.5±0.23 15.0±0.00 13.0±0.00 11.5±0.50 0
Sal. typhi 14.0±0.58 13.0±0.50 11.2±0.17 0 0 n=3
2
3.4.0 Results of antimicrobial screening of soluble fractions of T. schimperiana root bark against the test organisms
Separation of the crude ethanolic extract of T. schimperiana yielded five soluble fractions. The masses of the fractions obtained were: n-hexane-soluble fraction 77 mg, chloroform-soluble fraction 95 mg, ethylacetate-soluble fraction 15 g, acetone-soluble fraction 21 g and ethanol-soluble fraction 83 mg.
The results of the antimicrobial screening of the soluble fractions revealed that the ethylacetate and acetone - soluble fractions were active against the test organisms.
The n-hexane, chloroform and ethanol - soluble fractions did not show significant activity.
6
3.4.1 Effects of Ethylacetate-soluble Fraction of Terminalia schimperiana Root
Bark on the Test Organisms
Out of the five soluble extracts, the ethylacetate-soluble fraction was found to have the highest activity against P. aeruginosa isolates. Seven of the isolates were susceptible to the lowest concentration of the fraction used (1.25 mg/ml) while the rest were susceptible up to a concentration of 2.5 mg/ml. The inhibition zone diameters ranged from 10.0 mm at 1.25 mg/ml to 20 mm at concentration of 20 mg/ml (Table 12).
=
Table 12: Inhibition zone diameters (mm) exhibited by ethylacetate-soluble fraction of T. schimperiana root bark on the test organisms Test organism Concentration of fraction in mg/ml 20 10 5 2.5 1.25 P. aeruginosa 1 17.5±0.50 15.5±0.50 13.7±0.34 12.0±0.58 10.0±0.50
P. aeruginosa 2 16.5±0.87 14.5±0.50 12.5±0.50 11.0±0.23 9.5±0.50
P. aeruginosa 3 15.0±0.58 13.0±1.00 11.5±0.50 10.0±0.00 0
P. aeruginosa 4 17.5±0.23 15.7±0.50 13.3±0.67 11.5±0.50 10.0±0.58
P. aeruginosa 5 17.0±0.50 15.2±0.17 13.0±0.00 11.5±0.50 9.5±0.50
P. aeruginosa 6 16.0±0.58 4.0 ±.50 12.0±0.23 10.0±1.00 0
P. aeruginosa 7 16.5±0.87 14. ±0.50 12.5±0.50 10.5±0.50 0
P. aeruginosa 8 17.5±0.50 15. ±0.23 13.3±0.67 11.7±0.93 10.0±0.00
P. aeruginosa 9 16.2±0.17 14. 0±0.0 12.0±0.00 10.0±0.00 0
P. aeruginosa 10 16.0±1.00 13.5±0.50 12.0±1.00 10.5±0.50 0
P. aeruginosa 11 16.0±0.50 14.7±0.88 12.5±0.50 10.5±0.50 0
P. aeruginosa 12 20.0±0.58 17.5±0.50 16.0±0.00 14.0±0.00 11.7±0.17
P. aeruginosa 13 19.0±0.00 16.7±0.44 15.0±0.00 13.0±0.00 11.7±0.93
P. aeruginosa 14 16.0±0.23 14.0±0.00 12.3±0.34 10.2±0.17 0
P. aeruginosa 15* 16.0±0.00 14.0±1.00 12.2±0.17 10.3±0.67 0 n = 3, *ATCC Typed Sample
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3.4.2 Effects of Acetone- soluble Fraction of Terminalia schimperiana Root Bark on the Test Organisms
The acetone-soluble fraction was next to ethylacetate-soluble fraction in its activity against P. aeruginosa isolates. All the isolates were susceptible up to a concentration of 5.0 mg/ml, while seven were susceptible to the low concentration of
2.5 mg/ml and two isolates were susceptible to the lowest concentration of the fraction used (1.25 mg/ml). The inhibition zone diameters ranged from 10.0 mm at
1.25 mg/ml to 18 mm at concentration of 20 mg/ml (Table 13).
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Table 13: Inhibition zone diameters (mm) exhibited by acetone-soluble fraction of T. schimperiana root bark on the test organisms Test organism Concentration of fraction in mg/ml 20 10 5 2.5 1.25 P. aeruginosa 1 14.5±0.50 12.5±0.50 10.5±0.50 0 0
P. aeruginosa 2 13.0±1.00 11.0±1.00 10.0±0.00 0 0
P. aeruginosa 3 14.5±0.50 12.5±0.23 10.5±0.89 0 0
P. aeruginosa 4 17.5±0.50 15.3±0.34 13.5±0.23 11.8±0.44 10.0±0.58
P. aeruginosa 5 15.0±0.58 13.0±0.50 11.0±0.00 10.0±0.00 0
P. aeruginosa 6 15.0±0.56 13.0±0.50 11.0±1.00 10.0±0.50 0
P. aeruginosa 7 14.0±0.00 12.0±0.00 10.0±0.00 0 0
P. aeruginosa 8 15.0±1.00 13.0±1.00 11.0±0.56 10.0±0.50 0
P. aeruginosa 9 15.0±0.00 12.50.50 10.5±0.50 0 0
P. aeruginosa 10 16.0±0.58 14.0±0.50 12.0±0.23 10.0±1.00 0
P. aeruginosa 11 14.0±0.00 11.7±0.17 10.0±0.00 0 0
P. aeruginosa 12 16.0±0.00 14.0±0.00 12.3±0.34 10.2±0.17 0
P. aeruginosa 13 18.0±0.23 16.3±0.67 14.0±0.50 12.2±0.17 10.0±0.00
P. aeruginosa 14 14.0±0.50 12.0±0.50 10.5±0.50 0 0
P. aeruginosa 15* 14.0±0.00 12.0±0.00 10.2±0.17 0 0
n = 3, *ATCC Typed Sample
3.5.0 Results of Antimicrobial Screening of TLC Fractions of Ethylacetate- soluble Fraction against the Test Organisms
Due to its high antibacterial activity, the ethylacetate-soluble fraction was selected for further fractionation using Thin Layer Chromatography (TLC). The solvent system used was ethylacetale: n-hexane: water (6: 1 : 0.5). Fractionation of the ethylacetate–soluble fraction yielded twelve fractions labeled F1 to F12. After antimicrobial screening of these fractions, it was found that F1, F7 and F9 exhibited activity against the test organisms.
3.5.1 Effects of F1 Fraction on the Test Organisms
F1 inhibited the growth of P. aeruginosa isolates at the concentrations of 20 mg/ml to 5 mg/ml. The inhibition zone diameters (IZD) ranged from 10.0 mm to 14.0 mm. No inhibition occurred at concentrations of 2.5 mg/ml and 1.25 mg/ml. F1 also inhibited the growth of E. coli 1 and 2, with inhibition zone diameters ranging from
10.5 mm at 5.0 mg/ml to 14.3 mm at concentration of 20 mg/ml. F1 exhibited a high activity against S. aureus 1 and 2 with inhibition zone diameters ranging from 10.0 mm at 2.5 mg/ml to 16.3 mm at 20 mg/ml. F1 was most effective on Sal. typhi even at the lowest concentration used, with inhibition zone diameters ranging from 10.0 mm at 1.25 mg/ml to 18.5 mm at 20 mg/ml (Table 14).
Table 14: Inhibition zone diameters (mm) exhibited by F1 of Ethylacetate- soluble Fraction of T. schimperiana root bark on the test organisms Test organism Concentration of F1 in mg/ml 20 10 5 2.5 1.25 P. aeruginosa 1 11.5±0.23 10.2±0.17 0 0 0 P. aeruginosa 2 13.3±0.67 12.0±0.50 10.0±0.00 0 0 P. aeruginosa 3 13.0±0.50 11.8±0.44 10.3±0.34 0 0 P. aeruginosa 4 14.0±0.23 12.5±0.00 10.5±0.50 0 0 P. aeruginosa 5 10.5±0.50 0 0 0 0 P. aeruginosa 6 11.2±0.17 10.0±0.50 0 0 0 P. aeruginosa 7 10.7±0.88 10.0±0.50 0 0 0 P. aeruginosa 8 0 0 0 0 0 P. aeruginosa 9 12.7±0.34 11.0±0.50 0 0 0 P. aeruginosa 10 11.8±0.44 10.0±0.00 0 0 0 P. aeruginosa 11 12.5±.866 10.5±0.50 0 0 0 P. aeruginosa 12 11.3±0.34 10.3±0.67 0 0 0 P. aeruginosa 13 11.2±0.17 10.5±0.23 0 0 0 P. aeruginosa 14 10.0±0.00 0 0 0 0 P. aeruginosa 15* 12.0±0.00 10.5±0.23 0 0 0 E. coli 1 12.0±0.23 10.2±0.17 0 0 0 E. coli 2 14.3±0.34 12.5±0.50 10.5±0.50 0 0 S. aureus 1 16.3±0.34 14.5±0.23 12.5±0.50 10.5±0.50 0 S. aureus 2 16.0±0.00 13.8±0.44 12.0±0.23 10.0±0.17 0 Sal. typhi 18.5±0.50 16.5±0.58 14.5±0.50 12.0±0.23 10.0±0.00 n = 3, *ATCC Typed Sample
3.5.2 Effects of F7 Fraction on the Test Organisms
All the P. aeruginosa isolates were inhibited by F7 fraction at 20 mg/ml, thirteen isolates were inhibited at 10 mg/ml and four isolates were inhibited at 5.0 mg/ml. The inhibition zone diameters ranged from 10.2 mm to 13.7 mm. F7 slightly inhibited the growth of S. aureus 1 and 2 and Sal. typhi with inhibition zone diameters of 10.0 mm, 11.0 mm and 12.3 mm respectively at concentration of 20 mg/ml, and 10.0 mm to 10.3 mm at 10 mg/ml. The fraction did not inhibit the growth of E. coli. F7 fraction seems to be less effective on the isolates than the previous fractions tested (Table 15).
Table 15: Inhibition zone diameters (mm) exhibited by F7 of Ethylacetate- soluble Fraction of T. schimperiana root bark on the test organisms Test organism Concentration of F7 in mg/ml 20 10 5 2.5 1.25
P. aeruginosa 1 13.2±0.17 11.5±0.67 10.0±0.00 0 0 P. aeruginosa 2 12.3±0.67 10.8±0.44 0 0 0 P. aeruginosa 3 13.2±0.34 11.3±0.34 10.0±0.50 0 0 P. aeruginosa 4 13.7±0.17 12.0±.50 10.3±0.67 0 0 P. aeruginosa 5 10.5±0.50 0 0 0 0 P. aeruginosa 6 11.5±0.50 10.0±0.50 0 0 0 P. aeruginosa 7 12.0±0.50 10.5±0.50 0 0 0 P. aeruginosa 8 10.2±0.17 0 0 0 0 P. aeruginosa 9 13.0±.00 12.0±0.00 10.2±0.17 0 0 P. aeruginosa 10 12.0±0.50 10.3±0.67 0 0 0 P. aeruginosa 11 11.0±0.00 10.0±0.00 0 0 0 P. aeruginosa 12 11.5±0.50 10.5±0.50 0 0 0 P. aeruginosa 13 11.2±0.16 10.0±0.50 0 0 0 P. aeruginosa 14 10.8±0.44 10.0±0.23 0 0 0 P. aeruginosa 15* 12.5±0.50 10.5±0.50 0 0 0 E. coli 1 0 0 0 0 0 E. coli 2 0 0 0 0 0 S. aureus 1 11.0±0.50 10.0±0.23 0 0 0 S. aureus 2 10.0±0.00 0 0 0 0 Sal. typhi 12.3±0.34 10.3±0.17 0 0 0 n = 3, *ATCC Typed Sample
3.5.3 Effects of F9 Fraction on the Test Organisms
F9 inhibited the growth of twelve P. aeruginosa isolates at 20 mg/ml and 10 mg/ml, six isolates at 5.0 mg/ml and one isolate at 2.5 mg/ml. The inhibition zone diameters ranged from 10.5 mm to 15.0 mm. F9 is most effective on P. aeruginosa isolates than on the other test organisms. It exhibited a low activity against S. aureus
1 and 2 with inhibition zone diameters ranging from 10.7 mm at 10 mg/ml to 12.0 mm at 20 mg/ml and against Sal. typhi with inhibition zone diameters ranging from 10.5 mm at 10 mg/ml to 12.0 mm at 20 mg/ml. It did not inhibit the growth of E. coli
(Table 16).
2
Table 16: Inhibition zone diameters (mm) exhibited by F9 of Ethylacetate- soluble Fraction of T. schimperiana root bark on the test organisms Test organism Concentration of F9 in mg/ml 20 10 5 2.5 1.25
P. aeruginosa 1 13.5±0.50 11.8±0.4 10.5±0.50 0 0 P. aeruginosa 2 15.0±0.00 13.7±0.34 11.5±0.23 10.5±0.50 0 P. aeruginosa 3 14.0±0.58 12.3±0.67 10.3±0.67 0 0 P. aeruginosa 4 12.5±0.50 10.7±0.34 10.0±0.00 0 0 P. aeruginosa 5 0 0 0 0 0 P. aeruginosa 6 12.5±0.50 10.7±0.17 0 0 0 P. aeruginosa 7 13.5±0.23 11.8±0.44 0 0 0 P. aeruginosa 8 13.0±0.00 12.0±0.00 10.5±0.50 0 0 P. aeruginosa 9 10.0±0.00 0 0 0 0 P. aeruginosa 10 0 0 0 0 0 P. aeruginosa 11 13.5±0.50 11.8±0.44 10.0±0.23 0 0 P. aeruginosa 12 12.0±0.00 10.7±0.17 0 0 0 P. aeruginosa 13 11.5±0.50 10.5±0.50 0 0 0 P. aeruginosa 14 12.5±0.50 10.7±0.34 0 0 0 P. aeruginosa 15* 0 0 0 0 0 E. coli 1 0 0 0 0 0 E. coli 2 0 0 0 0 0 S. aureus 1 10.5±0.50 0.00±0.00 0 0 0 S. aureus 2 12.0±0.00 10.7±0.34 0 0 0 Sal. typhi 12.0±0.34 10.5±0.17 0 0 0 n = 3, *ATCC Typed Sample
6
3.6.0 Minimum Inhibitory Concentration (MIC) values of Crude Extracts and
Gentamicin Standard on Pseudomonas Isolates and other Bacteria
3.6.1 MIC values of Gentamicin Standard
The gentamicin standard exhibited activity against eight (8) out of 15 P. aeruginosa isolates. The minimum inhibitory concentration (MIC) values ranged from
0.200 µg/ml –1.995 µg/ml. MIC values for E. coli 1 and 2 were 0.955 µg/ml and
0.912 µg/ml respectively. MIC values of S. aureus 1 and 2 were 1.585 µg/ml and
1.047 µg/ml respectively. MIC value for Sal. typhi was 1.472 µg/ml (Table 17).
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Table 17: Minimum inhibitory concentrations (g/ml) of gentamicin standard on the test organisms. Test organism C MIC Organisms Mean SEM (Antilog C)
P. aeruginosa 1 -0.55 0.200 P. aeruginosa 0.521 0.08 P. aeruginosa 2 0.08 1.109 E. coli 0.933 0.01 P. aeruginosa 3 - NI S. aureus 1.316 0.55 P. aeruginosa 4 - NI Sal. typhi 1.472 - P. aeruginosa 5 - NI P. aeruginosa 6 0.09 1.160 P. aeruginosa 7 -0.16 0.691 P. aeruginosa 8 -0.16 0.692 P. aeruginosa 9 - NI P. aeruginosa 10 - NI P. aeruginosa 11 - NI P. aeruginosa 12 - NI P. aeruginosa 13 0.12 1.205 P. aeruginosa 14 -0.12 0.759 P. aeruginosa 15* 0.29 1.995 E. coli 1 -0.02 0.955 E. coli 2 -0.04 0.912 S. aureus 1 0.2 1.585 S. aureus 2 0.2 1.047 Sal. typhi 0.168 1.472 KEY: C- intercept on log drug conc axis, * ATCC Typed Sample, NI- No Inhibition
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3.6.2 MIC values of crude extracts of T. schimperiana root bark
For the crude ethanolic extract of T. schimperiana root bark, minimum inhibitory concentration (MIC) values for P. aeruginosa isolates ranged from 0.282 mg/ml to 2.512 mg/ml. MIC values for S. aureus 1 and 2 were 1.549 mg/ml and 0.689 mg/ml respectively, and MIC value for Sal. typhi was 0.689 mg/ml. However, no activity was exhibited agianst E. coli 1 and 2 (Table 18). For aqueous extract of T. schimperiana root bark, MIC values for P. aeruginosa ranged from 0.054 mg/ml to
1.622 mg/ml, indicating a very high activity (Table 19).
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Table 18: Minimum inhibitory concentrations (mg/ml) of ethanolic extract of T. schimperiana root bark on the test organisms. Test organism MIC Mean SEM C (Antilog C) Organisms MIC
P. aeruginosa 1 0.21 1.622 P. aeruginosa 1.529 0.15 P. aeruginosa 2 0.31 2.042 E. coli NI - P. aeruginosa 3 -0.03 0.933 S. aureus 1.119 0.43 P. aeruginosa 4 0.21 1.622 Sal. typhi 0.689 - P. aeruginosa 5 0.12 1.318 P. aeruginosa 6 0.32 2.089 P. aeruginosa 7 0.32 2.089 P. aeruginosa 8 -0.04 0.912 P. aeruginosa 9 0.4 2.512 P. aeruginosa 10 0.21 1.622 P. aeruginosa 11 0.32 2.089 P. aeruginosa 12 0 1.000 P. aeruginosa 13 -0.55 0.282 P. aeruginosa 14 0.19 1.549 P. aeruginosa 15* 0.097 1.250 E. coli 1 - NI E. coli 2 - NI S. aureus 1 0.19 1.549 S. aureus 2 -0.162 0.689 Sal. typhi -0.162 0.689 KEY: C- intercept on log drug conc axis, * ATCC Typed Sample, NI- No Inhibition
Table 19: Minimum inhibitory concentrations (mg/ml) of aqueous extract of T. shimperiana root bark on the test organisms. Organisms C MIC Organisms Mean SEM (Antilog of C) MIC
P. aeruginosa 1 0.175 1.496 P. aeruginosa 0.988 0.614 P. aeruginosa 2 -1.270 0.054 P. aeruginosa 3 -0.750 0.178 P. aeruginosa 4 0.190 1.549 P. aeruginosa 5 0.190 1.549 P. aeruginosa 6 -0.040 0.912 P. aeruginosa 7 -1.020 0.095 P. aeruginosa 8 -0.980 0.105 P. aeruginosa 9 0.016 1.038 P. aeruginosa 10 0.210 1.622 P. aeruginosa 11 0.200 1.585 P. aeruginosa 12 0.168 1.472 P. aeruginosa 13 -0.030 0.933 P. aeruginosa 14 -0.120 0.759 P. aeruginosa 15* 0.168 1.472 KEY: C- intercept on log drug conc axis, * ATCC Typed Sample, NI- No Inhibition
3.6.3 MIC values of crude ethanolic extract of T. schimperiana leaves
For T. schimperiana leaves, minimum inhibitory concentration (MIC) values for P. aeruginosa isolates ranged from 0.056 mg/ml to 1.585 mg/ml. MIC values for
E. coli 1 was 2.089 mg/ml. MIC values for S. aureus 1 and 2 were 0.832 mg/ml and
1.462 mg/ml respectively. MIC value for Sal. typhi was 0.912 mg/ml (Table 20).
Table 20: Minimum inhibitory concentrations (mg/ml) of ethanolic extract of T. shimperiana leaves on the test organisms. Test organism C MIC Organisms Mean MIC SEM (Antilog C) P. aeruginosa 1 0.2 1.585 P. aeruginosa 1.061 0.13 P. aeruginosa 2 0.165 1.462 E. coli 1.545 0.55 P. aeruginosa 3 -0.8 0.158 S. aureus 1.147 0.32 P. aeruginosa 4 -1.25 0.056 Sal. typhi 0.912 - P. aeruginosa 5 -0.16 0.692 P. aeruginosa 6 -0.165 0.684 P. aeruginosa 7 0.165 1.462 P. aeruginosa 8 0.165 1.462 P. aeruginosa 9 0.2 1.585 P. aeruginosa 10 0.2 1.585 P. aeruginosa 11 0.16 1.445 P. aeruginosa 12 0.02 1.047 P. aeruginosa 13 -0.05 0.891 P. aeruginosa 14 -0.045 0.902 P. aeruginosa 15* -0.045 0.902 E. coli 1 0.32 2.089 E. coli 2 - NI S. aureus 1 -0.08 0.832 S. aureus 2 0.165 1.462 Sal. typhi -0.04 0.912 KEY: C- intercept on log drug conc axis, * ATCC Typed Sample, NI- No Inhibition
3.6.4 MIC values of Crude extracts of A. cordifolia leaves
For ethanolic extract of A. cordifolia leaves, minimum inhibitory concentration (MIC) values for P. aeruginosa ranged from 0.603 mg/ml to 4.365 mg/ml. MIC values for E. coli 1 and 2 were 2.754 mg/ml and 3.020 mg/ml respectively. MIC value for Sal. typhi was 0.602 mg/ml. No activity was exhibited against S. aureus 1 and 2. For aqueous extract of A. cordifolia, MIC value for Sal. typhi was 0.912 mg/ml. No activity was exhibited against P. aeruginosa, E. coli and
S. aureus (Tables 21 and 22).
Table 21: Minimum inhibitory concentrations (mg/ml) of ethanolic extract of A. cordifolia leaves on the test organisms. Test Organism C MIC Organisms Mean SEM (Antilog C) MIC
P. aeruginosa 1 0.12 1.318 P. aeruginosa 2.733 0.30 P. aeruginosa 2 0.48 3.020 E. coli 2.887 0.13 P. aeruginosa 3 0.52 3.311 S. aureus NI - P. aeruginosa 4 0.16 1.445 Sal. typhi 0.602 - P. aeruginosa 5 0.64 4.365 P. aeruginosa 6 0.485 3.055 P. aeruginosa 7 0.48 3.020 P. aeruginosa 8 0.61 4.074 P. aeruginosa 9 0.482 3.034 P. aeruginosa 10 0.64 4.365 P. aeruginosa 11 -0.22 0.603 P. aeruginosa 12 0.486 3.062 P. aeruginosa 13 0.485 3.055 P. aeruginosa 14 0.29 1.950 P. aeruginosa 15* 0.12 1.318 E. coli 1 0.44 2.754 E. coli 2 0.48 3.020 S. aureus 1 - NI S. aureus 2 - NI Sal. typhi 0.24 0.602 KEY: C- intercept on log drug conc axis, * ATCC Typed Sample, NI- No Inhibition
2
Table 22: Minimum inhibitory concentrations (mg/ml) of aqueous extract of A. cordifolia leaves on the test organisms.
Test Organism C MIC (Antilog C) Organisms Mean MIC SEM P. aeruginosa - NI P. aeruginosa NI - E. coli 1 - NI E. coli NI - E. coli 2 - NI S. aureus NI - S. aureus 1 - NI Sal. typhi 0.912 - S. aureus 2 - NI Sal. typhi -0.04 0.912 KEY: C-intercept on log drug conc axis,* ATCC Typed Sample, NI- No Inhibition
6
3.6.5 MIC values of Crude extracts of S. mombin leaves
For ethanolic extract of S. mombin leaves, minimum inhibitory concentration
(MIC) value for Sal. typhi was 1.445 mg/ml. No activity was exhibited against P. aeruginosa, E. coli and S. aureus. For aqueous extract of S. mombin, MIC values for
S. aureus 1 and 2 were 0.331 mg/ml and 0.912 mg/ml respectively. MIC values for
Sal. typhi was 1.514 mg/ml. No activity was exhibited against P. aeruginosa and E. coli (Tables 23 and 24).
=
Table 23: Minimum inhibitory concentrations (mg/ml) of ethanolic extract of Spondias mombin leaves on the test organisms. Test C MIC Organisms Mean S EM Organism (Antilog C) MIC
P. aeruginosa - NI P. aeruginosa NI -
E. coli 1 - NI E. coli NI -
E. coli 2 - NI S. aureus NI -
S. aureus 1 - NI Sal. typhi 1.445 -
S. aureus 2 - NI
Sal. typhi 0.16 1.445
KEY: C- intercept on log drug conc axis,* ATCC Typed Sample, NI- No Inhibition
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Table 24: Minimum inhibitory concentrations (mg/ml) of aqueous extract of Spondias mombin leaves on the test organisms.
Test Organism C MIC Organisms Mean SEM (Antilog C) MIC P. aeruginosa - NI P. aeruginosa NI -
E. coli 1 - NI E. coli NI -
E. coli 2 - NI S. aureus 0.622 0.29
S. aureus 1 -0.48 0.331 Sal. typhi 1.514 -
S. aureus 2 -0.04 0.912
Sal. typhi 0.18 1.514
KEY: C- intercept on log drug conc axis, * ATCC Typed Sample, NI- No Inhibition
+
3.7.0 Minimum Inhibitory Concentration (MIC) values of T. schimperiana Root Bark Soluble Fractions. 3.7.1 MIC values of Ethylacetate-soluble fraction
For ethylacetate-soluble fraction, minimum inhibitory concentration (MIC) values ranged from 0.263 mg/ml to 1.000 mg/ml against P. aeruginosa isolates, indicating a very high antimicrobial activity (Table 25).
Table 25: Minimum inhibitory concentrations (mg/ml) of ethylacetate-soluble fraction of T. schimperiana root bark on the test organisms. Test Organism C MIC Organisms Mean SEM (Antilog C) MIC P. aeruginosa 1 -0.48 0.331 P. aeruginosa 0.690 0.09 P. aeruginosa 2 -0.44 0.363 P. aeruginosa 3 -0.04 0.912 P. aeruginosa 4 -0.4 0.398 P. aeruginosa 5 -0.4 0.398
P. aeruginosa 6 0 1.000 P. aeruginosa 7 0 1.000 P. aeruginosa 8 -0.4 0.398 P. aeruginosa 9 0 1.000 P. aeruginosa 10 0 1.000 P. aeruginosa 11 0 1.000 P. aeruginosa 12 -0.54 0.288 P. aeruginosa 13 -0.58 0.263 P. aeruginosa 14 0 1.000 P. aeruginosa 15* 0 1.000 KEY: C- intercept on log drug conc axis,* ATCC Typed Sample, NI- No Inhibition
3.7.2 MIC values of Acetone-soluble fraction
For acetone-soluble fraction, MIC values for P. aeruginosa ranged from 0.363 mg/ml to 1.660 mg/ml. These MIC values indicate a high antipseudomonas activity by this fraction (Table 26).
Table 26: Minimum inhibitory concentrations (mg/ml) of acetone-soluble fraction of T. schimperiana root bark on the test organisms.
Test Organism C MIC Organisms Mean SEM (Antilog C) MIC
P. aeruginosa 1 0.2 1.585 P. aeruginosa 1.275 0.12 P. aeruginosa 2 0.19 1.549 P. aeruginosa 3 0.2 1.585 P. aeruginosa 4 -0.44 0.363 P. aeruginosa 5 -0.02 0.955 P. aeruginosa 6 0.16 1.445 P. aeruginosa 7 0.2 1.585 P. aeruginosa 8 0.16 1.445 P. aeruginosa 9 0.22 1.660 P. aeruginosa 10 0 1.000 P. aeruginosa 11 0.2 1.585 P. aeruginosa 12 0 1.000 P. aeruginosa 13 -0.4 0.398 P. aeruginosa 14 0.14 1.380 P. aeruginosa 15* 0.2 1.585 KEY: C- intercept on log drug conc axis, * ATCC Typed Sample,
3.7.3 MIC values of F1 of Ethylacetate-soluble fraction
For F1, minimum inhibitory concentration (MIC) values for P. aeruginosa ranged from 1.318 mg/ml to 2.512 mg/ml with one strain showing resistance. MIC values for E. coli 1 and 2 were 1.585 mg/ml and 1,995 mg/ml respectively. MIC values for both S. aureus 1 and 2 was 1.00 mg/ml and MIC value for Sal. typhi was
0.437 mg/ml (Table 27).
Table 27: Minimum inhibitory concentrations (mg/ml) of F1 of ethylacetate-soluble fraction of T. schimperiana root bark on the test organisms.
Test Organism C MIC Organisms Mean S E M (Antilog C) MIC P. aeruginosa 1 0.12 1.318 P. aeruginosa 1.898 0.11 P. aeruginosa 2 0.199 1.581 E. coli 1.790 0.21 P. aeruginosa 3 0.185 1.531 S. aureus 1.000 0 P. aeruginosa 4 0.2 1.585 Sal. typhi 0.437 - P. aeruginosa 5 0.4 2.512 P. aeruginosa 6 0.32 2.089 P. aeruginosa 7 0.3 1.995 P. aeruginosa 8 - NI P. aeruginosa 9 0.32 2.089 P. aeruginosa 10 0.32 2.089 P. aeruginosa 11 0.32 2.089 P. aeruginosa 12 0.3 1.995 P. aeruginosa 13 0.3 1.995 P. aeruginosa 14 0.4 2.512 P. aeruginosa 15* 0.32 2.089 E. coli 1 0.3 1.995 E. coli 2 0.2 1.585 S. aureus 1 0 1.000 S. aureus 2 0 1.000 Sal. typhi 0.36 0.437 KEY: C-intercept on log drug conc axis, * ATCC Typed Sample, NI- No Inhibition
2
3.7.4 MIC values of F7 of Ethylacetate-soluble fraction
For F7, minimum inhibitory concentration (MIC) values for P. aeruginosa ranged from 1.445 mg/ml to 2.512 mg/ml. MIC values for S. aureus 1 and 2 were
2.089 and 2.512 mg/ml respectively and MIC values for Sal. typhi was 2.089 mg/ml.
E. coli 1 and 2 were resistant to F7 (Table 28).
6
Table 28: Minimum inhibitory concentrations (mg/ml) of F7 of ethylacetate- soluble fraction of T. schimperiana root bark on the test organisms.
Test Organism C MIC Organisms Mean SEM (Antilog C) MIC P. aeruginosa 1 0.2 1.585 P. aeruginosa 1.950 0.09 P. aeruginosa 2 0.16 1.445 E. coli NI - P. aeruginosa 3 0.19 1.549 S. aureus 2.301 0.21 P. aeruginosa 4 0.2 1.585 Sal. typhi 2.089 - P. aeruginosa 5 0.4 2.512 P. aeruginosa 6 0.32 2.089 P. aeruginosa 7 0.32 2.089 P. aeruginosa 8 0.4 2.512 P. aeruginosa 9 0.16 1.445 P. aeruginosa 10 0.32 2.089 P. aeruginosa 11 0.32 2.089 P. aeruginosa 12 0.32 2.089 P. aeruginosa 13 0.32 2.089 P. aeruginosa 14 0.3 1.995 P. aeruginosa 15* 0.32 2.089 E. coli 1 - NI E. coli 2 - NI S. aureus 1 0.32 2.089 S. aureus 2 0.4 2.512 Sal. typhi 0.32 2.089 KEY: C- intercept on log drug conc axis, * ATCC Typed Sample, NI- No Inhibition
=
3.7.5 MIC values of F9 of Ethylacetate-soluble fraction
For F9, minimum inhibitory concentration (MIC) values for 12 P. aeruginosa ranged from 0.912 mg/ml to 2.089 mg/ml while 3 isolates showed resistance. MIC values for S. aureus 1 and 2 were 2.089 mg/ml and 2.512 mg/ml respectively. MIC value for Sal. typhi was 2.089 mg/ml. E. coli showed resistance to F9 (Table 29).
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Table 29: Minimum inhibitory concentrations (mg/ml) of F9 fraction of ethylacetate fraction of T. schimperiana root bark on the test organisms.
Test Organism C MIC Organisms Mean SEM (Antilog C) MIC
P. aeruginosa 1 0.168 1.472 P. aeruginosa 1.488 0.11 P. aeruginosa 2 -0.04 0.912 E. coli - - P. aeruginosa 3 0.2 1.585 S. aureus 2.301 0.2 P. aeruginosa 4 0.162 1.452 Sal. typhi 2.089 - P. aeruginosa 5 - NI P. aeruginosa 6 0.32 2.089 P. aeruginosa 7 0.18 1.514 P. aeruginosa 8 0.16 1.445 P. aeruginosa 9 0.4 2.512 P. aeruginosa 10 - NI P. aeruginosa 11 0.2 1.585 P. aeruginosa 12 0.32 2.089 P. aeruginosa 13 0.32 2.089 P. aeruginosa 14 0.32 2.089 P. aeruginosa 15* - NI E. coli 1 - NI E. coli 2 - NI S. aureus 1 0.4 2.512 S. aureus 2 0.32 2.089 Sal. typhi 0.32 2.089 KEY: C- intercept on log drug conc axis, * ATCC Typed Sample, NI- No Inhibition
+
Summary of minimum inhibitory concentration (MIC) values of all the extracts for the test organisms A summary of the minimum inhibitory concentration (MIC) values of all the extracts and gentamicin is presented in Table 30. There is an inverse relationship between MIC values and antimicrobial activity. If MIC is low, it shows that activity is high. The results revealed that the extracts exhibited varying levels of activity against the test organisms. Extracts of T. schimperiana inhibited the growth of P. aeruginosa,
S. aureus and Sal. typhi but did not inhibit the growth of E. coli. Extracts of A. cordifolia only slightly inhibited the growth of P. aeruginosa, E. coli and S. aureus but significantly inhibited the growth of Sal. typhi. Extracts of S. mombin did not inhibit the growth of P. aeruginosa and E. coli but significantly inhibited the growth of S. aureus and Sal. typhi.
Figure 5 graphically portrays the MIC values of the four active extracts and gentamicin against 14 P. aeruginosa isolates and the typed sample. The figure clearly shows that aqueous extract of T. schimperiana root bark (Tsaq) had the lowest mean
MIC value, indicating the highest antimicrobial activity, while A. cordifolia ethanolic extract (Aset) had the highest mean MIC value which indicates the lowest activity among the extracts. Gentamicin standard exceeded the activity of the plant extracts.
Table 30: Summary of MIC values (mg/ml) of all the plant extracts (mg/ml) and gentamicin (µg/ml) on the test organisms
Test organism Tset Tsaq Tslet Acet Acaq Smet Smaq Gen
P. aeruginosa 1 1.622 1.496 1.585 1.318 NI NI NI 0.200 P. aeruginosa 2 2.042 0.054 1.462 3.020 NI NI NI 1.109 P. aeruginosa 3 0.933 0.178 0.158 3.311 NI NI NI NI P. aeruginosa 4 1.622 1.549 0.056 1.445 NI NI NI NI P. aeruginosa 5 1.318 1.549 0.692 4.365 NI NI NI NI P. aeruginosa 6 2.089 0.912 0.684 3.055 NI NI NI 1.160 P. aeruginosa 7 2.089 0.095 1.462 3.020 NI NI NI 0.691 P. aeruginosa 8 0.912 0.105 1.462 4.074 NI NI NI 0.692 P. aeruginosa 9 2.512 1.038 1.585 3.034 NI NI NI NI P. aeruginosa 10 1.622 1.622 1.585 4.365 NI NI NI NI P. aeruginosa 11 2.089 1.585 1.445 0.603 NI NI NI NI P. aeruginosa 12 1.000 1.472 1.047 3.062 NI NI NI NI P. aeruginosa 13 0.282 0.933 0.891 3.055 NI NI NI 1.205 P. aeruginosa 14 1.549 0.759 0.902 1.950 NI NI NI 0.759 P. aeruginosa 15* 1.250 1.472 0.902 1.318 NI NI NI 1.995 E. coli. 1 NI - 2.089 2.754 NI NI NI 0.955 E. coli. 2 NI - NI 3.020 NI NI NI 0.912 S. aureus 1 1.549 - 0.832 NI NI NI 0.331 1.585 S. aureus 2 0.689 - 1.462 NI NI NI 0.912 1.047 Sal. typhi 0.689 - 0.912 0.602 0.912 1.445 1.514 1.472 KEY: * ATCC Typed Sample, Tset- T. schimperiana root bark ethanolic extract, Tsaq- T. schimperiana root bark aqueous extract, Tslet- T. schimperiana leaf ethanolic extract, Acet- A. cordifolia leaf ethanolic extract, Acaq- A. cordifolia leaf aqueous extract, Smet- S. mombin leaf ethanolic extract, Smaq- S. mombin leaf aqueous extract, - Not tested, NI- Not inhibition.
Figure 5: Graphic representation of MIC values of the plant extracts and gentamicin for 14 P. aeruginosa isolates and the typed sample
Summary of minimum inhibitory concentration (MIC) values of the fractions for the test organisms A summary of the minimum inhibitory concentration (MIC) values of all the active fractions and gentamicin is presented in Table 31. The results revealed that the fractions exhibited varying levels of activity against the test organisms. Ethylacetate- soluble fraction and acetone-soluble fraction of T. schimperiana root bark significantly inhibited the growth of P. aeruginosa isolates (mean MIC= 0.690 mg/ml and 1.275 mg/ml respectively). The TLC fractions F1, F7 and F9 inhibited the growth of most of the P. aeruginosa isolates, S. aureus and S. typhi. Only F1 inhibited the growth of E. coli.
Figure 6 graphically portrays the MIC values of the five active fractions and gentamicin against 14 P. aeruginosa isolates and the typed sample. The figure clearly shows that (EASF) had the lowest mean MIC value, indicating the highest antimicrobial activity, while F7 had the highest mean MIC value which indicates the lowest activity among the fractions.
Table 31: MIC values (mg/ml) of ethylacetate-soluble fraction (EASF), F1, F7, F9 fractions of EASF, Acetone-soluble fraction and MIC of gentamicin (µg/ml) on the test organisms
Test organism EASF ASF F1 F7 F9 Gen
P. aeruginosa 1 0.331 1.585 1.318 1.585 1.472 0.200 P. aeruginosa 2 0.363 1.549 1.581 1.445 0.912 1.109 P. aeruginosa 3 0.912 1.585 1.531 1.549 1.585 NI P. aeruginosa 4 0.398 0.363 1.585 1.585 1.452 NI P. aeruginosa 5 0.398 0.955 2.512 2.512 NI NI P. aeruginosa 6 1.000 1.445 2.089 2.089 2.089 1.160 P. aeruginosa 7 1.000 1.585 1.995 2.089 1.514 0.691 P. aeruginosa 8 0.398 1.445 NI 2.512 1.445 0.692 P. aeruginosa 9 1.000 1.660 2.089 1.445 2.512 NI P. aeruginosa 10 1.000 1.000 2.089 2.089 NI NI P. aeruginosa 11 1.000 1.585 2.089 2.089 1.585 NI P. aeruginosa 12 0.288 1.000 1.995 2.089 2.089 NI P. aeruginosa 13 0.263 0.398 1.995 2.089 2.089 1.205 P. aeruginosa 14 1.000 1.380 2.512 1.995 2.089 0.759 P. aeruginosa 15* 1.000 1.585 2.089 2.089 NI 1.995 E. coli 1 - - 1.995 NI NI 0.955 E. coli 2 - - 1.585 NI NI 0.912 S. aureus 1 - - 1.000 2.089 2.512 NI S. aureus 2 - - 1.000 2.512 2.000 NI Sal. typhi - - 0.437 2.089 2.089 1.750 KEYS * ATCC Typed Sample EASF Ethylacetate-soluble fraction ASF Acetone-soluble fraction Gen Gentamicin F1 F7 Fractions of Ethylacetate-Soluble Fraction F9 - Not tested NI No inhibition
Figure 6: Graphic representation of MIC values of the fractions and gentamicin for 14 P. aeruginosa isolates and the typed sample
2
3.7.6 Mean MIC Values of the Extracts and Fractions for the test organisms
The mean minimum inhibitory concentration (MIC) values of the extracts and fractions for the test organisms were computed. The values represent means ± standard error of the mean (Tables 32 and 33). There is an inverse relationship between MIC values and antimicrobial activity. If MIC is low, it shows that activity is high. The mean values give more reliable information concerning the antimicrobial activities of the extracts and fractions. Figure 7 enables us to observe at a glance the relative activities of the extracts. Aqueous extract of T. schimperiana root bark (Tsaq) was the most effective of all the crude extracts tested and consequently had the lowest
MIC. This is followed by ethanolic extract of T. schimperiana leaves (Tslet), ethanolic extract of T. schimperiana root bark (Tset), and ethanolic extract of A. cordifolia leaves (Acet). Acet had the highest MIC which indicated the lowest activity against the test organisms. Figure 8 enables us to observe at a glance the relative activity of the fractions against the test organisms. For P. aeruginosa, ethylacetate- soluble fraction (EASF) had the lowest MIC indicating the highest activity, followed by the acetone-soluble fraction (ASF), F9, F1 and F7 in order of decreasing activity.
6
Table 32: Mean MIC values of the plant extracts (mg/ml) and of gentamicin (µg/ml) on the test organisms
Extract P. aeruginosa E. coli S. aureus S. typhi Tset 1.529±0.15 NI 1.119±0.43 0.689 Tsaq 0.988±0.16 NI NI - Tslet 1.061±0.13 1.545±0.55 1.147±0.32 0.912 Acet 2.733±0.30 2.887±0.13 NI 0.602 Acaq NI NI NI 0.912 Smet NI NI NI 1.445 Smaq NI NI 0.622±0.29 1.514 Gen 0.521±0.08 0.933±0.01 1.316±0.55 1.472 KEY: Tset- T. schimperiana root bark ethanolic extract, Tsaq- T. schimperiana root bark aqueous extract, Tslet- T. schimperiana leaf ethanolic extract, Acet- A. cordifolia leaf ethanolic extract, Acaq- A. cordifolia leaf aqueous extract, Smet- S. mombin leaf ethanolic extract, Smaq- S. mombin leaf aqueous extract, Gen- Gentamicin, - Not tested, NI- No inhibition.
=
Table 33: Mean MIC values of fractions (mg/ml) and of gentamicin (µg/ml) on the test organismss
Extract P . aeruginosa E. coli S. aureus Sal. typhi
EASF 0.690±0.09 - - - ASF 1.275±0.11 - - -
F1 1.831±0.11 1.790±0.21 1.000±0.00 0.437
F7 1.950±0.09 NI 2.301±0.21 2.089
F9 1.389±0.11 NI 2.301±0.21 2.089 Gen 0.521±0.11 0.750±0.01 1.316±0.55 1.472 KEYS EASF Ethylacetate-soluble fraction ASF Acetone-soluble fraction Gen Gentamicin F1
F7 Fractions of Ethylacetate-Soluble Fraction
F9 - Not tested NI No inhibition
?
*
a-5 aL/ <, , '=
*
+*
+ Tset Tsaq Tslet Acet
Extracts
Figure 7: Mean MIC values of extracts on P. aeruginosa isolates
KEYS Tset: Ethanolic extract of T. schimperiana root bark Tsaq: Aqueous extract of T. schimperiana root bark Tslet: Ethanolic extract of T. schimperiana leaves Acet: Ethanolic extract of A. cordifolia leaves
2+
)
l 2 m /
1g .8 m ( 1 .6 C I M 1 .4 n a
e 1.2 M 1
0.8
0.6
0.4
0.2
0
EASF ASF Series1 F1 F7 F9
Fractions
Figure 8: Mean MIC values of fractions on P. aeruginosa isolates
KEYS
EASF Ethylacetate-soluble fraction ASF Acetone-soluble fraction
F1 F7 TLC Fractions of Ethylacetate-Soluble Fraction F9
2
3.7.7 Comparison of Mean MIC values of Extracts and Fractions
3.7.7.1 Comparison of Mean MIC Values of Extracts and Gentamicin using
Analysis of Variance (ANOVA)
Using Analysis of Variance (Appendix 29) the mean minimum inhibitory concentration (MIC) values of the four extracts and gentamicin for the P. aeruginosa isolates were analyzed for statistically significant differences, based on 95% confidence limit (P<0.05). The results revealed that there were no significant differences (P>0.05) between the mean MIC values of the extracts in the same subset, but there were significant differences (P<0.05) between mean MIC values of the extracts in different subsets (Table 34). The MIC values of the extracts and gentamicin indicated that their antipseudomonas activities decreased in the order of gentamicin > Tsaq > Tslet > Tset > Acet. In other words, aqueous extract of T. schimperiana root bark (Tsaq) exhibited the highest activity in this study, while ethanolic extract of A. cordifolia leaves (Acet) exhibited the lowest activity.
Gentamicin exceeded the activity of the extracts.
3.7.7.2 Comparison of Mean MIC Values of Fractions and Gentamicin using
Analysis of Variance (ANOVA)
Using Analysis of Variance (Appendix 30) the mean minimum inhibitory concentration (MIC) values of the five fractions and gentamicin for the P. aeruginosa isolates were analyzed for statistically significant differences, based on 95%
2 confidence limit (P<0.05). The results revealed that there were no significant differences (P>0.05) between the mean MIC values of fractions in the same subset, but there were significant differences (P<0.05) between mean MIC values of the fractions in different subsets (Table 35). The MIC values of the fractions and gentamicin indicated that their antipseudomonas activities decreased in the order of gentamicin > EASF > ASF > F9 > F1 > F7. In other words, ethylacetate-soluble fraction (EASF) exhibited the highest activity in this study, while F7 exhibited the lowest activity. Gentamicin exceeded the activity of the fractions.
2
Table 34: Comparison of Mean MIC Values of Extracts and Gentamicin using Analysis of Variance (ANOVA).
Post Hoc Tests: Measurement for extracts Tukey HSD
Plant S u b s e t extract N 1 2 3
Gen 15 0.520733
Tsaq 15 0.987933 0.987933
Tslet 15 1.061200 1.061200
Tset 15 1.528733
Acet 15 2.733000
Sig. 0.322 0.322 1.000
Means for groups in homogeneous subsets are displayed. Based on observed means. The error term is Mean Square (Error) = 0.598. KEYS Gen: Gentamicin Tsaq: Aqueous extract of T. schimperiana root bark Tslet: Ethanolic extract of T. schimperiana leaves Tset: Ethanolic extract of T. schimperiana root bark Acet: Ethanolic extract of A. cordifolia leaves
2
Table 35: Comparison of Mean MIC Values of Fractions and Gentamicin using Analysis of Variance (ANOVA).
Post Hoc Tests: Measurement for Fraction
Tukey HSD
Subset
Fractions N 1 2 3 4
Gen 15 0.520733
EASF 15 0.690067 0.690067
ASF 15 1.274667 1.274667
F9 15 1.388867 1.388867
F1 15 1.831267 1.831267
F7 15 1.950067
Sig. 0.960 0.056 0.079 0.075
Means for groups in homogeneous subsets are displayed. Based on observed means. The error term is Mean Square(Error) = .308. KEYS: EASF Ethylacetate-soluble fraction ASF Acetone-soluble fraction F1 F7 TLC Fractions of Ethylacetate-Soluble Fraction F9
2
3.7.7.3 Comparison of Mean MIC values of Extracts and Fractions for
Gentamicin-Sensitive and Gentamicin-Resistant P. aeruginosa Isolates.
Using Independent Samples t-test (Appendices 31 and 32) the mean minimum inhibitory concentration (MIC) values of extracts and fractions for gentamicin- sensitive and gentamicin-resistant P. aeruginosa isolates were analyzed for statistically significant differences, based on 95% confidence limit (P<0.05). The results revealed that there were no significant differences (P>0.05) between the mean
MIC values of the extracts and fractions for gentamicin-sensitive and gentamicin- resistant P. aeruginosa isolates (Tables 36 and 37). Figures 8 and 9 graphically portray the relative mean MIC values of the extracts and fractions for gentamicin- sensitive and gentamicin-resistant P. aeruginosa isolates. These results signify that the gentamicin-resistant P. aeruginosa isolates can be equally susceptible to the plant extracts and fractions
22
Table 36: Comparison of mean MIC values of extracts for gentamicin-sensitive and gentamicin-resistant P. aeruginosa isolates, using Independent Samples t-test. Mean MIC SEM t Sig.(2 tailed) P-value Tset
Sensitive 1.479 0.227 -0.335 0.743
Resistant 1.585 0.216 -0.338 0.741
Tsaq
Sensitive 0.728 0.210 -1.907 0.079
Resistant 1.285 0.199 -1.924 0.077
Tslet
Sensitive 1.169 0.126 0.865 0.403
Resistant 0.938 0.247 0.832 0.427
Acet
Sensitive 2.601 0.345 -0.457 0.655
Resistant 2.884 0.532 -0.445 0.665
P > 0.05 = no significant differences P < 0.05 = shows significant differences KEYS Tset: Ethanolic extract of T. schimperiana root bark Tsaq: Aqueous extract of T. schimperiana root bark Tslet: Ethanolic extract of T. schimperiana leaves Acet: Ethanolic extract of A. cordifolia leaves
26
Table 37: Comparison of mean MIC values of active fractions for gentamicin- sensitive and gentamicin-resistant P. aeruginosa isolates using Independent Samples t- test.
Test Mean MIC SEM T Sig. (2-tailed) Organisms P-value EASF
Sensitive 0.669 0.126 -0.248 0.808 Resistant 0.714 0.126 -0.250 0.807 ASF Sensitive 1.372 0.142 0.925 0.372 Resistant 1.163 0.178 0.915 0.378
F1 Sensitive 1.698 0.273 -0.906 0.381 Resistant 1.984 0.127 -0.952 0.364
F7 Sensitive 1.987 0.117 0.421 0.681 Resistant 1.908 0.147 0.416 0.685
F9 Sensitive 1.451 0.254 0.882 0.394 Resistant 1.102 0.309 0.875 0.399 P > 0.05- no significant differences P < 0.05- shows significant differences KEYS EASF Ethylacetate-soluble fraction ASF Acetone-soluble fraction F1
F7 TLC Fractions of Ethylacetate-Soluble Fraction
F9
2=
Mean MIC(mg/ml)
*
{ % * w
+*
+ Ç Ç , Ç !
Extracts
Figure 9: Comparison of Mean MIC values of extracts for gentamicin-sensitive and gentamicin-resistant P. aeruginosa isolates KEYS Tset: Ethanolic extract of T. schimperiana root bark Tsaq: Aqueous extract of T. schimperiana root bark Tslet: Ethanolic extract of T. schimperiana leaves Acet: Ethanolic extract of A. cordifolia leaves
Mean MIC (mg/ml)
*=
*2
*
*
+*=
+*2
+*
+*
+ 9!{C
Fractions
Figure 10: Comparison of Mean MIC values of fractions f gentamicin-sensitive and gentamicin-resistant P. aeruginosa
KEYS EASF Ethylacetate-soluble fraction ASF Acetone-soluble fraction F1
F7 TLC Fractions of Ethylacetate-Soluble Fraction
F9
6+
3.8.0 Results of Acute Toxicity (LD50) Tests on Mice
3.8.1 Results of Acute Toxicity (LD50) Test with Crude Extract
The results of the acute toxicity test with crude ethanolic extract of T. schimperiana showed that no death occurred among mice dosed with 10 mg/kg and
100 mg/kg. The group dosed with 200 mg/kg had 33% death while the groups dosed
400 mg/kg, 800 mg/kg and 1000 mg/kg had 100% death (Table 38). The LD50 was obtained using the formula √A x B: where A = largest dose that causes 0% death and
B = smallest dose that causes 100% death.
From the data above LD50 = √100 x 400 = 200 mg/kg (Table 38) Signs of acute toxicity in the mice included excitement immediately after administration of extract, followed by reduced movement.
6
Table 38: Results of Acute Toxicity (LD50) test on mice with crude ethanolic extract of T. schimperiana root bark.
Dose of Animal Body Mass of extract Death %Death extract mass (g) administered (mg) pattern
10 mg/kg AI 21.0g 0.21mg ND 0% A2 23.0g 0.23mg ND A3 24.5g 0.25mg ND
100mg/kg B1 26.5g 2.65mg ND 0% B2 27.0g 2.70mg ND B3 27.5g 2.75mg ND
200mg/kg C1 18.0g 3.60mg ND 33% C2 19.0g 3.80mg ND C3 19.0g 3.80mg D
400mg/kg D1 24.3g 9.72mg D D2 27.0g 10.8mg D 100% D3 32.0g 12.8mg D
E1 20.0g 16.0mg D 100% 800mg/kg E2 23.0g 18.4mg D E3 19.0g 15.2mg D
1000mg/kg F1 27.5g 27.5mg D 100% F2 28.0g 28.0mg D F3 30.0g 30.0mg D KEY: ND- No Death, D- Death
6
3.8.2 Results of Acute Toxicity LD50 test with ethylacetate-soluble
fraction on mice
The results revealed that no death occurred among the mice up to the dose of
1000 mg/kg. Based on the result of the first step where no death occurred even with
1000 mg/kg, a second step was carried out in which doses of 5000, 7000 and 10,000 mg/kg were administered. In this second step, there were no death among mice dosed with 5000 and 7000 mg/kg. One death occurred among the group dosed with 10,000 mg/kg. From this result, LD50 was above 7000mg/kg (Tables 39 and 40).
6
Table 39: Results of Acute Toxicity (LD50) Test on mice with ethylacetate-soluble fraction (Step 1)
Dose of Animal Body Mass of extract Death % Death extract mass (g) administered (mg) pattern
10mg/kg A1 23.5g 0.24mg ND
,, A2 19.5g 0.20mg ND 0%
,, A3 23.5g 0.24mg ND
100mg/kg B1 19.5g 1.95mg ND
,, B2 22.5g 2.25mg ND 0%
,, B3 22.5g 2.25mg ND
1000mg/kg C1 21.5g 21.5mg ND
,, C2 21.5g 21.5mg ND 0% ,, C3 23.0g 23.0mg ND
KEY: ND -No Death
6
Table 40: Results of Acute Toxicity (LD50) test on mice with ethylacetate-soluble fraction (Step 2) Dose of Animal Body mass Mass of extract Death %Death extract (g) administered (mg) pattern 5000mg/kg A1 20.5g 102.5mg ND
,, A2 19.5g 97.5mg ND 0%
,, A3 21.5g 107.5mg ND
7000mg/kg B1 19.5g 136.5mg ND
,, B2 20.5g 143.5mg ND 0%
,, B3 22.5g 157.5mg ND
10,000mg/kg C1 20.5g 205.0mg D
,, C2 22.5g 225.0mg ND 33%
,, C3 23.5g 230.0mg ND
KEY: ND- No Death, D- Death
6
3.9.0 RESULTS OF PHYTOCHEMICAL ANALYSES
3.9.1 Phytochemical composition of T. schimperiana crude ethnanolic extract and the fractions
Table 41 shows the phytochemicals present in T. schimperiana crude ethanolic extract and also in its five soluble fractions. The compounds included alkaloids, glycosides, flavonoids, steroids, terpenoids, tannins, saponins, carbohydrates and resins. The soluble fractions contained these compounds in varying concentrations.
The ethylacetate – soluble fraction contained abundance of flavonoids and tannins and high concentrations of steroids, terpenoids and saponins. The acetone – soluble fraction contained abundance of saponins and high concentrations of steroids, terpenoids and saponins. The acetone – soluble fraction contained abundance of saponins and a high concentration of alkaloids, glycosides, flavonoids, tannins and carbohydrates. The ethanol – soluble fraction contained high concentrations of glycosides, flavonoid and tannins. The n-hexane – soluble fraction contained traces of alkaloids and moderate concentration of resins while chloroform-soluble fraction contained moderate concentrations of alkaloids and traces of steroids, terpenoids and resins while lacking the other compounds (Table 41).
62
Table 41: Phytochemical composition of T. schimperiana root bark crude ethnanolic extract and five soluble fractions Compound Crude n-hexane Chloroform Ethylacetate Acetone Ethanol extract fraction fraction Fraction Fraction fraction
Alkaloid + + + + + + + + + +
Glycosides + + + - - + + + + + + + +
Reducing sugar + - - - + +
Flavonoids + + + - - + + + + + + + + + +
Steroids + - + + + + + -
Terpenoids + - + + + + + -
Acidic + - - - - - compounds
Fats and oils ------
Tannins ++++ - - + + + + + + + + + +
Saponins ++++ - - + + + + + + + -
Carbohydrates + + + - - + + + + + +
Resins + + + + + - - + +
Key +++ + Abundantly present + + + Present in very high concentration + + Present in moderately high concentration + Present in small concentration - Not present
66
3.9.2 Phytochemical composition of A. cordifolia crude ethanolic extract and
S. mombin crude aqueous extract.
Table 42 shows the phytochemicals present in A. cordifolia leaf extract
(ethanolic) and S. mombin leaf extract (aqueous). Alkaloids, flavonoids, saponins, tannins and steroidal aglycone were present in high concentrations in A. cordifolia. S. mombin contained moderate concentrations of flavonoids, tannins and steroidal aglycones and traces of alkaloids and saponins. Also present in both extracts were glycosides, proteins, carbohydrates and reducing sugar in moderate and low concentrations. Cyanogenic glycosides were absent in both plants (Table 42).
6=
Table 42: Phytochemical composition of A. cordifolia leaf ethanolic extract and S. mombin leaf aqueous extract.
Test A. cordifolia S. mombin
Alkaloids
Dragendorff + + + + Mayers + + Wagner + + + Picric acid + + + + Flavonoids + + + + + Glycosides + + + + Proteins + + + CHO + + + Reducing sugars + ++ Saponins + + + + Tannins + + + + + 0-and C-glycosides - - Steroidal aglycone + + + + + Cyanogenic glycosides - - KEY + + + Present in high concentration + + Present in moderate concentration + Present in low concentration - Absent
6?
3.9.3 Ultraviolet (UV) Spectra of TLC Fractions (F1, F7 and F9)
Ultraviolet scanning of F1 fraction revealed three absorption wavelengths,
200.0 nm, 224.0 nm and 266.0 nm, with a high peak at 266.0 nm (Figure 10).
F7 fraction showed an absorption peak at wavelength 272.0 nm (Figure 11).
F9 fraction showed two absorption wavelengths, 272.0 nm and 368.0 nm, with a high peak at 272.0 nm (Figure 12).
=+
Figure 11: Ultraviolet absorption spectra of F1
Peak: WL01 = 200.0 nm Abs =0.125 WL02 = 224.0 nm Abs =0.301 WL03 = 266.0 nm Abs =2.381
=
Figure 12: Ultraviolet absorption spectrum of F7
Peak: WL01 = 272.0 Abs = 2.697
=
Figure 12: Ultraviolet absorption spectrum of F9
Peak: WL01 = 272.0 nm Abs = 2.304 WL02 = 368.0 nm Abs = 0.407
=
CHAPTER FOUR
DISCUSSION
The results of the antibiotic sensitivity tests revealed that all the test organisms exhibited multiple antibiotic-resistance ranging from 40% to 100%. Five of the P. aeruginosa isolates were resistant to all ten antibiotics studied. Infection of surgical wounds, burns, UTI and other systemic infections with ‘pan-resistant’ strains has led to treatment failure associated with high mortality. However, treatment of such resistant infections with medicinal plants has sometimes provided effective therapy and the development of new antimicrobials from plant extracts is presently the focus of many research efforts.
The results of the preliminary antimicrobial screening of plant extracts showed that the crude extracts exhibited good but varying antimicrobial activities against the test organisms. The aqueous extract of T. schimperiana root bark showed a significantly higher antipseudomonas activity (mean MIC=0.988 mg/ml) than its ethanolic extract (mean MIC=1.529 mg/ml) (Table 32). This is a significant finding, since the preparation of aqueous extract is more economical and it is expected that drugs developed with aqueous extracts will be cheaper than the drugs made from ethanolic extract. The ethanolic extract of T. schimperiana root bark and leaves were active against both the Gram-positive and Gram-negative organisms, indicating a possible broad spectrum activity. These results seem to confirm the effectiveness of T. schimperiana root bark in the treatment of burns wounds in Izzi Local Government
Area of Ebonyi State.
The activity of ethanolic extracts of T. schimperiana root bark and leaves
(mean MIC values = 1.119 mg/ml and 1.147mg/ml respectively) against S. aureus in
= this study is high and similar to the activity of its twig (chewing stick) reported by
Ogundiya et al., (2007) who demonstrated a high activity of extracts of chewing sticks from roots of T. schimperiana (T. glaucescens) against S. aureus, Strept. pyogenes and Strept. mutans. They recommended it as a suitable agent for better dental care. This agrees with reports from another study that the plant showed a wide spectrum activity against periodontopathic bacteria (Sote and Wilson, 1995). It also confirms the findings of Akande and Hayashi (1998) that extracts of the plant are effective against Staphylococcus. This portends well for the development of new germicidal tooth-cleaning agents using T. schimperiana extracts. T. schimperiana root bark ethanolic extract was also found to be highly active against the multiple antibiotic-resistant Salmonella typhi used in this study (MIC = 0.689 mg/ml). This is significant in view of the need to develop new drugs in the treatment of typhoid fever caused by resistant Sal. typhi.
A. cordifolia ethanolic leaf extract was active against P. aeruginosa, E. coli and Sal. typhi. The high susceptibility of Sal. typhi to this extract (MIC = 0.602 mg/ml) is significant in view of the multiple antibiotic-resistance shown by the bacterium in this study. The aqueous extract of A. cordifolia leaf was also active against Sal. typhi (mean MIC = 0.912mg/ml) but not against P. aeruginosa, E. coli and S. aureus
S. mombin leaf ethanolic extract did not exhibit significant activity against P. aeruginosa, E. coli and S. aureus but was active against Sal. typhi (MIC =
1.445mg/ml). Aqueous extract of S. mombin proved to be more active than the ethanolic extract, seemingly having broad spectrum activity. It exhibited a high activity against S. aureus 1 and 2 (MIC = 0.331mg/ml and 0.912 mg/ml respectively).
It was also active against Sal. typhi (MIC = 1.514). This is in consonance with the
= findings of Umeh et al., (2009), that extracts of S. mombin inhibited the growth of Sal. typhi and S. aureus with MIC values of 0.226 mg/ml and 0.289 mg/ml respectively.
The findings corroborate the reports of Oyewole and Shonukan (1999) that A. cordifolia ethanolic extract was active against P. aeruginosa and E. coli, and that S. mombin exhibits high activity against S. aureus. The activity of S. mombin against P. aeruginosa and S. aureus confirms its potency in the folkloric treatment of wounds, tonsilitis, sore throat and coughs (Abo et al., 1999; Database entry for Ubos, 2005).
However, the lack of activity of A. cordifolia aqueous extract against P. aeruginosa,
E. coli and S. aureus in this study does not seem to corroborate an earlier report of its broad spectrum activity (Okeke et al., 1999). The susceptibility of multiple antibiotic- resistant Sal. typhi to all the plant extracts used in this study portends success in the search for new drugs for the therapy of typhoid fever (Table 32).
The results of antimicrobial screening of the five soluble fractions of T. schimperiana root bark revealed that only two of the fractions exhibited activity against P. aeruginosa. Ethylacetate-soluble fraction (EASF) (mean MIC=0.690 mg/ml) was more active than the acetone-soluble fraction (ASF) (mean MIC = I.275 mg/ml) (Table 33).
The three active TLC fractions (F1, F7 and F9) obtained from ethylacetate- soluble fraction were active against P. aeruginosa and the other test organisms.
However, F1 was active against P. aeruginosa, E. coli, S. aureus and Sal. typhi (MIC values = 1.898mg/ml, 1.790mg/ml, 1.000mg/ml and 0.437 mg/ml respectively), while
F7 and F9 were active against P. aeruginosa, S. aureus and Sal. typhi but did not inhibit the growth of E. coli (Table 33). It was found in the study that the antipseudomonas activity of the fractions F1, F7 and F9 were lower than the activity of the ethylacetate-soluble fraction. This could indicate that the active principle was a
=2 substance which sublimed or a volatile compound which escaped, or it may be due to the method of analysis to which the fractions have been subjected. It may also be that the three active fractions have a synergistic effect when they act in combination.
Unlike the rural communities who use fresh/dried plant materials or their crude extracts, the industry lays importance on the isolation of active principles or standardized fractions, since crude extracts are not patentable. However, it is often seen that a crude extract is more active compared to the isolated active fraction. The purity of the fractions, F1, F7 and F9 was ascertained in this study using TLC analysis.
The single band formed by each fraction indicated its purity.
Inference from Analysis of Variance of antipseudomonas activities of extracts and fractions depict that there were no significant differences (P > 0.05) between the activities of gentamicin, aqueous extract of T. schimperiana root bark, and EASF belonging to subset 1 (Tables 34 and 35). Independent samples t-test analysis reveal that there were no significant differences in susceptibility to the plant extracts and fractions between the gentamicin-sensitive and gentamicin-resistant P. aeruginosa isolates. This implies that the antibiotic-resistant isolates were equally susceptible to the plant extracts and fractions (Tables 36 and 37). This result is of profound significance in the ongoing battle again drug-resistant organisms.
Results of the acute toxicity tests on the T. shimperiana crude ethanolic extract revealed that the LD50 was 200mg/kg, which indicates that it is too toxic for systemic use. This may explain why the traditional use of this plant root bark is limited to external application (on burns wounds), among the Izzi people of Ebonyi State. The
LD50 of the ethylacetate-soluble fraction was above 7000mg/kg (Tables 38 – 40).
According to Locke (1983), if no death occurs among the mice even at a dose of
5000mg/kg, the extract is not toxic and is therefore considered safe for use as
=6 medicine. The results obtained in this study indicate that the ethylacetate-soluble fraction has very low toxicity and therefore is safe for use by oral route. This result underscores the need to purify the crude extract by fractionation in order to reduce the toxicity and isolate the active fraction.
Results of phytochemical analysis of T. schimperiana crude ethanolic extract and its soluble fractions revealed an abundance of flavonoids, tannins and saponins, and a high concentration of alkaloids, glycosides, steroids, terpenoids and carbohydrates. This analysis also showed moderate concentrations of resins, and traces of reducing sugar and acidic compounds (Tables 41 and 42). This agrees with the findings of McGraw et al., (2001) who reported that trees of the genus Terminalia are a rich source of these secondary metabolites. A. cordifolia leaf ethanolic extract and S. mombin leaf aqueous extract were also found to contain alkaloids, flavonoids, tannins and steroidal aglycones. There was an absence of cyanogenic glycosides in the extracts of A. cordifolia and S. mombin which is an indication of low toxicity
(Ajao et al., 1985; Database entry for Ubos, 2005).
Earlier workers have proposed that the presence of flavonoids, alkaloids, tannins and saponins in plants confer antimicrobial activity on the plants (McGraw et al., 2001; Levan et al., 1979; Ibrahim et al., 1997). The presence of these antimicrobial compounds in the plants under study may be a confirmation of the antibacterial activity demonstrated in this study. This seems to lend credence to the claims of herbal healers and the local people that these plants are used effectively to cure diseases of bacterial origin. The presence of these phytochemicals in varying concentrations in the plants under study explain their varying antibacterial effects. A. cordifolia contained a higher concentration of the phytochemicals than did S. mombin and so exerted a broad spectrum antibacterial effect and a higher activity against Sal.
== typhi. The ethylacetate and acetone-soluble fractions of T. schimperiana root bark contained higher concentrations of the phytochemicals than n-hexane, chloroform and ethanol-soluble fractions and consequently exerted higher effects (mean MIC = 0.690 mg/ml and 1.275 mg/ml respectively) against the P. aeruginosa isolates. Conversely, n-hexane and chloroform-soluble fractions contained none or traces of these compounds and exerted little or no effect on the P. aeruginosa isolates.
The active TLC fractions F1, F7 and F9 obtained from EASF showed UV absorption wavelengths of F1 (266.0 nm), F7 (272.0 nm) and F9 (272.0 nm and 368.0 nm). These absorption wavelengths indicate that the TLC fractions are likely to be flavonoids which have been reported to have absorption maxima ranging from 248 nm to 299 nm (Seijas et al., 2006). Various workers have attempted to unravel the mode of action of flavonoids against bacterial pathogens. Flavonoids isolated from a medicinal plant- Smaller galangal- have been found to have inhibitory activity against penicillinase (Eumkel et al., 2011). It is therefore possible that the abundant flavonoid content of T. schimperiana root bark and its ethylacetate-soluble fraction (EASF), which were eventually isolated as TLC fractions F1, F7 and F9 acted against P. aeruginosa and S. aureus by inactivating their beta-lactamases. In a related research, it was found that flavonones significantly reduced the production of pyocyanin and elastase which are known to be virulent factors in P. aeruginosa (Vandeputte et al.,
2011).
The results of the antibiotic susceptibility tests revealed that the test organisms were resistant to the first generation antibiotics tested in this study (Tables 3 and 4).
All the 15 P. aeruginosa isolates showed 100% resistance to ampicillin, augmentin and nalidixic acid. This result is in consonance with the report of earlier workers
(Udo, et al., 1998; Kesah, et al., 1999). On the other hand, the relatively new
=? antibiotics such as the quinolones (ciprofloxacin, ofloxacin and perfloxacin) were found to be the most effective of all the antibiotics screened.
In conclusion, the results obtained in this study revealed that the plant extracts have antibacterial activity and this confirms the effectiveness of the plants in traditional medicine. It is notable in this study that P. aeruginosa strains that were resistant to the conventional antibiotics were susceptible to the plant extracts and fractions sometimes at the lowest concentration screened. Of particular interest is the high susceptibility of multiple antibiotic-resistant P. aeruginosa to 10 out of 12 extracts and fractions screened against it. Another interesting finding in this study is the high susceptibility of multiple antibiotic-resistant Sal. typhi to the 9 extracts and fractions screened against it. Also of interest is the susceptibility of multiple antibiotic-resistant S. aureus to 6 out of the 9 extracts and fractions screened against it especially the aqueous extract of S. mombin to which it was highly susceptible. E. coli exhibited the highest resistance in this study and was susceptible to only 3 out of 9 extracts and fractions tested against it. The phytochemical content of the plants were analyzed and they were found to contain compounds which confer antibacterial activity on plants. The plants analyzed in this study seem to have low toxicity to the human body as S. mombin and A. cordifolia are eaten or used in food processing. On its part, ethylacetate-soluble fraction of T. schimperiana was found to have very low toxicity as revealed by the acute toxity test. These findings underscore the possibility of using the active extracts and fractions in the developmnt of new antimicrobials for antibiotic-resistant infections.
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RECOMMENDATIONS
Pseudomonas aeruginosa isolates and other test organisms that showed resistance to the conventional antibiotics were found to be susceptible to the plant extracts and fractions used in the present study, sometimes at concentrations as low as
1.25 mg/ml. The acute toxicity tests also indicate the low toxicity of the ethylacetate- soluble fraction. In view of these encouraging results, it is recommended that the active extracts and fractions be used in the development of new drugs for antibiotic- resistant infections caused by P. aeruginosa, E. coli, S. aureus and Sal. typhi.
However, the chronic toxicity of the extracts needs to be investigated to make sure that the extracts will not have serious adverse side effects on the body when put into use.
In addition, there is need to study the antimicrobial activities of the extracts and fractions in vivo, as well as the bioavailability of the active constituents obtained in the present study using animal models.
Many of the plants in the tropical rain forest of Nigeria are reported to have medicinal properties. These represent a vast untapped source of medicines. Continued and further exploration of plant medicines should be sustained.
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REFERENCES
Abo, K.A., Ogunleye, V. O. and Ashidi, J. S. (1999). Antimicrobial potential of Spondias mombin, Croton zambesicus and Zygotritonia crocea. Phytotherapy Research. 13: 494-497.
Abraham, E. and Shah, S. (1992). Intranasal immunization with liposomes containing IL-2 enhances bacterial polysaccharide antigen-specific pulmonary secretory antibody response. The Journal of Immunology: 149 (11): 3719-3720.
Accelr8 (2010). History of antibiotic resistance (http://www.accelr8.com/index.php).
Adesina, S. K. and Sofowora, E. A. (1979). The isolation of an anticonvulsant glycoside from Tetrapleura tetraptera. Planta Medica: 36 (3): 270.
Adesina, S. K., Oguntimein, B. K. and Akinwusi, D. D. (1980). Phytochemical and biological examination of the leaves of Acalypha wilkesiana Muell. Arg. Quarterly Journal of Crude Drug; 3: 45 – 48.
Adjanahoun, E., Aliyu, M. R. A., Ake-Assi, L., Elewude, J. A. and Dramane, K. E. (1991). Traditional Medicine and Pharmacopoeia: Contribution to ethnobotanical floristic studies in Western Nigeria, Pub. Organization of African Unity, Scientific, Technical and Research Commission. Lagos, Nigeria. P. 420.
African Plants Database: Terminalia schimperiana. Accessed June, 6, 2007. http://www.ville-ge.ch/cjb/bd/africa/details.php?langue+andid+713.
Ajao, A. O., Shonukan, O. and Femi-Onadeko, B. (1985). Antibacterial effects of aqueous and alcoholic extracts of Spondias mombin and Alchornea cordifolia, two local antimicrobial remedies. Int. J. Crude Drug Res. 23: 67-72.
Akande, J. A. and Hayashi, Y. (1998). Potency of extract contents from selected tropical chewing sticks against Staphylococcus aureus and S. auricularis. World Journal of Microbiology and Biotechnology. 14(2):235–238. http://www.springerlink.com/contents/r875165841xr0533. Accessed February, 13, 2008.
Akinde, B. E., Okeke, I. and Orafidiya, O. O. (1999). Phytochemical and antimicrobial evaluation of Cassia alata leaves extracts. West Africa Journal of Pharmacy; 13(1): 36-40.
Akinsinde, K. A., and Olukoya, D. K. (1995). Vibriocidal activities of some local herbs. Journal of Diarrhoeal Diseases Research; 13(2): 127 - 129.
Akinyanju, J. A., Oluwole, J. A. and Okunla, E. O. (1986). Antimicrobial effect of leaf extract of Acallypha torta. The State of Medicinal Plants Research in Nigeria. Pp. 247-251.
?
Arbonnier, M. (2004). Trees, Shrubs and Lianas of West African Dry Zone. Margraf Publishers ISBN 3 8236-1419-3.
Arino, S., Marchal, R. and Vandecasteele (1998). Involvement of a rhamnolipid- producing strain of Pseudomonas aeruginosa in the degradation of polycyclic aromatic hydrocarbons by a bacterial community. Journal of Applied Microbiology. 84(5): 769 – 776.
Asuzu, I.U. (2001). From Plant to Drug. A paper presented at the workshop on medicinal plants of Nigeria organized by Association of Scientific Identification, Conservation and Utilization of Medicinal Plants of Nigeria (ASICUMPON) at Enugu, 25-27 June.
Baron, E. J. and Finegold, S. M. (1990). Method for testing antimicrobial effectiveness. In: Diagnostic Microbiology 8th ed. The C.V. Mosb Company.
Bauer, A. W., Kirby, W. M. M., Sherris, J. C. and Turck, M. (1966). Antibiotic susceptibility testing by a standardized single disk method. American Journal of Pathology. 45: 493 – 496.
Berk, R. S. (1963). Intracellular hemolysin of Pseudomonas aeruginosa. Journal of Bacteriology. 85: 522-526.
Berka, R. M., Gray, G. L. and Vasil, M. L. (1981). Studies of Phospholipase C. (Heat- labile hemolysin) in Pseudomonas aeruginosa. Infection and Immunity. 34 (3):1071-1074.
Biddlecom, S., Hass, M., Davies, J., Miller, G. H., Rane, D. F. and Daniels, P. J. L. (1976). Enzymic modification of aminoglycoside antibiotics: a new 3–N- acetylating enzyme from a Pseudomonas aeruginosa isolate. Antimicrobial Agents and Chemotherapy 9: 951-955.
Boffi, E., Chamot, E., Auckenthaler, R., Pechère, J. C. and Van Delden, C. (2000). The influence of preceeding antibiotic treatments on the resistance profile of Pseudomonas aeruginosa bacteremic strains. Clinical Microbiology and Infection. 6: 119. Accessed September 11, 2010.
Bressolle, F., Goncalves, F., Gouby, A. and Galter, M. (1994). Pefloxacin clinical pharmacokinetics. Clinical Pharmacokinetics. 27: 418-46.
Buell, R. C., Joarder, V., Lindeberg, M., Selengut, J., Paulsen, I. T., Gwinn, M. L., Dodson, R. J., Deboy, R. T. G., Durkin, A. S., Kolonay, J. R. and Madupu, R. (2003). The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae, pv. Tomato DC3000.PNAS, Vol. 100, No. 18 (1018 - 10186).
C L S I (2006). Performance Standards for Antimicrobial Disk Susceptibility Tests, C L S I. Vol. 26 No. 1 Jan. 2006.
?
Carmeli Y, Troillet N., Eliopoulos G. M., Matthew H. and Samore, M. H. (1999). Emergence of Antibiotic-Resistant Pseudomonas aeruginosa: Comparison of Risks Associated with Different Antipseudomonal Agents. Antimicrobial Agents and Chemotherapy. 43(6): 1379–1382. Accessed May 17, 2010.
Cheesbrough, M. (1994). Enteric Gram-negative rods and Gram-negative anaerobes. In: Medical Laboratory Manual for Tropical Countries. Volume 11: Microbiology Tropical Health Technology, Cambridgeshire. Pp 264 – 265.
CMPT M73-1 Urine: Pseudomonas aeruginosa (1997). http://www.interchange.ubc.ca/cmpt/cmpt_new/m73-1_nov97.h tm Accessed May 15, 2005.
Colsky, A. S., Kersna, R. S. and Kerdel, F. A. (1998). Analysis of antibiotic susceptibilities of skin wound flora in hospitalized dermatology patients. Achives Dermatology; 134(8):1-2. Accessed May, 4, 2006. http://archderm.ama-assn.org/issues/v134n8/abs/dob8010.html.
Cometta, A., Baumgartner, J. D., Lew, D., Zimmerli, W., Pittet, D., Chopart, P., Schaad, U., Herter, C., Eggimann, P. and Huber, O. (1994). Prospective randomized comparison of imipenem monotherapy with imipenem plus netilmicin for treatment of severe infections in nonneutropenic patients. Antimicrobial Agents and Chemotherapy. 38: 1309. Accessed May 17, 2010.
Corthout, J., Pieters, L., Claeys, M., VandenBerghe, D. and Vlietinct, A. J. (1987). Antivirally active constituents of Spondias mombin L. Pharm. Week bl. (Sci.ed.) 9: 222.
Cowan, S. T. and Steel, K. J. (1993). Manual For the Identification of Medical Bacteria. G.I. Barrow and R.K.A. Feltham. 3rd Ed. Cambridge University Press. Pp71-76.
Crouch, B. S., Wunderink, R. G. and Jones, C. B. (1996). Ventilator-associated pneumonia due to Pseudomonas aeruginosa. Chest: 109 (4): 1019 – 29 Medline.
Cryz, S. J. and Iglewski, B. H. (1980). Production of alkaline protease by Pseudomonas aeruginosa. Journal of Clinical Microbiology. 12(1): 131 – 133.
Database entry for Ageratum conyzoides (2005). http://www.rain-tree.com/ageratum.htm Accessed Aug. 18 2006.
Database entry for Ubos (2005). http://www.raintree.com/ubos.htm. Accessed May, 12 2006.
Davies, B. I., Maesen, F. P. V. and Van Norrd, J. A. (1983). Pseudomonas aeruginosa associated with recurrent chronic bronchitis. Journal of Antimicrobial Chemotherapy: 12 Suppl. A. De Pascual Teresa, J., Diaz, F., Sanchez, F. J., Hernandez, J. M. and Grande, M. (1980) Flavonoids from Digitalis thapsi leaves. Planta Medica: 17: 271 – 272.
?
Den Hollander, J. G., Horrevorts, A. M., Van Goor, M. P. J., Verbrugh, H. A. and Mounton, J. W. (1997). Synergism between tobramycin and ceftazidime against a resistant P. aeruginosa strain tested in an in vitro pharmacokinetic model. Antimicrobial Agents and Chemotherapy. 41(1): 95 – 100.
Di Sant’ Agnese, P. A. and Davis, P. B. (1979). Cystic fibrosis in Acults: 75 cases and a review of 232 cases in the literature. American Journal of Medicine. 66: 121- 132.
Domagala, J. M. (1994). Structure-activity and structure-side effect relationships for quinolone antibiotics. Journal Antimicrobial Chemotherapy 33: 685 – 706.
Durodola, J.J. (1977). Antibacterial property of crude extracts from herbal wound healing remedy – Ageratum conyzoides. Planta Medica: 32: 388 – 390.
Edmonds, R., Suskind, R. R., Macmillan, B. G. and Holder, I. A. (1972). Epidemiology of Pseudomonas aeruginosa in a Burns Hospital: evaluation of serological, bacteriophage and pyocin typing methods. Applied Microbiology: 24 (2) 213 –18.
Ekor, M, Ashorobi, R. B., Ibitoye, S. F. and Kasimu, L. S. (2005). Acute toxicity, analgestic potential and preliminary antimicrobial studies of the aqueous plant extract of Spilanthes filicaulis. Nigerian Journal of Health and Biomedical Sciences; 4(1): 30 – 34.
Eldere, J. V. (2003) Multicentre surveillance of Pseudomonas aeruginosa susceptibility patterns in nosocomial infections. Journal of Antimicrobial Chemotherapy. 51: 347-35. Accessed June 30, 2010.
Eumkel, G., Siriwong, S, Phitaktim, S., Rojtinnakorn, N. and Sakdarat, S. (2011). Synergistic activity and mode of action of flavonoids isolated from smaller galangal and amoxicillin combinations against amoxicillin-resistant Escherichia coli. Journal of Applied Microbiology. 112(1): 55-64.
Evans, L. R. and Linker, A. (1973). Production and characterization of the slime polysaccharide of Pseudomonas aeruginosa. Journal of Bacteriology. 116: 915-924.
Fakeye, T. O., Itiola, A. O. and Odelola, H. A. (2002). Evaluation of the antimicrobial property of the stem bark of Picralima nitida (Apocynaceae). Phytotherapy Research, 14: 368 - 370
Fauci, A. (1998). New and re-emerging diseases: the importance of biomedical research. Emerging Infectious Diseases. (www.cdc.gov/ncidod/EID/vo/4no3/fauci). 4:3
Fink, M. P., Snydman, D. R., Niederman, M. S., Leeper, K V. Jr., Johnson, R H., Heard, S. O., Wunderink, R G., Caldwell, J. W., Schentag, J. J,. Siami ,J. A., Zameck, R. L., Haverstock, D. C., Reinhart, H. H. and Echols, R. M. (1994). Treatment of severe pneumonia in hospitalized patients: results of a
?
multicenter, randomized, double-blind trial comparing intravenous ciprofloxacin with imipenem-cilastin. Antimicrobial Agents and Chemother. 38:547–557. Accessed May 20, 2010.
Forbes, B. A., Sahm, D. F. and Weissfeld, A. S. (2007). Bacterial Genetics, Metabolism and Structure. In: Diagnostic Microbiology. 12th Ed. Mosby. Elsevier. Pp 16-17.
Forbes, B. A., Sahm, D. F. and Weissfeld, A. S. (2007). Enterobacteriaceae. In: Diagnostic Microbiology. 12th Ed. Mosby. Elsevier. Pp 323-333.
Forbes, B. A., Sahm, D. F. and Weissfeld, A. S. (2007). Laboratory Methods and Strategies for Antimicrobial Susceptibility Testing. In: Diagnostic Microbiology. 12th Ed. Mosby. Elsevier. Pp 187-213.
Forbes, B. A., Sahm, D. F. and Weissfeld, A. S. (2007). Principles of Antimicrobial Action and Resistance. In: Diagnostic Microbiology. 12th Ed. Mosby. Elsevier. Pp 172-186.
Forbes, B. A., Sahm, D. F. and Weissfeld, A. S. (2007). Staphylococcus, Micrococcus and Similar organisms. In: Diagnostic Microbiology. 12th Ed. Mosby. Elsevier. Pp 254-263.
Fridkin, S. K. and Gaynes, R. P. (1999). Antimicrobial resistance in intensive care units. Clinics in Chest Medicine 20: 303–16. Accessed August 4, 2010.
Galentine, P. G., Cohen, E. J., Laibson, P. R., Adams, C. P., Michaud, R. and Arentsen, J. J. (1984). Corneal ulcers associated with contact lens wear. Archives of Ophthalmology. 102: 891-894.
Garber, N. and Friedman, J. (1970). Beta-lactamases and the resistance of P. aeruginosa to various penicillins and cephalosporins. Journal of General Microbiology. 64: 343-352.
Gills, L. S. (1992). Ethnomedical Uses of Plants in Nigeria. University of Benin Press. Pp. 220.
Gonzalez, I., Georgiou, M., Alcaide, F., Balas, D., Linares, J. and De la Campa, A. C. (1998). Fluoroquinolone-resistance mutations in the parC, parE and gyrA genes of clinical isolates of viridians group Streptococci. Antimicrobial Agents and Chemotherapy. 42(11): 2792-98.
Grishina, I. A. (1982). Safety, reactogenicity and efficacy of pyoimmunogen in immunizing burn patients. Zh Mikrobiol. Epidemiology and Immunobiology. (7):34-37.
Grutman, M. I., Par khomenko, L. V. and Par Khomenko, V. N. (2001). The defence of immune sera to complex surface antigens of Escherichia, Salmonella, Pseudomonas and Proteus under experimental infection. The 4th Annual
?2
Conference on Vaccine Research. Apr. 23-25, 2001. Hyatt regency crystal city, Arlington VA.
Gustafsson, L. L. and Wide, K. (1981).Marketing of obsolete antibiotics in Central America. Lancet. 1: 31-33.
Harbourne, J.B.C. (1973). Phytochemical Methods. Chapman and Hall, London. P. 279.
Harder S., Fuhr, U., Beermann, D. and Staib, A. H. (1990). Ciprofloxacin absorption in different regions of the human gastrointestinal tract. Investigations with the Lf- capsule. British Journal of Clinical Pharmacology. 30: 35 – 39.
Hatch, R. A. and Schiller, N. L. (1998). Alginate lyase promotes diffusion of aminoglycosides through the extracellular polysaccharide of mucoid P. aeruginosa. Antimicrobial Agents and Chemotherapy. 42(4): 974-977.
Hazlett, L. D., Berk, R. S. and Iglewski, B. H. (1981). Microscopic characterization of ocular damage produced by P. aeruginosa toxin A. Infection and Immunity. 34(3): 1025-1035.
Hirai, K., Susue, S., Irikura, T., Iyobe, S. and Mitsuhashi, S. (1987). Mutation producing resistance to norfloxacin in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy. 31: 582-586.
Hoiby, N. (1993). Antibiotic therapy for chronic infection of Pseudomonas in the lung. Annual Review of Medicine. 44: 1-10.
Holt, J. G., Krieg, N. R., Sneath, P. H. A., Staley J. T. and Williams, S. T. (1994). Genus Pseudomonas. In: Bergey’s Manual of Determinative Bacteriology. 9th Ed. Williams and Wilkins. Baltimore, Maryland 21202, USA. Pp 93-94.
HoogKamp-Korstanje and Van der Laag, J. (1983). Piperacillin and tobramicin in the treatment of Pseudomonas lung infections in cystic fibrosis. Journal of Antimicrobial Chemotherapy. 12: 175-183.
Horan, T., Culver, D. and Jarvis, W. (1988). Pathogens causing nosocomial infections. Antimicrobial Newsletter. 5(65-67).
Ibrahim, M. B., Owonubi, M. O. and Onaolapo, J. A. (1997). Antibacterial effects of extracts of leaf, stem and root bark of Aniogiessus leiocarpus on Staphylococcus aureus NCTC 6571, Streptococcus pyogenes NCTC 8198, Escherichia coli NCTC 10418 and Proteus vulgaris NCTC 4636. Journal of Pharmaceutical Research and Development. 2 (1) 20-26.
Insler, M. H. and Gore, H. (1986). Pseudomonas keratitis and folliculitis from whirlpool exposure. Am. J. Ophthalmol. 101: 41 – 43.
Iwalokun, B. A., Gbenle, G. O., Adewole, T. A. and Akinsinde, K. A. (2001). Shigellocidal properties of three Nigerian medicinal plants: Ocimum
?6
gratissimum, Terminalia avicennoides and Momordica balsamina. Journal of Health, Population and Nutrition. 19(4): 331-335.
Iwu, M. M. (1993). Handbook of African Medicinal Plants. CRC Press, Inc. Boca Raton, Fl. Pp 45-215.
Kalayi G. D. (2000). Burn wound sepsis in Zaria. Nigerian Journal of Surgery. 7(2).
Kesah, C. N., Egri-Okwaji, M. T. C., Odugbemi, T. O. and Iroha, E. O. (1999). Resistance of nosocomial bacterial pathogens to commonly used antimicrobial agents. The Nigerian Postgraduate Medical Journal. 6(2): 60-64.
Kohler, T., Kok, M., Michea-Hamzehpour, M., Plesiat, P., Gotor, N., Nishino, T., Curty, L. K. and Pechere, J.C. (1996). Multidrug efflux in intrinsic resistance to trimetoprim and sulfamethoxazole in P. aeruginosa. Antimicrobial Agents and Chemotherapy. 40: 2288-2290.
Kominos, S. D., Copeland, C. E., Grosiak, B. and Postic B. (1972). Introduction of P. aeruginosa into a hospital via vegetables. Applied Microbiology. 24(4): 567 - 570.
Kudi, A. C., Umoh, J. U., Eduvie, L. O. and Gefu, J. (1999). Screening of some Nigerian plants for antimicrobial activity. Journal of Ethnopharmacology. 67(2): 225 – 228.
Laurence, D. R., Benneth, P. N. and Brown, M. J. (1999). Chemotherapy of infections. In: Clinical Pharmacology. 8th Ed. Churchill Livingstone. Edinburgh. London, N.Y. Pp 187 – 218.
Lessie, T. G. and Phibbs, P. V. J. (1984). Alternative pathway of carbohydrate utilization in Pseudomonas. Annual Review of Microbiology. 38: 357 – 367.
Levan, M., Vanden Berghe, D., Marten, I., Vilientnich, A. and Lomweas, E. C. (1979). Screening of higher plants for biological activity. Planta Medica. 36: 311-312.
Li, X.Z., Nikaido, H. and Poole, K. (1995). Role of mexA-MexB OprM in antibiotic efflux in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 39: 1948-1953.
Lindberg, R. B., Moncrief, J. A., Switzer, W. E., Order, S. E. and Mills, N. Jr (1965). The successful control of burn wound sepsis. Journal of Trauma 5: 601-616.
Livermore, D. M. (1995). -lactamases in laboratory and clinical research. Clin. Microbiology Review. 8: 557-584.
Livermore, D. M. (1983). Kinetics and significance of the activity of the Sabbath and Abraham’s -lactamse of P. aeruginosa against cefotaxime and cefsuloxin. Journal of Antimicrobial Chemotherapy. 11: 169-179.
?=
Locke D. (1983). A new approach to practical acute toxicity testing. Arch. Toxicol. 54: 275-287.
Loudon, I. (1987). Puerperal fever, the Streptococcus and the sulphonamides. 1911- 1945. British Medical Journal. 295: 485.
Markley, K. and Smallman, E. (1968). Protection By Vaccination against Pseudomonas Infection after Thermal Injury. Journal of Bacteriology. 96(4): 867-874.
Martin, T., Parker, S. E., Hedstron, R., Le T., Hoffman, S. L, Norman, J., Hobart, P. and Lew, D. (1999). Plasmid DNA malaria vaccine: the potential for genomic integration after intramuscular injection. Hum – gene- Ther. 10 (5): 759-769.
Masuda, N., Sakagawa, E. and Ohya, S. (1995). Outer membrane proteins responsible for multiple drug resistance in P. aeruginosa. Antimicrobial Agents and Chemotherapy. 39: 645-649.
Mbam, Virginia. Herbal healer. Personal Communication (2007). Iboko, Izzi LGA. Ebonyi State.
McGraw, L. J., Rabe, T., Spary, S. G., Jager, A. K., Eloff, J. N. and Van Staden, J. (2001). An investigation of the biological activity of Combretum species. Journal of Ethnopharmacology. 75: 45-50.
Mclaughln-Borlace, L., Stapleton, F., Matheson, M. and Dart, J. K. G. (1998). Bacterial biofilm on contact lenses and lens storage cases in wearers with microbial keratilis. Journal of Applied Microbiology. 84 (5) 827-838.
Milatovic D, and Braveny I. (1987). Development of resistance during antibiotic therapy. European Journal of Clinical Microbiology. 6: 234–244. Accessed June 2, 2010.
Miller, G. H., Sabateli, F. J., Hare, R. S. and Waitz, J. A. (1980). Survey of aminoglycoside resistance patterns. Dev. Ind.Microbiol. 21: 91-104.
Morihara, K., Sanai, Y., Tsuzuki, H., Jyoyama, H., Hirose, K., Homma, J. Y. and Kato, I. (1981). Effects of proteases on the structure and activity of Pseudomonas aeruginosa exotoxin A. Infection and Immunity. 34: (2) 435- 440.
Mull, J. D and Callahan, W.S. (1963). Estimation of electrolytic activity of stains of Pseudomonas aeruginosa. Journal of Bacteriology. 85: 1178-1179.
Muller, M., Mester, I. and Reisch, J. (1979). Coumarins and alkaloids from Afraegle paniculata (Rutaceae). Planta Medica. 36: (3) 279.
Murray, M. (1995). The Healing Power of Herbs. Primer Publishing. Rocklin, Ca. Pp 162-171.
??
Nakano, M., Deguchi, T., Kawamura, T., Yasuda, M., Kimura, M., Yukio O. and Kawada, Y. (1997). Mutations in the gyrA and parC genes in fluoroquinolone –resistant clinical isolates of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy. 41(10): 2289-2291.
Naval Medical Research Center U.S.A. Silver Spring, Maryland. Immunology (2005): Genetic Immunization Against Biothreat Agents. http://www4.nationalacademies.org/rapNsfTitle/56.61.05.B5382? opendocument. Accessed July 5 2007.
Nelson, W. H. (1991). Physical Methods for Microorganisms Detecton. Nelson, W.H. ed. C.R.C press. Boca Raton. Ann Arbor, Pp 10 – 11.
Nester, E. W., Anderson, D.G., Roberts (Jr), C.E. Pearsall, N. N and Nester, M.T. (2001). Microbiology: A Human Perspective. 3rd Ed.McGraw Hill. NY P 116.
Nicas, T. I. and Iglewski, B.H. (1986). Production of elastase and other exoproducts by environmental isolates of Pseudomonas aeriuginosa. Journal of Clinical Microbiology. 23(5) 1967- 69.
Nikaido, H. (1994). Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science 264: 382-388.
O’ Brien, T. F. (1992) Global surveillance of antibiotic resistance. N. Engl. J. Med. 326: 339-40
Obaseiki-Ebor, E. E. (1985). Preliminary report on the in vitro antibacterial activity of Bryophyllum pinnatum leaf juice. African Journal of Medicines and Medical Sciences. 14 (3-4): 1992 – 202.
Odebiyi, O. O. (1999). Antibacterial principles from the stem of Jatropha podagrica Hook. 4th OAU/STRC Inter-African Symposium on Traditional Pharmacopoeia and African Medicinal Plants. Pp 505-508.
Oduyebo, O., Ogunsola, F. T. and Odugbemi, T. (1997). Prevalence of multi-resistant strains of Psedomonas aeruginosa isolated at the Lagos University Teaching Hospital Laboratory from 1994 to 1996. Nig. Ot. Journal Hospital Medicine. 7(4): 373-376.
Ofokansi, K. C., Adikwu, M. U. Esimone, C. O. and Nwodo, N. M. (2003). Antibacterial activity of the leaf extract of Ocimum gratissimum (fam. Labiatae). Journal of Biological Research and Biotechnology. 1(1): 35-42.
Ogundiya, M. O., Okunade, M. B. and Kolapo, A. L. (2007). Antimicrobial activities of some Nigerian chewingg sticks. http://www.siu.edu/ebl/leaflets/ogundya.htm. Accessed May 17, 2008.
Okafor, J.C. (2002). Predictive values of plant taxonomy in ethno-medicine: a paper presented at the workshop on medicine plants of Nigeria organized by ASICUMPIN at Enugu, 25-27 June.
++
Okeke, I. N., Ogundaine, A.O., Ogunbamila, F.O. and Lamikanra, A. (1999). Antimicrobial spectrum of Alchornea cordifolia leaf extract. Phytotherapy Research. 13: 67-69.
Olayinka, A. T., Onile, B. A. and Olayinka, B. O. (2004). Prevalence of multi-drug resistant (MDR) Pseudomonas aeruginosa isolates in surgical units of Ahmadu Bello University Teaching Hospital, Zaria, Nigeria: An indication for effective control measures. Annals of African Medicine. 3 (1): 13-16.
Olayinka, B. O., Olonitola, O. S., Olayinka, A. T. and Agada E. A. (2004). Antibiotic susceptibility pattern and multiple antibiotic resistance index of Pseudomonas aeruginosa urine isolates from a University Teaching Hospital. African Journal of Clinical and Experimental Microbiology. 5 (2): 198-202.
Onawunmi, G. C., Yisak, W. A. and Ogunlana, E. O. (1984). Antibacterial constituent in the essential oil of Cymbopogon citrates (DC) Stapf. Journal of Ethnopharmacology. 12 (3): 279 - 286.
Oyewole, O. B., and Shonukan, O. O. (1999). Antimicrobial effects of Alchornea cordifolia (Schum and Thonn) and Spondias mombin (Linn). The Bioprospector. 1 (2): 32 – 35.
Ozumba, U. C. (2003). Antibiotic Sensitivity of Isolates of Pseudomonas aeruginosa in Enugu, Nigeria. African Journal of Clinical and Experimental Microbiology: 4 (1): 48-51.
Poh, C. F. (2000). Antibacterial activity of Salacia pyriformis. Nigerian Journal of Natural Products and Medicine. 4: 65- 66.
Pollack, M. (1982). Toxoid from exotoxin A of Pseudomonas aeruginosa: Preparation and characterization. Journal Infectious Disease. 145 (5):688 – 98.
Pollack, M. (2000). Pseudomonas aeruginosa. In: Principles and Practice of Infectious Diseases. Mandell, G.L., Bennett, J.E., Dolin, R. eds. 5th ed. NY Churchill Livingstone: Pp. 2310 – 2327.
Pollack, M., Taylor, N. S. and Callahan L. T. (1977). Exotoxin production by clinical isolates of Pseudomonas aeruginosa. Infection and Immunity. 15: 776-780.
Poole, K., Tetro, K., Zhao, G., Neshat, S., Heinsichs, D. E. and Bianco, N. (1996). Expression of the multidrug resistance operon MexA-MexB-OprM in Pseudomonas aeruginosa: MexR encodes a regulator of operon expression. Antimicrobial Agents and Chemotherapy. 40: 2021 -2028.
Qarah, S., Cunha, B. A., Dua, P. and Lessnau, K. (2003). Pseudomonas aeruginosa infections. Eds: Marrie T.J., Talavera, F., Bronze M.S., Mylonakis E., Zevitz, M.E. 2003: 1-25. http:// www.emedicine.com/med/topic 1943. htm. Accessed April 30, 2005.
+
Ramirez-Ronda, C. H., Ramirez-Ramirez, C. and Vera, D. (1999). Change in resistance of aerobic Gram-negative bacilli is to ß-lactams after cefepime use. In Program and Abstracts of the Thirty-ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, USA. Abstract 1470, p. 163. American Society for Microbiology, Washington, DC, USA. Accessed July 17, 2010.
Rastegar – Lari, A. R., Alaghehbandan, R. and Akhlaghi, L. (2005). Burn wound infections and antimicrobial resistance in Tehran, Iran: an increasing problem. Annals of Burns and Fire Disasters; vol. xviii – n2.
Robbers, J., Speedie, M. and Tyler, V. (1996). Pharmacognosy and Pharmacobiotechnology. Williams and Wilkins, Baltimore Pp. 1 – 14.
Rosebury, T. (1962). Microorganisms indigenous to man: McGraw Hill Book Company Inc. NY; Pp. 121 – 130.
Sabath, L. D. Jago, M. and Abraham, E. P. (1965). Cephalosporinase and Penicillinase activities of a beta-lactamase from Pseudomonas aeruginosa. Biochemical Journal. 96: 739 – 752.
Sagardia, F., Regau, J. J., Martinez – Lahoz, A., Fuentes, F., Lopez, C. and Flores, W. (1975). Degradation of Benzothiophene and related compounds by a soil Pseudomonas in an oil-aqueous environment. Applied Microbiology. 29(6):722– 725.
Schentag, J. J., Plaut, M. A. and Cerra, F. B. (1981). Comparative nephrotoxicity of gentamicin and tobramycin: Phamacokinetic and clinical studies in 201 patients. Antimicrobial Agents and Chemotherapy 19: 859 – 66.
Schwarzmann, S. and Boring III, J. R. (1971). Anti-phagocytic effect of slime from a mucoid strain of Pseudomonas aeruginosa. Infection and Immunity. 3:762- 767.
Simpson, J. A., Smith, S. E. and Dean, R. T. (1988). Alginate inhibition of the uptake of Pseudomonas aeruginosa by macrophages. Journal of General Microbiology. 134: 29 – 36.
Seijas, J. A., Vazquez-Tato, M. P. and Carballido-Reboredo (2006). Prediction of flavone UV-Vis spectrum: semiempirical versus ab initio methods. 10th International Electronic Conference on Synthetic Organic Chemistry (ECSOC-10). 1-30 Nov. 2006. http://www.mdpi.org/ecsoc-10/
Singleton, P. (1999). Bacteria. In : Biology, Biotechnology and Medicine. 4th ed. Wiley. Pp.107-112.
Sofonara, (1982). Food and Agricultural Organization the United Nations. Appendix 5– Medicinal Plants in Common Use in West Africa. http: //www.fao.org/docrep/t9450eof.htm. Accessed July 4, 2007.
+
Soission, A., Masterson, C. P., Tom, T. D., McNally, M. T., George, H. L. and Strand, M. (1992) Induction of protective immunity in mice using a 62 – KDa recombinant fragment of a Schistosoma mansoni surface antigen. The Journal of Immunology. 149 (11): 3612– 20.
Sokol, P. A., Iglewski, B. H., Hagar, T. A., Sadoff, J. C., Cross, A. S., McManus, A. Farber, B. F., and Iglewski, W. J. (1981). Production of exoenzymes by clinical isolates of Pseudomonas aeruginosa. Infection and Immunity. 34 (1): 147 – 153.
Sokomba, E., Wambebe, C., Chowdhury, B. K., Iriah, J., Ogbeide, O. N. and Orkor, D. (1986). Preliminary phytochemical, pharmacological and antibacterial studies of the alkalodal extracts of the leaves of Synclisia scarbida Miers. Journal of ethnopharmacology. 18: 173 – 185.
Sote, E. O. and Wilson, M. (1995). In vitro antibacterial effects of extracts of Nigerian tooth-cleaning sticks on periodontopathic bacteria. Afr. Dent. J. 9:15 – 9.
Stanislavskii, E. S. (1982). Experimental immunological effectiveness and safely of pyimmunogen vaccine against Pseudomonas aeruginosa infection. Zh Mikrobiol. Epidemiol. Immunobiol (5): 70 – 75.
Starr, M.P., Stolp, H., Truper, H.G., Balow A. and Schlegel, H.G. (1981). Human and animal pathogenic members of the genus Pseudomonas. In: The Prokaryotes. Vol. 1. Springer-Verlag. Berlin, Heidelberg, New York. Pp. 655 – 700.
Taylor, L. (2005). Rainforest facts. Raintree Nutrition, Inc., Carson City, NV 89701.
Thomas, E. T., Jones, L. F., Simao, E., Sole-Vernin, C. and Famer III, J. J. (1975). Epidemiolgy of Pseudomonas aeruginosa in a general hospital: a four year study. Journal of Clinical Microbiology. 2(5); 397 – 402.
Trease, G. E. and Evans, W. C. (1989). Textbook of Pharmacognosy. 14th Ed. W.B. Sanders, London. Pp. 46 – 47, 832.
Udo, S. M., Ebana, R. A. and Madunagu, B. E. (1998). Study of normal faecal flora of humans as reservoir for multiple-drug resistant microorganisms. Mary Slessor Journal of Medicine, 1 (1) 16 – 19.
Ugwuozor, (2006). Personal Communication. Nsukka, Nigeria.
Umeh, E. U., Igoli, J. O., Agada, E. O. and Usman, S. S. (2009). Evaluating Extracts of Spondias mombin for Antimicrobial Activities. Bio-Research, 7(2): 505- 508.
Van Asperon, I. A., DeRover, C. M., Schijven, J. F., Octomo, S. B., Schellekens, J. F. P., Van Leeuven, N. J., Colle’, C., Havelaar, A. H., Kromhout, D. and Sprengen M. W. J. (1995). Risk of Otitis externa after swimming in recreational fresh water lakes containing Pseudomonas aeruginosa. British Medical Journal. 311 (7017): 407-1410.
+
Van Boxtel, R. A. J. and De Klundert, J. A. M. (1998). Expression of the Pseudomonas aeruginosa gentamicin resistant gene aacC3 in Escheichia coli. Antimicrobial Agents and Chemotherapy. 42: 3173 - 3178.
Vera, R. (1993). Chemical composition of the essential oil of Ageratum conyzoides, L. (Asteraceae) from Reunion. Flavour Fragrance Journal. 8: 256 – 260.
Van der Auwera, P. and Schuyleneer, F. (1983). In vitro susceptibility of Pseudomonas aeruginosa to old and new -lactam antibiotics and aminoglycosides. Journal of Antimicrobial Chemotherapy. 11: 511 – 515.
Vandeputte, O. M., Kiendrebeogo, M., Rasamiravaka, T., Stevigni, C., Zuez, P., Rajaonson, S., DIALLO, B., Mol, A., Baucher, M. and El Jaziri, M. (2011). The flavonone naringenin reduces the production of quorum sensing- controlled virulence factors in P. aeruginosa PAO1. Microbiology. 157 (7): 2120-2132.
Vliegenthart, J.S., Van Gaalen, K. and Van de Klundert, J.A.M. (1991). Nucleotide sequence of the aacC3 gene, a gentamicin resistance determinant encoding aminoglycoside–(3)–N-acetyltransferase III expressed in Pseudomonas aeruginosa but not in Escherichia coli. Antimicrobial Agents and Chemotherapy. 35: 892 – 897.
Wain, J., Hoa, N. T. T, Chinh, N. T., Vinh, H., Everett, M. J., Diep, T. S., Day, N. P. J., Solomon, T., White, N. J., Piddock, L. J. V. and Parry, C. M. (1997). Quinolone-resistant Salmonella typhi in Viet Nam : molecular basis of resistance and clinical response to treatment. Clinical Infectious Diseases, 25 (6): 1404-1410.
Weinstein, R. A. (1991). Epidemiology and control of nosocomial infections in adult intensive care units. American Journal of Medicine. 91: 1795 – 1845.
Wikipedia, (2007). Terminalia schimperiana. Accessed March, 13 2008. http://en.wikipedia.org/wiki/Terminalia.glaucescens.
Wilson, L. A., and Ahearn, D. G. (1977). Pseudomonas induced corneal ulcers associated with contaminated eye mascaras. American Journal of Ophthalmology. 84: 112 – 119.
Wilson, L. A., Schlitzer, R. L. and Ahearn, D. G. (1981). Pseudomonas corneal ulcers associated with soft contact-lens wear. American Journal of Ophthalmology. 92: 546 – 554.
Wood, D. E. Schaffer, M. S., Rabin, H. R., Campbell, G. D. and Sokol, P. A. (1986). Phenotypic comparism of Pseudomonas aeruginosa strains isolated from a veriety of clinical sites. Journal of Clinical Microbiology. 24 (2): 260 – 264.
Wood, P., Caldwell, D. E., Evans, E., Jones, M., Korber, D. R., Wolfhaardt, G. M., Wilson, M. and Gilbert, P. (1998). Surface – catalyzed disinfection of thick
+
Pseudomonas aeruginosa biofilms. Journal of Applied Microbiology. 84(6): 1092 – 1098.
World Health Organization. The world drug situation. Geneva: WHO; 1988.
Yagi, T., Kukokawa, H., Senda, k., Ichiyama, S., Ito, H., Ohsuka, S., Shibayama, K., Shimokata, K., Kato, N., Michito, O. and Arakawa, Y. (1997). Nosocomial spread of cephem-resistant Escherichia coli strains carrying multipleToho-1- Like - lactamase genes. Antimicrobial Agents and Chemotherapy. 41 (12): 2606-2611.
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APPENDICES
Appendix 1
Composition of King’s Medium B
Formula
Proteose Peptone Number 3- 2.0 g
Glycerol 1.0 g
K2HPO4 0.15 g
MgSO4. 7H2O 0.15 g
Agar 1.5 g
Distilled water 100 ml
+2
Appendix 2
Composition and preparation of Nutrient Agar (Fluka)
Formula g/l
Peptone 5.0
Beef extract 3.0
Sodium chloride 8.0
Agar 12.0
Preparation
Twenty eight grams (28g) of the powder was suspended in 1 litre (or equivalent w/v concentrations for lower volumes) of distilled water. This was boiled over a Bunsen burner flame to dissolve completely and subsequently sterilized by autoclaving at 121oC for 15 min. After cooling to about 47oC, the sterile, molten medium was distributed, about 20ml each, into sterile Petri dishes.
+6
Appendix 3
Preparation of Blood Agar
Preparation
Nutrient agar was prepared as in Appendix 2 above. This was removed from the autoclave and cooled to 45oC - 50oC (It should not be too hot on your cheek). Five per cent (5%) human blood was asceptically added and the flask closed with cotton wool and foil. It was subsequently rotated to mix very well and to avoid air bubbles and then poured into sterile Petri dishes.
+=
Appendix 4
Composition and preparation of MacConkey Agar (ANTEC)
Formula g/l
Peptone 20.0
Lactose 10.0
Bile salts 5.0
Sodium chloride 5.0
Neutral red 0.075
Agar 12.0
Preparation
This was prepared as in Appendix 2 above using (w/v) concentration equivalent of 52 g of the dehydrated medium in one litre of distilled water according to the manufacturer’s instructions.
+?
Appendix 5
Composition and preparation of Mueller - Hinton Agar (Oxoid)
Formula g/l
Beef infusion solids 2.0
Acid hydrolysed casein 17.5
Starch 1.5
Agar 17.0
Calcium ions 5.0 mg/ml
Magnesium ions 20.0 mg/ml
Preparation
Thirty eight (38) grams of dehydrated medium was dispersed in 1 litre of distilled water (or equivalent w/v concentrations when smaller volumes were used).
The mixture was dissolved, sterilized and poured into sterile Petri dishes.
+
Appendix 6
Composition and preparation of Peptone Water (LAB M)
Formula g/l
Peptone 5.0
Tryptone 5.0
Sodium chloride 5.0
pH 7.2
Preparation
Fifteen grams of powder was weighed and dispersed in 1 litre of deionised water (or equivalent w/v concentrations when smaller volumes were used). This was allowed to dissolve and then distributed into final containers and sterilized at 121oC for 15 minutes. Glucose, sucrose, lactose and a pH indicator were added for studying fermentation reactions.
Appendix 7
Method of Gram Staining 1. A wire loop was sterilized by flaming and used to pick a bacterial colony from a 24 hr growth on nutrient agar. This was emulsified in a drop of sterile distilled water on a clean grease-free slide, and spread to make a thin smear, allowed to dry and then heat-fixed by waving it close to a flame three times until it dries. 2. The smear was flooded with crystal violet (primary stain) for 60 seconds. The slide was placed on a rack and rinsed in slow running water to remove excess dye. 3. The smear was flooded with Gram’s iodine for 60 seconds and again rinsed with water. (Iodine is a mordant that increases the affinity of cellular components to the dye). 4. The smear was then washed with acetone-alcohol (decolorizer), the slide being held in a slanting position, until the dye no longer runs off the slide. The decolorizer will wash off the dye-iodine complexes from gram-negative but not from gram-positive organisms. Care must be taken not to over decolorize the cells. 5. The smear was again flooded with safranin (counterstain) for 30 seconds, rinsed with water and blotted with paper towel. It was air-dried and viewed under the microscope with oil immersion lens (×100).
Result: Gram-positive organisms stain purple while gram-negative organisms stain reddish pink.
Appendix 8
Colony characteristics and biochemical reactions of P. aeruginosa isolates
Isolate Growth On Media Pigment Growth Temp (oC) Gm Oxidase Glu Suc Lac rxn MA BA NA BG 37 42 45 A G Pale - Cream colonies haem Discrete P. aerug.1 + + + + + + − − + + − − −
P. aerug.2 + + + + + + − − + + − − −
P. aerug.3 + + + + + + − − + + − − −
P. aerug.4 + + + + + + − − + + − − −
P. aerug.5 + + + + + + − − + + − − −
P. aerug.6 + + + + + + − − + + − − −
P. aerug.7 + + + + + + − − + + − − −
P. aerug.8 + + + + + + − − + + − − −
P. aerug.9 + + + + + + − − + + − − −
P.aerug. 10 + + + + + + − − + + − − −
P. aerug.11 + + + + + + − − + + − − −
P. aerug.12 + + + + + + − − + + − − −
P. aerug.13 + + + + + + − − + + − − −
P. aerug.14 + + + + + + − − + + − − −
P. aerug15* + + + + + + − − + + − − −
KEY: MA- MacConkey agar, BA-Blood agar, NA- Nutrient agar, BG- Blue-green, Gm rxn-Gram reaction, Glu- glucose, Suc- sucrose, Lac- lactose, A- acid, G- gas, - haem- beta haemolysis. * ATCC Typed sample.
Appendix 9
Preparation of McFarland Nephelometer Standards
Tube N u m b e r
0.5 1 2 3 4 5 6 7 8 9 10
Barium Chloride(ml) 0.05 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Sulphuric acid(ml) 9.95 9.9 9.8 9.7 9.6 9.5 9.4 9.3 9.2 9.1 9.0
Approx. Cell density 1.5 3 6 9 12 15 18 21 24 27 30 (×108 cfu/ml)
(Baron and Finegold, 1990)
Sulphuric acid was 1% while Barium Chloride (BaCl. 2H2O) was 1.17%
Appendix 10
Zone Diameter Interpretive Chart for Antibiotic susceptibility Testing
Antibiotic and Disc R I S Code Content
Ampicillin (PN) 10 g ≤ 13 14 – 16 ≥17
Augmentin (AU) 30 g ≤ 13 14 − 17 ≥18
Ceporex (CEP) 10 g ≤ 17 18 – 20 ≥21
Cephaplexin (CP) 30 g ≤ 14 15 – 17 ≥18
Ciprofloxacin 5 g ≤ 15 16 – 20 ≥21 (CPX)
Clindamycin (CD) 2 g ≤ 16 17 – 20 ≥21
Cotrimoxazole 30 g ≤ 15 16 – 18 ≥19 (Septrin)(SXT)
Erythromycin (E) 15 g ≤ 13 14 – 22 ≥23
Gentamicin (GN) 10 g ≤ 12 13 – 14 ≥15
Nalidixic acid 30 g ≤ 13 14 – 18 ≥19 (NA)
Ofloxacin (OFX) 5 g ≤ 14 15 – 17 ≥18
Perfloxacin (PEF) 10 g ≤ 12 13 – 16 ≥17
Streptomycin (S) 10 g ≤ 11 12 –14 ≥15
Appendix 11
Extracts and their Solubilities in different Solvents
Extracts Solvents
DMSO/Nutrient Tween80/Nutrient Propylene glycol Distilled Broth(1 in 4) Broth(1 in 10) water Ethanolic extracts
A. cordifolia L + NT NT NT
N. latifolia R Deep yellow - - NT Suspension N. latifolia L + NT + NT
S. mombin L - + - NT
T.schimperiana RB + NT NT NT
T.schimperiana L NT NT + NT
Aqueous extracts
A. cordifolia L + + NT NT
N. latifolia R NT NT NT +
N. latifolia L NT NT NT +
S. mombin L NT + NT -
T. schimperiana RB NT NT NT +
Ethylacetate-soluble + + NT NT Fraction
Acetone-solution fraction + + NT NT
F1 NT + NT NT
F7 NT + NT NT
F9 NT + NT NT
KEY: L- Leaves, RB- root bark. R- root, + Soluble, - Insoluble, NT- Not tested
2
Appendix 12
Dilution of Gentamicin Stock Solution (Control Antibiotic)
Injection ampoule contains 280 mg/2 ml (140mg/ ml).
Using the dilution formula: C1V1 = C2V2
C1=140 mg/ ml C2 = 1 mg/ ml V1 = 1 ml V2 = ?
V2 = C1 V1 = 140 × 1 = 140 ml C2 1
Therefore 139 ml of sterile distilled water + 1ml of gentamicin stock solution
= 1 mg/ml
1 mg/ml = 1000 µg/ml
1 ml of 1000 µg/ml + 99 ml of distilled water = 10 µg/ml
Serial dilution proceeds.
6
Appendix 13
Preparatory Thin Layer Chromatography (Prep. TLC)
Application of Samples on TLC plates Strips of silica precoated (Silica Gel 60 F254 Merck KGA Germany) chromatography plates, 1.5 by 5 cm dimension were used. A needle point was used to mark the starting line i.e. the place where the sample will be spotted, 0.5 cm from the lower edge of the plate. Another line was made for the solvent front, 3.75 cm from the starting line indicating the distance the solvent is allowed to travel. A small amount of the sample was dissolved in absolute ethanol in a test tube. A 1 ml syringe with needle was used to apply a small drop of the solution onto the plates. The drop was allowed to dry. The spotted plates were then placed separately in a separating chamber (a small jar) containing varying proportions of solvents. The jar contained an amount of the solvent system to cover the bottom up to a height of 0.3 cm, covered with a glass plate and allowed to stand for about 10 minutes for chamber saturation. The solvent systems surveyed in this Prep TLC were: n-Hexane: Ethylacetate, 1 : 10 to 1 : 1 Methanol: Chloroform, 1 : 2 n-Hexane: Acetone 1 : 5 Ethanol: Ethylacetate 1 : 1 Ethanol: Ethylacetate: Haxane 1 : 1 : 0.5 Ethanol: Ethylacetate: Water 0.1 : 1.75 : 1 Ethylacetate: n-Hexane: water 6 : 1 : 0.5
After development, the plates were allowed to dry and an iodine tank was used to develop the colour of the bands. The plate on which the components have separated as distinct bands (spots) was used to establish the best solvent system to be used for Thin Layer Chromatography.
=
Appendix 14
300 y = 207.45x - 43.954 2 250 R = 0.9277
200
150 2 Mean IZD
100
50
0 -0.5 0 0.5 1 1.5
-50
-100 Log drug concentration
Graph of square IZD against log drug concentration of ethanolic extract of T. schimperiana root bark on P. aeruginosa 1
?
Appendix 15
250
y = 187.52x - 60.223 200 R2 = 0.7998
150
2 Mean IZD 100
50
0 -0.5 0 0.5 1 1.5
-50
-100 Log drug concentration
Graph of square IZD against log drug concentration of ethanolic extract of T. schimperiana root bark on P. aeruginosa 2
+
Appendix 16
300 y = 210.53x + 7.3478 R2 = 0.9523 250
200
150 2 Mean IZD
100
50
0 -0.5 0 0.5 1 1.5
-50 Log drug concentration
Graph of square IZD against log drug concentration of ethanolic extract of T. schimperiana root bark on P. aeruginosa 3
Appendix 17
300 y = 220.16x - 47.336
250 2 R =2 0.9482
200
150 2 Mean IZD
100
50
0 -0.5 0 0.5 1 1.5
-50
-100 Log drug concentration
Graph of square IZD against log drug concentration of ethanolic extract of T. schimperiana root bark on P. aeruginosa 4
Appendix 18
300
y = 224.23x - 30.28 2 250 R = 0.8357
200
2 Mean IZD 150
100
50
0 -0.5 0 0.5 1 1.5
-50 Log drug concentration
Graph of square IZD against log drug concentration of ethanolic extract of T. schimperiana root bark on P. aeruginosa 5
Appendix 19
350
y = 266.75x - 47.201 300 2 2 R = 0.9158 250
200
2 Mean IZD 150
100
50
0 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4
-50
-100 Log drug concentration
Graph of square IZD against log drug concentration of aqueous extract of T. schimperiana root bark on P. aeruginosa 1
Appendix 20
y = 209.95x + 266.85 600
2 R = 0.9291
500
400
2 Mean IZD 300
200
100
0 -2 -1.5 -1 -0.5 0 0.5 1 1.5
-100 Log drug concentration
Graph of square IZD against log drug concentration of aqueous extract of T. schimperiana root bark on P. aeruginosa 2
Appendix 21
350
y = 147.49x + 109.01
300 2 2 R = 0.9242
250
200 2 Mean IZD
150
100
50
0 -1.5 -1 -0.5 0 0.5 1 1.5
-50 Log drug concentration
Graph of square IZD against log drug concentration of aqueous extract of T. schimperiana root bark on P. aeruginosa 3
2
Appendix 22
300 y = 227.61x - 42.871
2 R = 0.9301 250
200
150 2 Mean IZD
100
50
0 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4
-50
-100 Log grug concentration
Graph of square IZD against log drug concentration of aqueous extract of T. schimperiana root bark on P. aeruginosa 4
6
Appendix 23
300 y = 235.19x - 42.743
2 R 250 = 0.9219
200
150 2 Mean IZD
100
50
0 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4
-50
-100 Log drug concentration
Graph of square IZD against log drug concentration of aqueous extract of T. schimperiana root bark on P. aeruginosa 5
=
Appendix 24
350 y = 169x + 77.784
2 300 R = 0.9924
250
200
2 Mean IZD 150
100
50
0 -1.5 -1 -0.5 0 0.5 1 1.5
-50
-100 Log drug concentration
Graph of square IZD against log drug concentration of ethylacetate-soluble fraction of T. schimperiana on P. aeruginosa 1
?
Appendix 25
300 y = 150.57x + 64.759
2 250 R = 0.9771
200
150 2 Mean IZD
100
50
0 -1.5 -1 -0.5 0 0.5 1 1.5
-50
-100 Log drug concentration
Graph of square IZD against log drug concentration of ethylacetate-soluble fraction of T. schimperiana on P. aeruginosa 2
+
Appendix 26
250 y = 172.41x + 4.7419 2 R = 0.9539 200
150
100
2 Mean IZD 50
0 -1.5 -1 -0.5 0 0.5 1 1.5
-50
-100
-150
-200 Log drug concentration
Graph of square IZD against log drug concentration of ethylacetate-soluble fraction of T. schimperiana on P. aeruginosa 3
Appendix 27
350 y = 172.91x + 71.344
2 300 R = 0.9848 2
250
200 2 Mean IZD 150
100
50
0 -1.5 -1 -0.5 0 0.5 1 1.5
-50
-100
-150 Log drug concentration
Graph of square IZD against log drug concentration of ethylacetate-soluble fraction of T. schimperiana on P. aeruginosa 4
Appendix 28
350 y = 162.86x + 67.267
2 300 R = 0.9868 2
250
200
2 Mean IZD 150
100
50
0 -1.5 -1 -0.5 0 0.5 1 1.5
-50
-100 Log drug concentration
Graph of square IZD against log drug concentration of ethylacetate-soluble fraction of T. schimperiana on P. aeruginosa 5
Appendix 29
Univariate Analysis of Variance for Plant Extract
Between-Subjects Factors Value Label N Test organism 1 P. aeruginosa 1 5 2 P. aeruginosa 2 5 3 P. aeruginosa 3 5 4 P. aeruginosa 4 5 5 P. aeruginosa 5 5 6 P. aeruginosa 6 5 7 P. aeruginosa 7 5 8 P. aeruginosa 8 5 9 P. aeruginosa 9 5 10 P. aeruginosa 10 5 11 P. aeruginosa 11 5 12 P. aeruginosa 12 5 13 P. aeruginosa 13 5 14 P. aeruginosa 14 5 15 P. aeruginosa 15 5 Plant extract 1 Tset 15 2 Tsaq 15 3 Tslet 15 4 Acet 15 5 Gen 15
KEY Tset: Ethanolic extract of T. schimperiana root bark Tsaq: Aqueous extract of T. schimperiana root bark Tslet: Ethanolic extract of T. schimperiana leaves Acet: Ethanolic extract of A. cordifolia leaves
Descriptive Statistics Dependent Variable:Measurement Test organism Plant extract Mean Std. Deviation N P. aeruginosa 1 Tset 1.622000E0 . 1 Tsaq 1.496000E0 . 1 Tslet 1.585000E0 . 1 Acet 1.318000E0 . 1 Gen .200000 . 1 Total 1.244200E0 .5954160 5 P. aeruginosa 2 Tset 2.042000E0 . 1 Tsaq .054000 . 1 Tslet 1.462000E0 . 1 Acet 3.020000E0 . 1 Gen 1.109000E0 . 1 Total 1.537400E0 1.1002740 5 P. aeruginosa 3 Tset .933000 . 1 Tsaq .178000 . 1 Tslet .158000 . 1 Acet 3.311000E0 . 1 Gen .000000 . 1 Total .916000 1.3869551 5 P. aeruginosa 4 Tset 1.622000E0 . 1 Tsaq 1.549000E0 . 1 Tslet .056000 . 1 Acet 1.445000E0 . 1 Gen .000000 . 1 Total .934400 .8300496 5 P. aeruginosa 5 Tset 1.318000E0 . 1 Tsaq 1.549000E0 . 1 Tslet .692000 . 1 Acet 4.365000E0 . 1 Gen .000000 . 1 Total 1.584800E0 1.6666327 5
P. aeruginosa 6 Tset 2.089000E0 . 1 Tsaq .912000 . 1 Tslet .684000 . 1 Acet 3.055000E0 . 1 Gen 1.160000E0 . 1 Total 1.580000E0 .9823627 5 P. aeruginosa 7 Tset 2.089000E0 . 1 Tsaq .095000 . 1 Tslet 1.462000E0 . 1 Acet 3.020000E0 . 1 Gen .691000 . 1 Total 1.471400E0 1.1492586 5 P. aeruginosa 8 Tset .912000 . 1 Tsaq .105000 . 1 Tslet 1.462000E0 . 1 Acet 4.074000E0 . 1 Gen .692000 . 1 Total 1.449000E0 1.5458451 5 P. aeruginosa 9 Tset 2.512000E0 . 1 Tsaq 1.038000E0 . 1 Tslet 1.585000E0 . 1 Acet 3.034000E0 . 1 Gen .000000 . 1 Total 1.633800E0 1.1998392 5 P. aeruginosa 10 Tset 1.622000E0 . 1 Tsaq 1.622000E0 . 1 Tslet 1.585000E0 . 1 Acet 4.365000E0 . 1 Gen .000000 . 1 Total 1.838800E0 1.5749040 5 P. aeruginosa 11 Tset 2.089000E0 . 1 Tsaq 1.585000E0 . 1 Tslet 1.445000E0 . 1 Acet .603000 . 1
2
Gen .000000 . 1 Total 1.144400E0 .8335951 5 P. aeruginosa 12 Tset 1.000000 . 1 Tsaq 1.472000E0 . 1 Tslet 1.047000E0 . 1 Acet 3.062000E0 . 1 Gen .000000 . 1 Total 1.316200E0 1.1154511 5 P. aeruginosa 13 Tset .282000 . 1 Tsaq .933000 . 1 Tslet .891000 . 1 Acet 3.055000E0 . 1 Gen 1.205000E0 . 1 Total 1.273200E0 1.0516360 5 P. aeruginosa 14 Tset 1.549000E0 . 1 Tsaq .759000 . 1 Tslet .902000 . 1 Acet 1.950000E0 . 1 Gen .759000 . 1 Total 1.183800E0 .5386917 5 P. aeruginosa 15 Tset 1.250000E0 . 1 Tsaq 1.472000E0 . 1 Tslet .902000 . 1 Acet 1.318000E0 . 1 Gen 1.995000E0 . 1 Total 1.387400E0 .3986362 5 Total Tset 1.528733E0 .5909871 15 Tsaq .987933 .6143358 15 Tslet 1.061200E0 .5096896 15 Acet 2.733000E0 1.1601584 15 Gen .520733 .6274955 15 Total 1.366320E0 1.0452513 75
6
Tests of Between-Subjects Effects Dependent Variable:Measurement Type III Sum Source of Squares df Mean Square F Sig. Corrected 47.351a 18 2.631 4.398 .000 Model Intercept 140.012 1 140.012 234.067 .000 Organism 4.669 14 .333 .558 .886 Extract 42.682 4 10.671 17.839 .000 Error 33.498 56 .598 Total 220.861 75 Corrected Total 80.849 74 a. R Squared = .586 (Adjusted R Squared = .452)
Post Hoc Tests
Measurement Tukey HSD
Plant Subset extract N 1 2 3 Gen 15 .520733 Tsaq 15 .987933 .987933 Tslet 15 1.061200 1.061200 Tset 15 1.528733 Acet 15 2.733000 Sig. .322 .322 1.000 Means for groups in homogeneous subsets are displayed. Based on observed means. The error term is Mean Square (Error) = .598. KEY Gen: Gentamicin Tset: Ethanolic extract of T. schimperiana root bark Tsaq: Aqueous extract of T. schimperiana root bark Tslet: Ethanolic extract of T. schimperiana leaves Acet: Ethanolic extract of A. cordifolia leaves
=
Appendix 30
Univariate Analysis of Variance for Fractions
Between-Subjects Factors Value Label N Test organism 1 P. aeruginosa 1 6 2 P. aeruginosa 2 6 3 P. aeruginosa 3 6 4 P. aeruginosa 4 6 5 P. aeruginosa 5 6 6 P. aeruginosa 6 6 7 P. aeruginosa 7 6 8 P. aeruginosa 8 6 9 P. aeruginosa 9 6 10 P. aeruginosa 10 6 11 P. aeruginosa 11 6 12 P. aeruginosa 12 6 13 P. aeruginosa 13 6 14 P. aeruginosa 14 6 15 P. aeruginosa 15 6 Fractions 1 EASF 15 2 ASF 15
3 F1 15
4 F7 15
5 F9 15 6 Gen 15
?
Descriptive Statistics Dependent Variable:Measurement for Fraction Test organism Fractions Mean Std. Deviation N P. aeruginosa 1 EASF .331000 . 1 ASF 1.585000E0 . 1
F1 1.318000E0 . 1
F7 1.585000E0 . 1
F9 1.472000E0 . 1 Gen .200000 . 1 Total 1.081833E0 .6412081 6 P. aeruginosa 2 EASF .363000 . 1 ASF 1.549000E0 . 1
F1 1.581000E0 . 1
F7 1.445000E0 . 1
F9 .912000 . 1 Gen 1.109000E0 . 1 Total 1.159833E0 .4709779 6 P. aeruginosa 3 EASF .912000 . 1 ASF 1.585000E0 . 1
F1 1.531000E0 . 1
F7 1.549000E0 . 1
F9 1.585000E0 . 1 Gen .000000 . 1 Total 1.193667E0 .6403929 6 P. aeruginosa 4 EASF .398000 . 1 ASF .363000 . 1
F1 1.585000E0 . 1
F7 1.585000E0 . 1
F9 1.452000E0 . 1 Gen .000000 . 1 Total .897167 .7202054 6 P. aeruginosa 5 EASF .398000 . 1 ASF .955000 . 1
F1 2.512000E0 . 1
+
F7 2.512000E0 . 1
F9 .000000 . 1 Gen .000000 . 1 Total 1.062833E0 1.1758426 6 P. aeruginosa 6 EASF 1.000000 . 1 ASF 1.445000E0 . 1
F1 2.089000E0 . 1
F7 2.089000E0 . 1
F9 2.089000E0 . 1 Gen 1.160000E0 . 1 Total 1.645333E0 .5064894 6 P. aeruginosa 7 EASF 1.000000 . 1 ASF 1.585000E0 . 1
F1 1.995000E0 . 1
F7 2.089000E0 . 1
F9 1.514000E0 . 1 Gen .691000 . 1 Total 1.479000E0 .5479420 6 P. aeruginosa 8 EASF .398000 . 1 ASF 1.445000E0 . 1
F1 .000000 . 1
F7 2.512000E0 . 1
F9 1.445000E0 . 1 Gen .692000 . 1 Total 1.082000E0 .9054411 6 P. aeruginosa 9 EASF 1.000000 . 1 ASF 1.660000E0 . 1
F1 2.089000E0 . 1
F7 1.445000E0 . 1
F9 2.512000E0 . 1 Gen .000000 . 1 Total 1.451000E0 .8815083 6 EASF 1.000000 . 1 P. aeruginosa 10 ASF 1.000000 . 1
F1 2.089000E0 . 1
F7 2.089000E0 . 1
F9 .000000 . 1 Gen .000000 . 1 Total 1.029667E0 .9345118 6 P. aeruginosa 11 EASF 1.000000 . 1 ASF 1.585000E0 . 1
F1 2.089000E0 . 1
F7 2.089000E0 . 1
F9 1.585000E0 . 1 Gen .000000 . 1 Total 1.391333E0 .7921416 6 P. aeruginosa 12 EASF .288000 . 1 ASF 1.000000 . 1
F1 1.995000E0 . 1
F7 2.089000E0 . 1
F9 2.089000E0 . 1 Gen .000000 . 1 Total 1.243500E0 .9500587 6 P. aeruginosa 13 EASF .263000 . 1 ASF .398000 . 1
F1 1.995000E0 . 1
F7 2.089000E0 . 1
F9 2.089000E0 . 1 Gen 1.205000E0 . 1 Total 1.339833E0 .8504757 6 P. aeruginosa 14 EASF 1.000000 . 1 ASF 1.380000E0 . 1
F1 2.512000E0 . 1
F7 1.995000E0 . 1
F9 2.089000E0 . 1 Gen .759000 . 1
Total 1.622500E0 .6840371 6 P. aeruginosa 15 EASF 1.000000 . 1 ASF 1.585000E0 . 1
F1 2.089000E0 . 1
F7 2.089000E0 . 1
F9 .000000 . 1 Gen 1.995000E0 . 1 Total 1.459667E0 .8290152 6 Total EASF .690067 .3335436 15 ASF 1.274667E0 .4331898 15
F1 1.831267E0 .6076756 15
F7 1.950067E0 .3491071 15
F9 1.388867E0 .8169224 15 Gen .520733 .6274955 15 Total 1.275944E0 .7588717 90
Tests of Between-Subjects Effects Dependent Variable:Measurement for Fraction Type III Sum Source of Squares df Mean Square F Sig. Corrected 29.692a 19 1.563 5.073 .000 Model Intercept 146.523 1 146.523 475.680 .000 Organism 4.354 14 .311 1.010 .454 Fractions 25.338 5 5.068 16.451 .000 Error 21.562 70 .308 Total 197.777 90 Corrected Total 51.254 89 a. R Squared = .579 (Adjusted R Squared = .465)
Post Hoc Tests Fractions
Measurement for Fraction Tukey HSD
Fractio Subset ns N 1 2 3 4 GEN 15 .520733 EASF 15 .690067 .690067 ASF 15 1.274667E0 1.274667E0
F9 15 1.388867E0 1.388867E0
F1 15 1.831267E0 1.831267E0
F7 15 1.950067E0 Sig. .960 .056 .079 .075 Means for groups in homogeneous subsets are displayed. Based on observed means. The error term is Mean Square(Error) = .308.
KEYS EASF Ethylacetate-soluble fraction ASF Acetone-soluble fraction
F1
F7 Fractions of Ethylacetate-Soluble Fraction
F9
Appendix 31
Independent Samples t-test: Group Statistics Comparing Mean MIC of Extracts for Gentamicin-Sensitive and Gentamicin-Resistant P. aeruginosa Groups N Mean Std. Deviation Std. Error Mean Tsaq Sensitive 8 0.728 0.593 0.210 Resistant 7 1.285 0.527 0.199
Tset Sensitive 8 1.479 0.643 0.227 Resistant 7 1.585 0.571 0.216 Tslet Sensitive 8 1.169 0.356 0.126 Resistant 7 0.938 0.652 0.247 Acet Sensitive 8 2.601 0.974 0.345 Resistant 7 2.884 1.408 0.532
Levene’s Test for Equality of Variances F Sig.
Tsaq Equal variances assumed 0.367 0.555 Equal variances not assumed Tset Equal variances assumed 0.146 0.708 Equal variances not assumed
Tslet Equal variances assumed 4.601 0.051 Equal variances not assumed Acet Equal variances assumed 0.592 0.455 Equal variances not assumed KEY Tset: Ethanolic extract of T. schimperiana root bark Tsaq: Aqueous extract of T. schimperiana root bark Tslet: Ethanolic extract of T. schimperiana leaves Acet: Ethanolic extract of A. cordifolia leaves
t-test for Equality of Means t df Sig. (2- Mean tailed) Difference Tsaq Equal variances assumed -1.907 13.000 0.079 -0.556 Equal variances not -1.924 12.992 0.077 -0.556 assumed Tset Equal variances assumed -0.335 13.000 0.743 -0.106 Equal variances not -0.338 12.992 0.741 -0.106 assumed Tslet Equal variances assumed 0.865 13.000 0.403 0.230 Equal variances not 0.832 9.009 0.427 0.230 assumed Acet Equal variances assumed -0.457 13.000 0.655 -0.282 Equal variances not -0.445 10.501 0.665 -0.282 assumed
t-test for Equality of Means 95% Confidence Interval of the Difference Std. Error Lower Upper Difference Tsaq Equal variances assumed 0.292 -1.186 0.074 Equal variancesnot assumed 0.289 -1.181 0.069 Tset Equal variances assumed 0.316 -0.789 0.577 Equal variances not assumed 0.313 -0.783 0.571
Tslet Equal variances assumed 0.266 -0.345 0.805 Equal variances not assumed 0.277 -0.396 0.856 Acet Equal variances assumed 0.618 -1.618 1.053 Equal variances not assumed 0.634 -1.686 1.121 KEY Tset: Ethanolic extract of T. schimperiana root bark Tsaq: Aqueous extract of T. schimperiana root bark Tslet: Ethanolic extract of T. schimperiana leaves Acet: Ethanolic extract of A. cordifolia leaves
2
Appendix 32
Independent Samples t-test: Group Statistics Comparing Mean MIC of Fractions for Gentamicin-Sensitive and Gentamicin-Resistant P. aeruginosa Groups N Mean Std. Deviation Std. Error EASF Sensitive 8 0.669 0.355 0.126 Resistant 7 0 . 7 1 4 0.333 0.126 ASF Sensitive 8 1.372 0.401 0.142 Resistant 7 1.163 0.472 0.178
F1 Sensitive 8 1.698 0.773 0.273 Resistant 7 1.984 0.336 0.127
F7 Sensitive 8 1.987 0.332 0.117 Resistant 7 1.908 0.390 0.147
F9 Sensitive 8 1 . 4 5 1 0.719 0.254 Resistant 7 1.102 0.817 0.309
Levene’s Test for Equality of Variances
F Sig. EASF Equal variances assumed 1.075 0.319 Equal variances not assumed ASF Equal variances assumed 0.953 0.347 Equal variances not assumed
F1 Equal variances assumed 2.205 0.161 Equal variances not assumed
F7 Equal variances assumed 0.836 0.377 Equal variances not assumed
F9 Equal variances assumed 0.474 0.503 Equal variances not assumed
6
t-test for Equality of Means T df Sig. Mean (2-tailed) Difference EASF Equal variances assumed -0.248 13.000 0.808 -0.044 Equal variances not assumed -0.250 12.920 0.807 -0.044
ASF Equal variances assumed 0.925 13.000 0.372 0.208 Equal variances not assumed 0.915 11.910 0.378 0.208
F1 Equal variances assumed - 0 . 9 0 6 13.000 0.381 -0.287 Equal variances not assumed -0.952 9.810 0.364 -0.287
F 7 Equal variance assumed 0 . 4 2 1 13.000 0.681 0.078 Equal variances not assumed 0.416 11.925 0.685 0.078
F9 Equal variances assumed 0 . 8 8 2 13.000 0.394 0.350 Equal variances not assumed 0.875 12.122 0.399 0.350
=
t-test for Equality of Means Std. Error 95% Confidence Difference Interval of the Difference Lower Upper EASF Equal variances assumed 0.179 -0.430 0.342 Equal variances not assumed 0.178 -0.429 0.340 ASF Equal variances assumed 0.225 -0.278 0.695 Equal variances not assumed 0.228 -0.288 0.705
F1 Equal variances assumed 0 . 3 1 7 -0.971 0.397 Equal variances not assumed 0.301 -0.960 0.387
F7 Equal variances assumed 0 . 1 8 6 -0.324 0.481 Equal variances not assumed 0.188 -0.332 0.489
F9 Equal variances assumed 0 . 3 9 6 -0.507 1.206 Equal variances not assumed 0.400 -0.521 1.220 KEYS EASF Ethylacetate-soluble fraction ASF Acetone-soluble fraction
F1
F7 Fractions of Ethylacetate-Soluble Fraction
F9