Chapter One INTRODUCTION The primary function of normal intact skin is to control microbial populations that live on the

skin surface and prevent the underlying tissue from becoming colonized and invaded by potential pathogens. The exposure of subcutaneous tissue following a loss of skin integrity (i.e., a wound) provides a moist, warm, and nutritious environment that is conducive to microbial colonization and proliferations. (Bowler, 2001) A bridge in the skin or exposure of the subcutaneous layer of the skin usually occurs as a result of injuries commonly referred to as wound. Wound can be defined as a break on the skin or the body resulting in blood loss. It can also be described as injury on the skin in which there is a breakage of the external part of the skin. The internal part of the body may also have an injury or ulcer (Wikipedia). Wound could be due to several factors which include; (1) road accident (2) knife cut (3) pressure (4) surgery, (5) bullet (6) amputation (7) crushing (8) burnt (9) laceration (10) bite Wound can be chronic or acute and may also be referred to as closed wound or open wound. A chronic wound is a kind of wound that fails to heal in an orderly stage and in a predictable amount of time, the way most wounds heal. It usually arises as a result of endogenous compromise of both epidermal and dermal tissues. Wounds that usually will not heal up within three months are often referred to as chronic wounds. (Mustae, 2005) Chronic wound is often associated with one or more delayed phases of wound healing, for example chronic wound often remains in the inflammatory stage for long. (Syder, 2004, Taylor, et al, 2005).


Chronic wound usually involves Collagen fibres of the epithelial tissue. (Edwards, 2004) While acute wound on the other hand, is such type of wound which is associated with the interrupted wound healing and have a regular course of healing, it usually heal within a limited period of time. In acute wounds, there is a precise balance between production and degradation of molecules such as collagen. Wound may also be classified as open wound or closed wound. An open wound is a break
in the external skin resulting in bleeding (Jones and Ballet 2010), an open wound may be

chronic or acute. A closed wound is a type of wound in which the skin remains intact, without any damage to the skin. Closed wounds typically are caused by a blunt impact, which ruptures a blood vessel or capillary under the skin. The blood pools out, but typically stops bleeding within about 30 seconds after the injury occurred. As the body reabsorbs the blood, the skin will be discolored changing from black and blue to green then yellow. Chronic wounds ranges from bruises to hematoma, to crushing injury. Classification of Wounds Wounds can be further classified into various types, Open wound for example can be further classified into a) Incision or Incised wound, b) Laceration, c) Abrasions d) Puncture wound e) Penetration wound f) Gunshot wound. While closed wound can be classified into contusion, b) Hematoma, c) crush wound d) chronic and acute wound Closed wound on its own can be classified into a) Contusions b) Hematomas c) Crush injury d) Traumatic wounds Chronic wound can be classified into three which are venous ulcers, diabetic, and pressure ulcers(Moreo 2005, Mustoe 2004), other types of chronic wounds which do not fall on any of the above mentioned categories may be due to Ischemia or radiation poisoning.(Mustoe,2004).

Types of Open wounds

Incised wound or Incision: This type of wound is caused by a clean, sharp-edged object such as a knife, a razor or a glass splinter. It tends to have smooth edges and resembles a paper or surgical cut. The amount of bleeding from this type of wound depends on the depth, the location and the size of the wound (Jones and Bartlett 2010).

Laceration: This is a wound made by tearing or cutting of body tissue, it is usually manifested as a wound with jagged, irregular edges. It is often caused by forceful tearing away of skin tissue Abrasion: is a superficial wound in which the topmost layer of the skin is cut off, with little or no blood lost. It may be painful as it often involves the abrasion of nerve ending along with the epidermis. It usually arises from sliding fall onto a rough surface. Abrasion can be very serious if it covers a large surface area or have foreign matter embedded in it. Abrasion can also be called scrape road rash and rug burn. Puncture wound: is usually a deep narrow wound in the skin and underlying organs, such as stab wound from a nail or knife. This type of wound gives room for a high risk of infection. The object causing the injury may have the remains of the object responsible for the puncture within the wound. Penetration wound or Avulsion: Penetration or avulsion wound is a type of wound which occurred as a result of an object such as knife or nail entering or piercing the skin and coming out. It may result in a piece of skin getting torn and hanging on the skin or may be removed completely. This type of wound is commonly associated with the hand, finger, and ear.


Gun-shot wound: caused by a bullet or similar projectile driving into or through the body. There may be two wounds, one at the site of entry and one at the site of exit, generally referred to as a "through-and-through."

Types of closed wound Contusions, more commonly known as bruises, caused by a blunt force trauma that damage tissue under the skin. Hematomas: also called a blood tumor, caused by damage to a blood vessel that in turn causes blood to collect under the skin. Crush injury, caused by a great or extreme amount of force applied over a long period of time.
Chronic and Acute: Acute or traumatic wounds are the result of injuries that disrupt

the tissue. Chronic wounds are those that are caused by a relatively slow process that leads to tissue damage. Chronic wound can be categorized into Venous ulcers Diabetic ulcers Pressure ulcers

Venous and arterial ulcers
Venous ulcers, which usually occur in the legs, account for about 70% to 90% of chronic wounds (Synder, 2005) and mostly affect the elderly. They are thought to be due to venous hypertension caused by improper function of valves that exist in the veins to

prevent blood from flowing backward. Ischemia results from the dysfunction and, combined with reperfusion injury, causes the tissue damage that leads to the wounds.

Diabetic ulcers
Another major cause of chronic wounds, diabetes, is increasing in prevalence. (Synder 2005) (Velander 2004) Diabetics have a 15% higher risk for amputation than the general population (Synder 2005) due to chronic ulcers. Diabetes causes neuropathy, which inhibits nociception and the perception of pain.(Synder 2005) Thus patients may not initially notice small wounds to legs and feet, and may therefore fail to prevent infection or repeated injury.(Mustoe, 2004)Further, diabetes causes immune compromise and damage to small blood vessels, preventing adequate oxygenation of tissue, which can cause chronic wounds.(Mustoe 2004) Pressure also plays a role in the formation of diabetic ulcers.(Moreok 2004)

Pressure ulcers Pressure is another important cause of chronic wound; it usually occurs in people with critical health cases such as paralysis. Pressure ulcers are caused by ischemia that occurs when pressure on the tissue is greater than the pressure in capillaries, and thus restricts blood flow into the area.(Boyce 2005) Muscle tissue, which needs more oxygen and nutrients than skin does, shows the worst effects from prolonged pressure.(e medicine) As in other chronic ulcers, reperfusion injury damages tissue. Wound infections
The skin is meant to protect the body from microbial infections but any bridge on the skin surface usually gives room for the invasion of the internal layer of the skin by microorganisms. The presence of microbial normal flora on the skin surface provides an opportunity for the easy access of microbial population into the abridged skin or the skin which has lost its integrity. The 5

abundance and diversity of microorganisms in any wound will be influenced by factors such as wound type, depth, location, and quality, the level of tissue perfusion, and the antimicrobial efficacy of the host immune response. The population of microorganisms in clean surgical wounds is usually smaller, but in traumatic wounds the population of microorganisms present is usually influenced by the presence of devitalized tissue, and the presence of foreign materials (Robson 1997). Wound infections usually occur when the virulence factors expressed by one or more microorganisms in a wound out-compete the host natural immune system and subsequent invasion and dissemination of microorganisms in viable tissue provokes a series of local and systemic host responses (Peel, 1992). Wound colonization is most frequently polymicrobial involving numerous microorganisms that are potentially pathogenic (Brook, 1998, Lydon, 1989, Mousa 1987, Summannen, 1995). Hence any wound is at the risk of being infected by microorganisms. An infected wound usually manifests by the failure of such wound to heal in time, causing a lot of pain to the patient and leading to an increase in treatment cost and general wound management practices become more resources demanding (Bowler 2001). Colonization of wound by microbes usually precedes the incidence of infection in a wound. Several microorganisms have been found associated with wound infections, these include both aerobic and an aerobic bacteria. They include Staphylococcus epidermidis, micrococci, skin diphtheroids, propionibacteria, Bacteroides, Prevotella, Porphyromonas, and Peptostreptococcus spp. These are mostly microorganisms which are at closer proximity to the location of the wounds The ability of microorganisms to invade a wound, colonize the wound and be able to overcome the host immune responses is referred to as wound infection. Wound infection usually results in the prolong healing of wounds, increased cost of treatment for the affected patients and the general management of wound become more resource demanding. (Bowler 2001)


It has been observed that wound infections account for one of the cause of prolong hospitalization. Most of the cases recorded in one of the hospital study were found to be due to wound infection and that the cost of treating a patient amounted to about $3,937 per patient in the US (Zoutman,, 1998). Wound infections are usually characterized by Traumatic pain, inflammation, purulent discharge or painful spreading erythema indicative of cellulitis around a wound (Peel, 1992). Treatment of wound will require the reduction of the microbial load in a wound, this can be achieved through the use of several antimicrobial agents, but a major constrain is increasing tendency of microbial resistance to ensue, most especially with the use of topical agents. As a result of the possibility of inducing resistance associated with the use of topical agents, antiseptics are often used in the topical treatment of wounds (Bowler, 2001). Broad spectrum antimicrobial agents are often used either as a prophylaxis in the treatment of wound that are likely to be heavily infected from surgery as a therapeutics for wound that have been clinically infected. The common antibiotics used in wound infections include ceftriazone, co-trimoxazole, amoxycillin, ciprofloxacin, pefloxacin, and Azithromycin.

High vaginal swab Swabs are clinical materials used for the collection of microorganism from the interior part of the body such as ear, nose and eye, cervix and the vaginal. Swab usually is a rolled stick with clean thin cotton wool on its head. High vagina swab is a type of swab that is often used for the collection of bacteria and other microorganisms from the vaginal region of the woman. Microorganisms for cultural identification are mostly extracted from the vagina region through the use of high vaginal swab.

The vagina The vagina is an available space which attaches the lower part of the uterus to the outside environment (Michael and Mickey, 2010). The vagina can be divided into three regions, the upper region, the middle region and the lower regions; the upper region is the part of the vagina close to the cervix, the middle region of the vagina is below the urethrovessical junction and it ends at the lower level margin of the pubis symphysis, the lower region of the vagina is located around the rectum and urethra with which it shares a common wall, and it is associated with the vulva, an organ with which it is attached at the lower part forming the vulvo- vaginal vestibule( Michael And Mickey 2010). Vaginal infection may be defined as the presence of microorganisms in a vaginal discharge other than the normal microbial flora of the vagina; it can also be defined in terms of the reduction in the number of normal microbial flora of the vagina resulting in a variety of manifestations such as inflammation of the vagina, odor and pain sensation. Vaginal infection is also known as vaginitis. Vaginitis is a collective term for all the inflammation of the vagina which creates discharge causes pain, odour and itching (Melissa, 2006). Vaginitis mostly arises from the alteration of the vagina ecosystem, resulting in a change of composition or property of the vaginal ecosystem or the vaginal normal flora (Melissa, 2006, Cooper 2003). Apart from the Lactobacillus which constitute the normal flora of the vagina, vagina also contain other microorganisms in small proportion such as Staphylococcus, Ureaplasma, Corynebacterium, Streptococcus, Peptostreptococcus, Gardnerella, Bacteroides, Mycoplasma, Enterococcus, Escherichia, Veillonella, Bifidobacterium and Candida (Larsen & Monif, 2001; Marrazzo 2002; Redondo-Lopez et al, 1990; Zhou, 2004), If any of these organisms overgrow in the vagina they could cause vaginitis. The vaginal microbial floras have been known for their ability to prevent the invasion of the vagina by other microbes which can cause infectious diseases including those

responsible for bacterial vaginosis (Donders et al., 2000; Gupta et al., 1998; Sobel, 1999; van-Wijgert et al, 2000; Zhou,, 2004) The change in the vaginal ecosystem could have resulted from the use of antibiotics, Hormonal disorder sometimes during menopause, Sexual intercourse, douches, vaginal medications, sexually transmitted diseases and change of sexual partner or having multiple sexual partners (Melissa 2006). This change in the vaginal normal flora may in turn be responsible for vaginitis. This could have resulted from the influence of any of the factors mentioned above on the population of the vaginal normal flora. Vaginitis is a common infection among women of all ages but it is mostly common in females experiencing their menstrual cycle and pregnant women in which there is a possibility of microbial contamination from the menstrual flow and low level of immunity (Brian,). It is the most common infection which takes women to the hospital in the USA (Brian, Vaginitis is common mostly among women of reproductive age although women of all ages are susceptible to the disease (centre for diseases control 2010, Yudin and Money, 2008). It can be divided into two: Infectious vaginitis and vaginitis due to hormonal changes and stress or specific and non-specific vaginitis. Infectious vaginitis could be caused by either Trichomonas vaginalis, Candida particularly albicans, and bacteria particularly Gardnerella vaginalis Mycoplasma and other anaerobes, however bacterial vaginosis had been described to have arisen from more than a group of bacteria (Brian 2008). Staphylococcus aureus may also be isolated from vaginal swab sample, if there is alteration of the vaginal normal flora; this may be common in women having their menstrual cycle (Charles,, 1982, Foley,, 1991).Staph aureus has been identified as the possible cause of bacterial vaginitis whenever there is an overgrowth of them in the vaginal (Cook, 1992). Staph species may be transmitted or transferred to the vagina through the use of tampon, or from those present in the cervical wall as blood flows from there through the vaginal (Smith, 1982). The presence of Staphylococcus aureus in a vaginal discharge could be indicative of a possible risk of

toxic shock syndrome (Centre for disease control UK, 1980, Davis,, and Shands 1980). Toxic shock syndrome (TSS) is a toxin-mediated multisystem disease precipitated by infection with either Staphylococcus aureus or group A Streptococcus (GAS), also called Streptococcus pyogenes (Sharma, 2006) Bacterial vaginosis usually result in dreadful contrary health conditions such as Preterm labour and delivery (Gravett,, 1986, Hiller,, 1995, Brian,, 2008). It may also lead to the development of pelvic inflammatory diseases (PID) (Sweet, 1995), and can lead to the acquisition of sexually transmitted diseases such as human immune deficiency virus (Sha, 2005, Schand,, 2000, Sewankambo,, 1997,). Other complications associated with bacterial vaginosis include a high risk of post abortionuterine infection, breast abscess, cervical intraepithelial neoplasia, and post hysterectomy cuff infection. The vaginal pathogens are capable of doing so because of their ability to overcome the normal microbial flora of the vagina. Also the possession of some virulent factors enables the pathogens to colonize the vagina and initiate an infection, these microorganisms e.g. Gardnerella could form biofilm (Moreo, 2010) which may prevent them from being overcome by vagina immunity. Treatment of vaginitis can be achieved through the use of various antimicrobial agents. The antibacterial agents used for the treatment of bacteria vaginosis or vaginitis and other vaginitis include; Metronidazole and clindamycin, the activities of some first generation cephalosporin have been identified effective in the treatment of vaginitis (Wathne, 1989). Though there have been available treatments for bacteria vaginitis and other form of vaginitis, there are report of bacteria resistance to antimicrobial treatment of this infection. This had been identified with the recurrent cases of bacteria vaginosis after its treatment with the said antimicrobial agents. The presence of Staph aureus in the vagina may influence the resistant witness to the antimicrobial agents of choice in the treatment of vaginitis owing to several reports of resistance associated with Staphylococcus aureus (Katherine, 2006).

Bacteria could acquire resistance to antimicrobial agent through the acquisition of transmissible resistance gene from resistant species or by chromosomal mutation due to selective pressure by the use of antibiotics. Schaberg 1991 showed the resistance of bacteria to the most recent beta lactam antibiotics, while the resistance of bacteria to the most recent cephalosporin has also been identified (Jacoby, 1994). Likewise resistance to amoxicillin, sulphamethazole to aminoglycosides and even the newly coming fluoroquinolones have been reported (Alos and Chacon, 1988, Hardy, 1980, Hooper, 2001, Katherine, 2006).


Chapter two LITERATURE REVIEW 2.1. Wound infection overview. Wound infection occurs when the virulence factor expressed by a microorganism in a wound outcompete the host natural immunity, in which subsequent invasion and microbial proliferation in the wound induces a series of local and systemic responses from the host tissue (Bowler 2001). Wound infection can thus be defined as the multiplication and invasion of an infectious agent within the body tissues causing signs and symptoms of disease. Wound infection is one of the most common infections which cause a high rate of morbidity, an increase in the cost of healthcare delivery to those affected and a more resource demanding wound management (Bowler, 2001). Wound infections are associated with the largest percentage of nosocomial infection in the US (Bowler, 2001, Zoutman, McDonald, and Vethanayagan, 1998). Wound infections can occur in people of all gender and different ages and can be acquired both from the community and from the hospital. Wound infection usually results in an increase in the days of hospitalization in the hospital. In the US, a study of post-surgical wound infections following head and neck surgery shows an average increase in the days of hospitalization from 14days to 24days, following an infection of this region (Bowler, 2001, Johnson and Yu, 1991). Another study similar to this on post-surgical wound infection of 108 patients in US shows a treatment cost increase to about $3,937 per infected patient (Zoutman,


1998). The characteristic local response to microbial invasion and proliferation include purulent discharge and painful spreading erythema which is a feature of cellulitis around a wound (Peel, 1992). The tendency of a wound to become infected depends on several microbial and host factors, which include size, depth, type and site of the wound, the extent of nonviable exogenous contamination of the wound, level of blood flow to the site, the general health and immune status of the host, the microbial load, and the combined level of virulence expressed by the types of microorganisms involved (Bowler,, 2001). Wound is easily colonized by mixed population of both aerobic and anaerobic bacteria that occupy the region close to the site of tissue abridgement. These bacteria include Coagulase-positive Staphylococcus aureus and Coagulase negative Staphylococcus aureus, Peptostreptococcus asaccharolyticus, Micrococcus spp, Peptostreptococcus anaerobius, Peptostreptococcus magnus, micros, Beta-hemolytic Streptococcus (group C), Streptococcus (group G Peptostreptococcus Streptococcus spp, Beta-hemolytic

Peptostreptococcus prevotii Streptococcus spp. (fecal) Peptostreptococcus indolicus, (viridans) Peptostreptococcus sp, Clostridium cadaveris, Corynebacterium xerosis, Escherichia coli, Clostridium Streptococcus intermedius, Corynebacterium sp, Clostridium perfringens, Bacillus sp. Clostridium clostridioforme, histolyticum, baratii, Escherichia hermanii, Clostridium septicum, Serratia liquefaciens, Clostridium Klebsiella pneumoniae, Clostridium tertium, Enterobacter aerogenes, Clostridium difficile, Citrobacter freundii Clostridium bifermentans, Proteus mirabilis, Clostridium limosum Proteus vulgaris, Eubacterium limosum, Providencia stuartii, Propionibacterium acnes, Morganella morganii, Acinetobacter calcoaceticus, Bacteroides Bacteroides ureolyticus, Stenotrophomonas fragilis, Pseudomonas aeruginosa, maltophilia, Bacteroides ovatus,

Sphingobacterium multivorum, Bacteroides uniformis, Bacteroides stercoris, Candida parapsilosis, Bacteroides capillosus, Candida krusei, Bacteroides thetaiotaomicron, Bacteroides caccae, Prevotella oralis, Prevotella oris, Prevotella disiens, Prevotella bivia, Prevotella buccae, Prevotella sp, Prevotella corporis, Prevotella intermedia, Prevotella melaninogenica, Porphyromonas


Gram-negative pigmented bacillus Fusobacterium necrophorum,

Veillonella spp (Bowler,2001, Brook, 1989, Johnson,,1995, and Mousa,1997). The classification of wound infections depends on the anatomical position of the wound. Surgical wound infection; the risk of a surgical wound becoming infected depends on the susceptibility of the surgical wound to microbial contamination (Raahave,, 1986). Clean surgical wound are less prone to microbial contamination, (a risk of about 1-5% postoperative infection had been identified with clean surgical infections) while dirty surgical operations have a high risk of contamination of about 27% of the available wound pathogens (Nichols,1998.). Surgical wound is characterized by poly microbial contamination, due to the presence of the normal microbial flora in the regions close to the location of the wound. Surgical wound could involve various parts of the body, such as the skin and other internal organs. The surgical wound infection can thus be classified as being incisional (involving the skin, the subcutaneous tissue, and some muscle tissue) and other internal organ or the anatomical region such as the large intestine, the head and the neck region (Mangram,, 1999). In the latter region there is the possibility of a high risk of microbial contamination. Acute soft tissue infections include those infection that affect the body causing cutaneous abscess, traumatic wound and necrotizing infections (Bowler, 2001). Necrotizing infection are those infections which affect the skin and other parts of the body resulting in cellulitis. Classification of necrotizing systemic infection is complex and it is based on the type and level of tissue involved the rate of progression, the causative organism, initial clinical findings and type of therapy required. Bite wound infections, is a kind of wound infection associated with the bite of an organism either by human being or animals like Cat and Dog. Infection associated with human bite had been estimated to be between the range 10 to 50% depending on the severity and location of the wound; infection of wound in relation to the bite of cat and had been found to be between 20% to 30% and 50% (Bowler 2001). Brook et al (1987) reported that 74% of 39 human and animal bites are infected with poly microbial aerobic and anaerobic microorganisms.


Burn wound infections is an infection associated with wounds caused as a result of burnt. Burn wound infections have been recorded to be the major cause of about 75% of the death associated with burn wound (Bowler, 2001). Diabetic foot ulcer is a kind of wound infection associated with diabetes mellitus; it is also called plantar ulcer infection. This type of ulcer is highly susceptible to microbial infection due to the high incidence of mixed wound microflora (Diamantopoulos, 1998) and the inability of the polymorphonuclear cell to prevent the invasion of microbial pathogens effectively (Armstrong,,. 1995). Leg and Pressure ulcer; Leg ulcer infection has to do with infections found in leg ulcer and pressure sore. Pressure sores arises from continued skin pressure over bony prominences. Several microorganism are usually present around leg ulcer and pressure ulcer, these organisms exist as microbial flora in this regions. This group of microorganisms includes both aerobe and anaerobe (Robson, 1997). Microbial infection of leg and pressure ulcer is characterized by a synergistic relationship between the anaerobic and aerobic microbes around the ulcer or wound. Davies and Bowler, (1999) suggested the synergistic relationship between aerobes and anaerobes as the possible source of infection in leg and pressure ulcer. Wound can also be classified as slow healing wound and minor healing wound on the basis of the microbial load, present in the wound. Slow healing wound has in it a large amount of microbial load capable of causing delayed healing of wound, while minor healing wounds are characterized by the presence of smaller number of the body normal microbial flora, and are thus less prone to infections (Bowler, 2001). The exposure of the subcutaneous tissue usually provides an enabling environment, for the microbial infection of such tissues. And once the host immune system become compromised and can no longer overcome the virulent factors being released by the pathogens, an infection ensues (Bowler,, 2001). A wound can become contaminated through any of the following sources; the environment also known as exogenous source, involving microorganisms present in the region closer to the site of infection, and may be introduced by object causing the wound in the case of traumatic injury; The surrounding skin, involving the surrounding skin normal microbial flora such as Staphylococcus aureus, Staphylococcus epidermidis,

Pseudomonas aeruginosa and Micrococci introducing an infection in the wound. And endogenous source; the endogenous source of infection involves the mucous membrane (Duerden, 1994). Wound infection can result in serious complications which could range from localized infection to systemic infections such as sepsis and shock. No matter the complication a wound is only considered infected when the microbial load in the wound has been found to exceed certain critical cfu/ml (colony forming unit/ ml). Bendy (1964), reported that a pressure ulcer can only be considered infected when the microbial load in the wound is ≥ 105 cfu/ml or 106 cfu/g tissue biopsy. Using wound swab for the quantification of microbes a microbial load of 106 cfu/ml is considered possible to initiate an infection. A colony forming unit of 106 per ml was described suitable to cause a microbial infection, this has also been described suitable to initiate an infection in Pressure ulcer, delay closure of some surgical wound, and infection in skin graft (Krizek,, 1967, Robson, 1968, Robson and Hegger, 1969, and, Robson and Hegger, 1979). Levine,, (1976) demonstrated that the infecting cfu/ml of microbes in a burnt wound swab must be ≥ 106, similarly Brenderbach and Triger (1995) demonstrated that a critical level of bacteria ≥ 104 in tissue must be reached before microbes can cause infection in such tissue wound. The result of Velvet pad imprint technique suggests a minimum infective cfu/cm2 of 4.6 × 105 for a wound infection. 5 x104 cfu/ cm2 were identified as the sufficient microbial load that can cause an infection in skin grafting process. Significantly, microbial contamination in a wound must exceed 105 cfu/ml before the wound is said to be infected. This however depends on the method of wound sampling, whether a swab or tissue biopsy.


2.1.1 Clinical presentation of wound infection The presenting sign and symptoms of wound infection can easily be identified by certain characteristic local responses like purulent discharge, localized pain , erythema, heat, or local warmth, Swelling, Pyrexia (in the case of surgical wound), Delayed healing, Abscess, Malodour (Kownhar, 2008, Peel, 1998). Further extension of Erythema can occur in spreading infections, it may also involve lymphangitis, crepitus in soft tissue and wound break down (Keryln, 2011). In full thickness burn wound infection and skin graft pain may not be a characteristic symptom. Induration, extension of the wound, unexplained increased white cell count or signs of sepsis may be signs of deep wound (ie sub-fascia) infection (Keryln, 2011). Various types of wound manifest various types of symptoms; surgical site infection could manifest with any of the following symptoms (Keryln, 2011): Purulent drainage from superficial incision Localized swelling, redness and heat Wounds are usually open in superficial incision The manifestation of chronic wounds classically include Delayed (or stalled) healing, Peri-wound oedema, Bleeding or friable (easily damaged) granulation of tissue, Distinctive malodour, or change in odour, wound bed discolouration, Increased or altered/purulent exudate, Induration Pocketing Bridging, for localized wound infections. Spreading chronic infection are characterized by the following symptoms, • •

Wound breakdown Erythema extending from wound edge Crepitus, warmth, induration or discoloration spreading into peri-wound area Lymphangitis Malaise or other non-specific deterioration in patient’s general condition

• •

In addition, chronic wound may however shows certain modifications to the symptoms described above in immunocompromised patients, for example, in a diabetic patient with foot ulcer pain may not be a prominent symptom. Also in the case of arterial ulcer a previously dried wound may become wet if infected (Keryln, 2011). Inflammation is not a necessary symptom of infection in diabetic foot ulcer. Wound infection may result in

serious clinical condition like sepsis and septic shock once a systemic infection has occurred and may manifest with any of these symptoms; infection with pyrexia or hypothermia, tachycardia, tachypnoea, raised or depressed white blood cell count, and multiple organ dysfunction, hypotension despite adequate volume resuscitation and death. It should however be noted that wound infection should only be considered as the cause of sepsis or septic shock only when other possible source had been excluded (Keryln, 2011). Further severe manifestation of wound infection could include necrotizing fasciitis in the case of soft tissue infection. Necrotizing infection occur with varying degree of severity and progression, ranging from subcutaneous aerobic and anaerobic cellulitis to muscle fasciitis (Brook, 1998). 2.2 Diagnosis of wound infection An important feature in the diagnosis of wound infection is the identification of the significant microbial load in the wound, so as to distinguish wound contamination from infection. The process of wound infection involves several phases, it is therefore imperative that infection of wound is established in diagnosis (Bowler, 2001). The diagnosis of wound is complicated and depends on the quantitative culture of viable wound tissue or wound fluid. The diagnostic method for wound infection is dependent on the type of wound being examined. One basic quantitative technique available for wound diagnosis is wound sampling (of wound fluid or tissue) (Bowler, 2001). Wound sampling involves various techniques of isolating microorganism from infected wound; the available sampling techniques include wound swabbing, wound biopsy, aspirate collection, and dermabrasion (Finegold, 1989, Marwimuro, 1997, Thomson and Smith, 1994, Pappasian and Kragel, 1997). Wound swabbing is a diagnostic technique used for the investigation of microbial load in a superficial wound and tissue debris. Wound swabbing involves the use of a cotton tipped swab, to sample superficial wound fluid or tissue. An alginate swab can also be used for the collection of microbes from wound fluid or tissue; this is because swab will provide a more quantitative analysis of the microbial load in the wound. Other methods used for wound swabbing include the use of velvet pad which imprints the microbial content of the wound, filter paper disc and cylinder swabbing (Bowler, 2001).

Wound fluid sampling; in a wound with profuse flow of fluid needle aspiration is the best technique for sampling the microbial bioburden of the wound. Needle aspiration can also be used for the sampling of deeper pocket of fluid beneath superficial debris. It is the best method of investigating the microbial bioburden in a purulent wound from intact cutaneous abscess. Aspiration can be done for cavity wound by irrigation with sterile saline and gentle massaging, so that fluid will be used for aspiration. Wound biopsy; this involve the acquisition of deep tissue after debridement and cleansing of superficial debris have been carried out (Finegold, 1989 Thomson and Smith, 1994, and Marwimuro, 1997). Biopsy is usually carried out by aseptically removing the tissue from wound, and then weighed and homogenized and serially diluted. This is then cultured in a selective and non–selective agar medium under both aerobic and anaerobic conditions. The culture is done in both aerobic and anaerobic medium in other to have quality information on the wound. The sampling of diabetic ulcer can be investigated by the removal of superficial devitalized tissue using curettage (Pappasian and Kragel, 1997). The surplus of technique available for the sampling of wound often generate confusion as regard the best practices in terms of microbial load that can be recovered, and the best cleansing procedure before sampling of wound is done, however each of these techniques has its own benefit (Johnson,, 1995, Hansson, 1995, Rudensky, 1992, Perry, 1992, Sapisco, 1984). Based on this there is no single procedure that is considered the best but each of the sampling procedures, is employed in different types of wound. For example wound biopsy is considered the most appropriate technique for identifying the causative pathogen in surgical wound infection, closure wound and skin graft (Robson, 1999). Needle aspiration is considered the best method for the sampling of the microbial population in close or open lesion and excised uncontaminated tissue or exudates from wound (Bowler,, 2001). Although wound swabbing has been described to have certain limitations such as the possibility of giving false information about the microbial load in a wound, David and Bowler (1999) were able to show that superficial wound swabbing can be a suitable procedure for the investigation of any type of wound (David and Bowler, 1999)


Once the samples are collected, they are carried in a transport medium to the laboratory for further investigations. Once the sample reaches the laboratory, the presence of necrosis associated with malodour is indicative of a possible microbial infection. The use of Gas- Liquid chromatography will give a rapid confirmation of pathogenic anaerobic bacteria. The use of GLC however suffers a limitation of being very expensive and that only wound with purulent discharge can benefit from it (Bowler, 2001). This limitation thus made the other method of investigation more acceptable. 2.2.1 Gram staining Gram staining is an old technique of staining microorganism, used for the rapid detection of bacteria in life threatening wound infection condition, although its use is still debatable for some clinical infection it has been found to be potent for some wound infection (Popescu and Doyle 1996).Gram staining can be done for an infected wound for the rapid detection of the microbial load in the wound. Gram staining of microorganism from wound swab for an open wound can give a significant microbial load which correlates with the number of microorganisms in the wound (Hegger, 1969). This procedure has also given significant information on the number of microbes from a wound swab of a surgical wound (Levine, 1976). Although Gram staining provides a rapid basis for the detection of microbes in a wound its role in facilitating the timely and suitable treatment of wound infection is dependent primarily on the type of wound. Gram stain reliably indicates sterile and mixed abscesses, as well as those containing pure S. aureus (Meislin et al, 1978). Gram staining may also enhance the identification of the etiological agents of wounds infection following clean surgery, in the presence of a higher ratio of one microorganism. However, in most other wound types that are characterized by a complex aerobic-anaerobic microflora, the Gram stain has little value, although the combined presence of leukocytes and bacteria is likely to be a good indicator of infection, as reported by Hussey et al. (Husey,, 1998) in studying rapid diagnostic tests for intra-amniotic infection. With the exception of Grampositive spore-forming anaerobes such as Clostridium perfringens, differentiation between aerobic and anaerobic bacteria is difficult and is further complicated by the fact that many gram-positive anaerobes become Gram variable on exposure to oxygen (Johnson,, 1995)

2.2.2 Culture of wound specimen Culturing is a routing technique used by microbiologist for the identification of the microbial species responsible for an infection in wound. Culturing can be done both on selective and non-selective agar media. Microbes are cultured in the laboratory for the purpose of the quantitative identification of the microbes in wounds (Bowler,, 2001). The culturing of the isolates may last for between 24hrs to 7 days depending on the organism. It is essential that wound culture is done for wound specimen both aerobically and anaerobically. The procedure should involve the use of one selective medium and a non-selective medium; the addition of a 5-mg metronidazole disk to an agar plate is often used for the detection of anaerobic bacteria in the wound specimen. The production of zone of inhibition, around the metronidazole disk is indicative of the possible presence of anaerobes. The presence of facultative anaerobes like Staph aureus will encourage the growth of black pigmented species such as Prevotella or Porphyromonas spp (Bowler, 1999) Once the causative agents of wound infection have been identified, antibiotic sensitivity testing should be carried out. There are various techniques for performing antibiotic sensitivity testing; but the two most common methods are Kirby-Bauer method and the tube dilution techniques, such as Vitek and Microsan, have been introduced that can rapidly and efficiently provide susceptibility information. Antibiotic sensitivity gives the microbiologist and the clinician an idea of the most appropriate antibiotic that can be used for the treatment of the infection. There are more rapid methods for the diagnosis of wound infection such as radiological diagnosis of osteomyelitis, and the use of hematologic and biochemical markers (William, 2004). The microbiological analysis of wound can be described as semi quantitative or quantitative. The semi quantitative analysis of wound usually grade bacterial growth as scanty, light, moderate, or heavy. The semi quantitative analysis of wound is bias towards motile and fast growing organisms (Healy and Freedman, 2006). Semi quantitative analysis of wound may underestimate fastidious organism such as Staph aureus. The quantitative microbiological analysis of wound usually gives an estimate of the microbial load in a wound.

2.2.3 Treatment of wound infection The treatment of wound infection involves the use of both antimicrobial agent and other non-antimicrobial agent (Bowler, 2001). The treatment or management of wound with antibiotic is faced with the challenges of the anatomical position of the wound. Different site of wound infection require different method of treatments, with antimicrobial agent, some infections and as such the choice of treatment depends on the anatomical position of the wound infection (Healy, and Freedman, 2004). Antibiotic treatment of wound could be through topical application or systemic administration of antibiotics. Although systemic antibiotic therapy is essential for advancing cutaneous infections and those that involve deeper tissues, wounds that exhibit only localized signs of infection or are failing to heal but do not have clinical signs of infection (i.e., heavy colonization) may initially be treated with topical agents. Topical antimicrobial agents include the use of both antiseptics and antibiotics (Bowler, 2001). Other treatment options such as Hyper basic oxygen therapy, which facilitates the host immune response and may also have a direct antimicrobial effect against some anaerobic bacteria (e.g., C. perfringens), antimicrobial peptides, and botanical extracts may also have roles to play in wound management and are worthy of consideration. Surgical debridement can also be carried out for the treatment of healing and non- healing wound (Bowler, 2001). Antimicrobial treatment of wound can be a prophylactic treatment or therapeutic treatment. The prophylactic treatment is done mostly in surgical wound to prevent microbial infection of the wound. Most uncomplicated surgical or traumatic wounds heal normally without the need for prophylactic antimicrobial treatment (Phillips, and Davey, 1997.). Some of the antimicrobial agents used in the treatment of wound include cefoxitin, imipenem, or ticarcillin-clavulanate, cefuroxime or cefotaxime as sole agents, with other agents being used in combination, including metronidazole or clindamycin and gentamicin. The use of systemic antimicrobial agents for pressure sore is not recommended by the European pressure ulcer association (European Pressure Ulcer Advisory Panel. 1999). However topical antibiotics are advised by the European pressure ulcer advisory panel, for use in such wound infection. Broad spectrum topical antibiotics are the antibiotic

employed for topical administration. However the antibiotic of choice in the treatment of wound infection is related to the type of wound concern, as indicated in the Table below. The use of the under listed drugs in the Table is subject to the directive of a physician. Type of wound infection Surgical wound Infection Bite wound Diabetic Ulcer Necrotizing Fasciitis MRSA suspected infection Recommended Antibiotic of Choice Co-Amoxiclav, cefuroxime, metronidazole Co- amoxiclav Co-amoxiclav, ciprofloxacin, clindamycin, High dose of benzyl penicillin, Vancomycin, and Linezolid

In the treatment of wound it is essential to optimize the host response to the possibilities of infections. These can be achieved by any of the following method, Optimization of management of co morbidities, e.g. optimizing glycemic control in diabetic patients and enhance tissue perfusion/oxygenation. Eliminating the risk factors for infection where feasible Nutritional status and hydration should be optimized. Other sites of infections should be treated e.g. Urinary tract infection It is also essential to reduce the microbial load in the wound, which can be achieved through any of the methods described below. Prevent further wound contamination or cross contamination – e.g. – Infection control procedures and protecting the wound with an appropriate dressing Facilitate wound drainage as appropriate Optimize wound bed: – By removing necrotic tissue and slough (debridement) – increase frequency of dressing change as appropriate – cleanse wound at each dressing change – manage excess exudates – manage malodour (Sussman & Fletcher, 2011) The use of antiseptics is another method of treating wound infection; Antiseptics are antimicrobial non-selective agents which are applied to inhibit the growth of

microorganism or kill the microorganisms. The choice of antiseptic is influenced by any of the following: clinical familiarity, availability, cost and reimbursement issues, ease of use and implications for pattern of care efficacy and safety. The type of wound that enjoys topical application of antiseptics includes traumatic wound and chronic wound. Antiseptics can be applied to wound with or without clinical sign of infection. The commonly used antiseptics are iodine releasing agents such as povidone iodine [PVP-I] and cadexomer iodine, Chlorine releasing solutions (e.g. Dakin’s solution and sodium hypochlorite solution), hydrogen peroxide, chlorhexidine, silver-releasing agents, and acetic acid. 1% acetic acid has been identified to be very effective against Pseudomonas aeruginosa but its activity is limited to a few organisms (Bowler, 2001, Philip and David, 1997). Other antimicrobial technique used in the treatment of wound include, the use of botanical agents and other natural products such as honey, Australian plant (Melaleuca alternifolia) oil. Hyper basic oxygen (HBO) therapy is another method available for the treatment of wound infection in wound with ischemic condition. The principle of this therapy is based on the tendency of aerobic respiration to reduce the proliferation of anaerobes in a wound (Bowler, 2001). Apart from antimicrobial related therapy for wounds there are also the non-antimicrobial related therapies for wound. This includes debridement, pressure reduction in wounds, and infection control. Debridement is the removal of devitalized and contaminated tissue, from wounds to expose healthier tissue and facilitate wound healing (Vowden and Vowden, 1999). Devitalized tissue usually provides an environment for microbial proliferation, its removal will therefore remove the microbial load in the wound, hence, minimize the infection. There are various types of debridement; surgical debridement, Autolytic and enzymatic debridement, Bio surgical debridement. Surgical debridement is the backbone of all treatments for wound infections of various causes. Its principle is based on providing enabling environment for the migration of epithelia tissue and reducing the bacterial bio burden in the wound (Armstrong, and Athanasiou, 1998, Steed, 1996).

Autolytic and enzymatic debridement; the idea of autolytic and enzymatic debridement came from the principle that some dressings and topical modalities provide an enabling environment, for the activation of endogenous enzymes, which causes the autolysis of fibrin and encourages extracellular matrix turn over, and maintenance (Cherry,1984,Mulder and Walker, 1989, Mulder, 1993). Bio surgical debridement; it involves the use of biological agents especially animals such as fly larvae and maggot in wound for the enzymatic degradation of devitalized tissue (Thomas,, 1999; Thomas and Jones. 1998, Livingston, 1936.Livingstone, 1938 Graner, 1997). It is an ancient method debridement. It has its origin to the period of the Second World War (Thomas,, 1999, Thomas and Jones 1998). Pressure reduction is another method that had been put into use for the control of microbial infection of wound without an antimicrobial agent. The reduction on pressure and the duration of pressure plays a key role in the pathogenesis of a large number of wound being treated (bowler, 2001). Wounds in which the pressure has not been reduced are known to take a longer time before they are healed and thus provide a greater risk of microbial contamination of such wounds. Thus it can be inferred that reduction in pressure is capable of preventing the proliferation of microbes in wounds (Laing, P. 1994). There are two methods that have been employed in the reduction of pressure in wound; Pressure off- loading and use of vacuum closure. Vacuum assisted wound closure helps to enhance wound healing and prevent wound infection through the application of sub-atmospheric pressure which ensures the reduction of interstitial pressure that delays wound healing, thereby preventing the proliferation of wound microbes that may likely cause infection. The method of vacuum assisted wound closure involves the use of foam dressing in external sterile open wound, thereby reducing the pressure in the wound (Morykwas and Argenta. 1997). Infection control is a method that dependent on the dressing for the control of infection of wounds. Dressing plays significant role in preventing to physically prevent the dissemination of microorganisms in wound. Some wound dressing that can maintain moist wound environment has been found to prevent the dispersal of microbes in burnt

wounds (Lawrence,1994), as well as the rate of infections (Boulton,, 1999, , Lawrence, 1994 Laing, 1994 Hutchinson, and Lawrence. 1991). Treatment of wound can be done using antimicrobial agent to prevent microbial proliferation and possible infection. The antimicrobial agent could be an antibiotic which may be used singly or as a combination therapy, either in synergy with other antibiotics e.g. the combination of aminoglycosides and metronidazole. It may be applied topically or used systemically in the case of systemic infection. Apart from the antimicrobial agent there are also other antimicrobial methods of treating wound infection such as hyper baric oxygen method of treatment. The antimicrobial agents may also be an antiseptic which is not selective in its activities. Treatment may also involve the use of non – antimicrobial agents such as the use of pressure reduction. 2.3. Vaginitis Vaginitis refer to the series of infection which affects the vaginal resulting in a variety of responses ranging from inflammation through painful discharge from the vaginal, to severe clinical conditions such as urinary infections, high risk pelvic inflammatory disease, Post abortion uterine infection, preterm labour, breast abscess, cervical intraepithelial neoplasia, and post hysterectomy cuff infection (Polycar, 1996, Sobel, 2002, Schaaf,, 1990, Bornstein,, 2001). Vaginitis is usually due to the ability of opportunistic pathogens, which are usually aerobes, anaerobes, or facultative anaerobic microorganisms to overcome the vaginal microbial population or outgrowth the vaginal normal microbial flora and initiate an infection of the vaginal. This disruption caused by the growth of these non- inhabitant of the vaginal community, result in Vaginitis. Vaginitis had been identified to have its aetiology to three groups of microorganisms which are member of the protozoan, the bacteria and the fungi. The protozoan species is known as Trichmonas vaginalis, the bacteria cause includes aerobe and anaerobic bacteria as well as the facultative anaerobes. The commonest fungal cause of Vaginitis is Candida albicans (Schwiertz,, 2006 Larson 2005, Ferris,, 2006, Burton,, 2004).

Vaginitis is the commonest cost of hospital visit among women in the US and the commonest vaginal infection in women (Schwiertz, 2006, Cook, 1992). Vaginitis can be classified into infectious and non infectious vaginitis. Infectious Vaginitis are those vaginal infections due to the activities of microorganisms, while the non infectious vaginitis are such vaginal infections which do not involve the activity of microorganism but as a result of response to a product or injury such as sexual activities, pain (Annex teen clinic). Another method of classifying vaginitis is its classification based on the aetiological agents. That is vaginitis can be classified as Trichomonas vaginitis, Candidiasis, and Bacterial vaginitis. Bacteria vaginitis is the commonest of these three types of vaginitis. It is also known as bacteria vaginosis or non specific vaginosis and can be caused by various groups of aerobic and anaerobic bacteria such as Gardnerella spp, Peptostreptococcus sp, and Escherichia coli, and other facultative bacteria. Bacteria vaginitis has been identify with the ability to increase the risk of susceptibility of infected patient to sexually transmitted diseases (Cohen et al., 1995; Martin et al., 1999; Sewankambo et al., 1997)

2.3.1 Clinical presentation of vaginitis The clinical manifestation of vaginitis is presented by certain characteristic symptoms such as coloured vaginal discharge, which may be greyish or whitish distinguishing it from the normal vaginal discharge. The flow may increase with some characteristic fishy like odour and become thinner than usual. These symptoms are usually not associated with itching, soreness or irritation. In most women however vaginitis is asymptomatic (Hay, 2006,, 2009). It may be associated with some complications such as
Pelvic Inflammatory Diseases (PID), pre term labour in pregnant women, preterm premature

rupture of membranes, and postpartum endometritis (Hay, 1998, guidelines for treatment of sexually transmitted diseases 1998, Watts, 1990, Goldenberg, 2000). Bacteria vaginitis has been found associated with an increase in the incidence of vaginal cuff cellulitis, and abscess formation following trans-vaginal hysterectomy (III) (Soper, 1993).

2.3.2 Diagnosis of vaginitis The diagnosis of the vaginitis is begins with the identification of the sign and symptoms of the diseases, with the recognition of the swab will be used to collect vaginal discharge from the vaginal for further identification. The detail diagnosis of vaginitis in can be carried out by either of the methods described by Amsel or Hay. According to Hay the presence of a whitish, thin homogenous vaginal discharge, with the presence of clue cells from the microscopy of a wet mount or wet prep of vaginal swab sample may be indicative of vaginal infection or vaginitis. Also when the pH of the vaginal fluid is discovered to be greater than or equal to 4.5 with a characteristic fishy smell vaginitis may be diagnosed. Gram staining of the vaginal discharge must also be carried out. The Hay criteria for the diagnosis of vaginitis are based on the type of microorganism isolated from the vaginal fluid culture. According to his method, the presence of a few population of lactobacillus with a predominant population of Gardnerella or Mobiluncus indicates the presence of vaginitis (Hay, 2006, Schwiertz, 2006).

2.3.3 Treatment of vaginitis. The first line of treatment for bacteria vaginitis is the use of oral metronidazole 500mg twice a day for seven days, Intra vaginal metronidazole, and 0.75g gel twice a day for 5days, oral clindamycin 300 mg twice a day for 7 days, and 2 percent intravaginal clindamycin cream at bedtime for 7 days. The second line therapy is oral metronidazole in a 2 gm stat dose or oral amoxicillin/clavulanic acid, 500 mg three times a day for 7 days. The third line therapy is oral ampicillin/amoxicillin, oral cefadroxil, oral ofloxacin, or intravaginal triple sulfa cream. A comparison of metronidazole doses (stat dose versus 7-day regimen) shows that single dose therapy has a lower clinical cure rate (47–85%) than the 7-day course (82–89%) (Eschenbach,, 1983, Hovik, 1983, Jerve, 1984 and Swedberg,, 1985) Side effects of therapies include candidiasis, nausea and vomiting, and vaginal irritation 2.4 Incidence of the microbiology aetiology of wound and vaginitis Microbial aetiologies of wound infection

Wound infection can be caused by a wide variety of microorganisms like bacteria fungi, protozoa and sometimes viruses (Cooper 2003, Zafar, 2007). Different microorganisms can exist in polymicrobial community especially near a wound margin or in chronic wound (Cooper,, 2003) these organisms may be aerobes or anaerobes. The different site of wound present different group of microorganism associated with microbial infection in such wound site. Though the microbial aetiology of most wound infection gives a representation of similar groups of bacteria, each of the sites differs in the significant population of microorganisms that are associated with the observed infection. The microbiology of 20 diabetic wound ulcer following scraping reveals Bacteroides spp, Proteus spp and Peptostreptococcus spp as the predominant species (Louis 1976). In a study of chronic leg and foot ulcer in 2005 Howell-Jones, discovered Staphylococcus aureus as the predominant organism isolated from a foot and leg ulcer. Also Staphylococcus epidermidis has been found to be pre-dominant, organism that is isolated from venous leg ulcer having a percentage of about 14% (Bowler and David, 1999). The study of Sapico, indicate Bacteroides, coliform and Pseeudomonas aeruginosa as the predominant species of bacteria present in Pressure sore. Similarly, Staphylococcus aureus, Bacteroides spp and Prepstreptotococcus spp were identified as the predominant species in decubitus ulcer, animal and human bite wound and leg ulcer (Brook,, 1987, 1990, 1991). In Nigeria the study of Adejumo, 2011 Staphylococcus aureus was identified as the predominant pathogen isolated from a wound infection, following a post operation caesarean section in a hospital in Oyo state Nigeria. Out of a total number of 72 infected wound observed in children in a bacteriological study of wound infection in the children hospital in Lahore Staphylococcus aureus was identified as the predominant organisms in various wound infections, with a frequency of 41.28% other microorganisms isolated include, E. coli (13.76%), Klebsiella spp. (12.84%), P. aeruginosa 5.50% P. flourescene 4.54%, other Pseudomonas spp. (8.26%), CoNS (5.50%), Proteus spp. (4.59%), Streptococcus spp. (2.75%) and Serratia spp.

(0.92%) from all the 109 microbial isolates from various children wound (ZAFAR, 2007). Mumtaz et al. (2002) has also found S. aureus as the most common pathogen (49%) followed by E.coli (25.9%) Klebsiella spp, (9.5%), P. aeruginosa (8.6%) and Proteus spp (4%) isolated from various wound samples. In general gram positive cocci and gram negative bacilli constitute the predominant pathogen that are commonly being isolated from wound, considering the work of Zafar 2007, the four major species isolated from various wound infection in children are Staph aureus, Pseudomonas aeruginosa, Klebsiella, and Escherichia coli. This is similar to the observation of Gales et al, 1997. The incidence of Staphylococcus aureus as the leading pathogen in wound infection had been reported in several clinical conditions in Nigeria. The study of Idowu (2011) reveals Staph aureus as the highest microbes that can be isolated from wound with a frequency of 51.3 % (Idowu, 2011). The presence of Staphylococcus aureus had been reported in both infectious and Noninfectious leg/ chronic ulcer. Staphylococcus aureus .in an infected wound was reported to be about 43% of the microbial population in that ulcer while about 88% of the microbial population of the non- infected ulcer, has Staphylococcus aureus present in it (Bowler and David, 1999). Although Staphylococcus aureus and other species of Staph may not be isolated from all wound, it has thus far remain the most common organism isolated from wound infection and to a greater length responsible for several Nosocomial infection, arising from wound infection (Adejuwon,, 2011, Monica, 2002). The Microbial aetiologies of vaginitis Several microorganisms are associated with infectious or non- specific vaginitis, this microorganisms include member of the fungi, the bacteria, protozoa, and viruses (Otunoye,, 2004). Bacterial vaginosis (bacterial vaginitis) has a wide range of microorganisms associated with its incidence in an individual; they include Gardnerella, Bacteroides, Peptostreptococcus, Mobiluncus, M. hominis (Eschenbach, 1994). The study of otunoye in 2004 however include Staphylococcus aureus as a member of the microorganism isolated from bacteria vaginitis, the outcome of their finding shows

that these gram-positive cocci alongside with Streptococcus spp and Neisseria spp constitute the largest percentage(51.2%) of bacteria isolated from patient with bacteria vaginosis in Nigeria(otunoye,, 2004). Similarly the work of Chen, indicates the presence of Staphylococcus aureus, having the highest percentage in the vagina of pregnant women in New York USA (Chen, 2006). Also in the 90s a drastic increase in the staphylococcal colonization of the vagina had been recorded. In Japan an increase in the population of staphylococcus aureus which colonizes the vaginal was identified from about 5% (Lasen, 1982, 1980) in the 70s to 7.5% in the 90s (Mitsuda,, 2005), similar observation was also found among the British women where the staph aureus population of the vaginal increased to 8% in the 90s (Dancer and Noble, 2005). The study of 3000 American women shows 9% vagina colonization of staphylococcus aureus and about 4.3% staph aureus anal colonization (Parsonnet,, 2005). The investigation of cook in 2004 on the microbiology and the biochemical factor of recurrent bacterial vaginosis identified three groups of bacteria as the organism associated with this clinical condition; the aerobe the anaerobes and the facultative an anaerobes. These groups include various bacteria at different or varied frequencies; they are Fusobacterium spp, 58% Bacteroides spp 58%, Eubacterium spp 3%, Actinomyces spp 6%. Peptostreptococcus spp, 52% Veillonella spp 6% Gardnerella vaginalis 87% Lactobacillus spp 48%, Corynebacterium spp 84%, Streptococcus spp 77%, Staphylococcus spp 65%. Micrococcus spp 33%, aerobic gram-negative rods, 29%. The presence of certain fungi such as Candida albicans and Torulopsis spp were also indicated as part of their finding for recurrent vaginitis. Despite the paucity of literature on the association of Staphylococcus aureus with bacteria vaginosis, its frequent isolation from clinical manifestation could be indicative of an aetiological agent for this disease condition. More so, with an established fact that bacterial vaginosis cause is poly microbial (Holmes, 1984, Cook,, 2005, Oakley, 2008), Staph aureus could be a major contributor. 2.5 The prevalence of Staphylococcus aureus and other staph species in wound and vaginitis.

Wound Several literatures have indicated the presence of staphylococcus aureus and some coagulase negative staph as one of the commonly isolated organisms from wound related infections. However the degree of expression of these organisms in different wound varied with wound types and clinical conditions, as various types of wound present different microbial flora (bowler,, 2001). Idowu, (2011) reported Staphylococcus aureus as the leading etiological agent of pus producing infections of the eye, ear and wound as it accounts for about 51.7% of all the isolates found in this clinical condition. They also identify Staphylococcus aureus as the only organism isolated from the wound sample used in their study (Idowu, 2011) This observation is in line with the work of Shittu (2003) and Bisi, (2005) who also described Staphylococcus as the leading aetiological agent in wound infection. Okesola, (2009) found Staphylococcus aureus as the predominant pathogen from wound samples at the university college hospital with a percentage of 44.8 of the total isolates. This further emphasizes the predominant role of Staphylococcus aureus in wound infection. These Observations contradicts the work of Louis, 1976 where Bacteroides were identified as the predominant microorganism isolated from wound infection, and that of brook, that identified Staph epidermidis as the most commonly isolated organism from burn wound infection. Similarly the work of Sapico, 1986 reveals Bacteroides as the most common pathogen from pressure sore. Although Staphylococcus aureus is not the most commonly isolated organism in the work of these scientists, it still form a member of the predominant species found in all these wounds. Staph aureus however still remain the most commonly reported bacteria that have been isolated from various wound infection. According to the work Shittu (2003), Bisi,, (2005), Okesola, (2009) Akinjogunla,, (2009) Idowu, (2011), 44.8%, 51.6% of the isolates from wound. Vaginitis There is fewer literature which talks about the significance of staph as a causative agent in vaginitis. Fewer discussions have been said concerning the presence of Staph in bacteria vaginosis isolates. The work of otunoye 2011 however reveals Staph aureus

as the predominant agent isolated from bacteria vaginosis in women in Nigeria with a percentage of 40% of the total isolates. The health effect of Staphylococcus aureus was also described by Lihares, which described staphylococcus aureus as one of the microorganism that can be found present in the vagina when vagina normal microbial flora has been disrupted (Linhares,, 2010). The work of ogunshe 2009 identified the presence of Staphylococcus aureus as one of the pathogen that can be isolated from a non HIV patient, although at a lower percentage of 2.3% of the total population of microbes from non HIV patient in Ibadan. In a study in Ghana staph aureus form the fourth major organism isolated from infectious condition in two different hospitals. Parts of the body, from which the organisms were isolated, include the high vaginal region, and the evaluation of all the microbes isolated shows that the frequency of isolation for Staph aureus is 19% (Edoh and Mensah, 2005). Brian, 2008 recongnises Staphylococcus aureus as one of the organisms associated with the bacterial community of the clinical defined vaginitis. Although not so much relationship has been described between bacteria vaginosis and Staphylococcus aureus, the association of Staph with sexually transmitted diseases affecting the reproductive region may be an index for relating Staphylococcus aureus with bacteria vaginosis since it is known to be caused by various microorganisms. The presence of Staphylococcus aureus in the vagina had been traced to toxic shock syndrome in people using tampon and other vagina devices; if a strain of Staphylococcus aureus producing toxic shock toxin colonizes the vagina (Todd, 2005)

2.6 General characteristics of Staphylococcus aureus Staphylococci belong to the family Staphylococcaceae, but were initially classified as a member of the family Micrococcaceae. They are differentiated from the other common general of their initial family by their ability to ferment glucose under aerobic and anaerobic condition hence are facultative anaerobe. Staphylococci are gram positive non motile and non-spore forming bacteria with a diameter between 0.5and 1.5µm which

divide in more than one plane to form a grape like cluster. One distinguishing factor between the staphylococci and streptococci is production of catalase from hydrogen peroxide while streptococci will not produce catalase from hydrogen peroxide. Catalase is an important virulence factor because hydrogen peroxide is microbicidal and its degradation limits the ability of neutrophil to kill pathogenic bacteria. Member of this genus are all catalase positive and indole negative (Harris, 2002). Staphylococci are tolerant to high concentration of salt (Wilkinson, 1997, Harris, 2002) and exhibit resistant to heat (Kloos and Lambe1991, Harris, 2002). Most species have complex nutritional requirements but they all utilize an organic source of nitrogen, which is provided by five to twelve essential amino acids (like Arginne and, Valine) and Vitamin B e.g. Thiamine and Nicotinamide (Klose and Schleifer 1986, Wilkinson, 1997, Harris, 2002). Staphylococci are resistant to lysis by lysostaphin (Cullimore,, 2000, Olarinde, 2010 (Unpublished)). They are resistant to bacitracin (0.04U) which is indicated by the absence of inhibition halo, measuring up to 9mm in diameter, but susceptible to furazolidone (100µg) (Baker, 1984). Staphylococci grow in a media containing 7.5 to 10% Nacl and this form the basis for the use of manitol salt agar in their isolation from a mixed culture or sample containing mixed organism. Hence growth in 15% Nacl is also used in distinguishing Staphylococcus from other genera in the family Micrococcaceae (Wellstood, 1992). On blood agar, nutrient agar, milk agar, and chocolate agar the colonies usually appear as a circular slightly raised shining colonies after 18- 24hrs of incubation at 37 with a diameter between 1-2mm. these colonies are usually pigmented, with either golden, yellow or orange colour in some strain, white or cream for Staph aureus . The coloured colonies are due to the ability of the organisms to produce carotenoid pigment. Staph epidermidis and Staph albus produce white colonies on blood agar and other agar because they lack the ability to produce this pigment. There are thirty-two species of Staphylococci that have been recognized and eight subspecies, many of which preferentially colonize the human body (Kloos and Bannerman, 1994).Staphylococcus aureus is a coagulase positive staphylococci species which produces a series of other enzymes and toxins .Staphylococcus is the commonest of the

genus staphylococci and has been implicated in a variety of infection and intoxication including both immunocompetent and immunocompromised individuals resulting in high rate of morbidity and with complications which constitute problem to health care system (Odugbemi and Coker, 1987) . The production of coagulase is a distinguishing feature of pathogenic staph from non pathogenic staph and hence clots blood (Kloos and Musselwhite, 1975), and thus distinguishes Staph aureus a human pathogen, S. intermedius and S. hyicus from others which are coagulase negative such as Staph epidermidis (Kloos and Musselwhite, 1975). Coagulase negative Staph was often regarded as Saprophytic or rarely pathogenic species until recently when some of them were being isolated from clinical condition (Kloss and Scheifer, 1975). However, over the last decade coagulase negative staph has been implicated, as aetiological agent of a series of infectious process, representing the microorganism commonly isolated from blood cultures (Huebner and Goldman, 1999). The species of coagulase negative staph that have reported in association with wound and vagina infection is Staph epidermidis (Bowler, 2001, Brook, 1999). Staph epidermidis was discovered as the second most isolated organisms from vaginitis by Hoelst in 1987. The isolation of Staph epidermidis could have arisen from the use of invasive procedures such as Intrauterine device. In relation to the identified pathogenic potentials of coagulase negative staphylococci within the hospital environment, various interest have been developed in relation to the pathogenic species as regard their infection, toxigenic and virulence potential over the last decade. This has led to the publication of various studies on these aspects. However, despite the growing characteristics of coagulase negative staphylococci infections, these organisms are not identified in clinical microbiology laboratories (Cunha,, 2004). The scheme proposed by Kloss and Scheifer (1975) and modified by Bannerman (2003) is the method conventionally used; however, this method requires that a large number of biochemical tests be carried out. Identification of staphylococci based on the morphology and cultural identity, such as Gram reaction, catalase, and coagulase, which are commonly employ in the lab, may not be enough to differentiate the species of Staphylococci. Identification of each species is achieved by combining a number of tests as stated above with others including especially

carbohydrate fermentation/oxidation test and antibiotic susceptibility. Usually while most strains of Staph aureus ferment manitol while the other two, Staph epidermidis and Staph saprophyticus, do not. Among these three Staph saprophyticus is resistant to the antibiotic novobiocin (5µg), the susceptibility of Staph epidermidis to Novobiocin is a distinguishing features for them. Mc Taggart and Elliot, 1989, reported 100% and 96% susceptibility of Staph epidermidis however further test is recommended for its final identification, such test include alkaline phosphate; fermentation of sugars such as trehalose, mannose, urease test and growth in PYR broth. 2.7 Antibiotic Antibiotics are the core agents for the treatment of microbial infections including wounds and vaginitis which may be severe or may result in death. Antibiotics are substances that inhibit or disrupt the growth of microorganisms and sometimes kill them, by taking advantage of the prokaryotic nature of its target. Antibiotics are metabolic product of microorganism whether synthetic or semi-synthetic which in a very small quantity is capable of inhibiting the growth of microorganisms or even kills the microbes. This definition was proposed by Waksman in 1945. The term antibiosis had been in use before this definition, Vuillemin in 1889 defined antibiosis as a condition in which one organism destroys the life of another organism in order to sustain its own, the first being entirely active and the other being passive (Pelczar,, 1993). All the definitions given above do not give the best interpretation of antibiotics with the various antimicrobial agents available, which are not of microbial origin. Antibiotic in its broader sense can thus be defined as a substance produced by a microorganism or to similar substances produced wholly or partially by chemically synthesis, which in low concentration will inhibit the growth of other microorganisms or even kill them. Chloramphenicol though now produced totally by chemical synthesis was an early example. Antimicrobial agents such as Sulphonamides and the 4- quinolone produced wholly by chemical synthesis are often referred to as antibiotics (Russell, 2004). Antibiotics are examples of secondary metabolites that play no critical role in cells life. In other words, the characteristic of microorganisms to produce antibiotic is a dispensable

function. The definition excludes primary metabolic products such as acid, alkali, and enzymes or toxins which may show inhibition against bacteria. There are many antibiotics in existence but only a few had found clinical relevance. Some have shown good in vitro activity and but their high toxicity in vivo has prevented their systemic usage for the treatment of diseases in human. Others that show very good activity in vitro and in vivo initially after their introduction have become disfavoured by chemotherapeutists in recent time due to the incidence of resistant strains. Microorganisms have developed resistance to a good number of antibiotics as a result of their long term usage. This situation remains a major challenge to physicians and microbiologists as it keeps them on their toes searching for a better alternative to the agents to which these organisms are resistant. The problem is made worrisome as well because the process by which microbes become resistant is considered a never ending; it is propelled by evolutionary forces which is beyond human control (Okore, 2005) 2.7.1 Bacteria resistance to antibiotics A bacterial strain is said to be resistant to an antimicrobial agent when the minimum inhibitory concentration (MIC) or the minimum bactericidal concentration (MBC) against the organism is so high that the usual effective dose or concentration of the antimicrobial agent is no longer effective (Okore, 2005). Bacterial resistance may be dependent on the gene of the microbes or not. Resistance could either be intrinsic or acquired. Intrinsic resistance is common in Gram negative bacteria because of the variation in their cell envelope when compared to Gram positive bacteria. This is typified in Pseudomonas aeruginosa, which is known to resist many antibiotics. On the other hand, the ability of bacteria to grow and multiply in the presence of antibacterial agent may be acquired. This shows a difference in the genetic composition of the resistant strains and those that are sensitive. This is different from resistance or reduced susceptibility known to result from bacterial adaptation to specific environment. The latter type is phenotypic in nature because the ability of microorganism to resist antimicrobial agent in the environment can be reverted upon subculture in conventional laboratory media and genetic mutant can be isolated (Smith, 2004)

37 Genetic basis of resistance Resistance of microorganisms to antibiotics is often as a result of genetic change and considering it on this basis gives insight to the evolutionary origin of resistance (Okore, 2005). It is accepted that gene evolve with the determinants changing in stages until they specify characters that may be remotely related to those of the original species; this process of sudden change in sequence of nucleotide/gene is called mutation. Mutation could occur spontaneously or by induction. A single mutation usually confers resistance to only one antibiotic or to closely related antibiotics which have similar target site. Spontaneous Mutation Spontaneous mutation in microorganism is such mutation in microbial population which is not traceable to any known or specific effect. It is acquired without the influence of the drug. The resultant mutant is able to grow at the expense of susceptible organisms. Spontaneous mutation occurs randomly at a frequency of 10-3 to 10-9, and thus are the sporadic cause of emergence of clinical drug resistance in a given patient (Pelczar,, 1993, Hodges, 2004) Resistance by acquisition of genetic material Resistance in bacteria could also be due to the acquisition of pieces of genetic material from the environment or other bacteria. The genetic material might have evolved or reformed from elsewhere. This confers additional resistance mode of genetic variation on the bacterial species. The acquired genetic material may confer on the bacteria species resistance to a number of antibiotics on the bacteria species. If the gene conferring resistance on the chromosome is located on the chromosome of the bacterial species, the resistance is said to be chromosomal mediated. However if the gene conferring resistant on the bacteria is not located on the original chromosome of the bacteria, the resistance is said to be extra-chromosomal based resistance. Plasmids also known as R- factor are extra chromosomal factor which confer resistance on bacteria to antibiotics. Plasmids are like chromosome because they are capable of independent replication but they differ from bacterial chromosome because they are

smaller with an approximate size of between 0.1- 10% of the chromosome in bacteria. Plasmids do not play any role in the normal functioning of bacteria cell but may confer on the bacteria character that can give the bacteria some survival advantage in certain condition such as the ability to survive in an unfavourable condition like in the presence of an antibiotic. For example the ability of Staphylococcus aureus to resist the activity of Methicillin is associated with the presence of a beta lactamase plasmid which made this possible. Similarly, a R- factor, pSH6, found in Staph aureus encodes resistance to gentamicin, trimethoprim and Kanamycin (Lambert, 2004) Plasmid may also code for other properties in bacteria such as the ability to produce toxin, utilize or ferment unusual sugar or food source e.g. camphor, production of pili for the attachment of a cell to substrate (e.g. intestinal epithelial). Plasmid transfer occurs readily from one organism to another and between species, thereby increasing the spread of resistance. Some exhibit a marked degree of host specificity and may be transferred between different strains of the same species. Although others, particularly associated with the Gram-negative bacteria may cross between different species in the same genus or in different genera. It is however certain that plasmids are transferred among bacteria species by various mechanisms including;

Conjugation This is a process of unilateral transfer of genetic material among bacteria of the same or different species. It involves direct cell to cell contact among bacteria. It involves the transfer of R-plasmid through a cytoplasmic bridge from a donor cell to a recipient cell. It is mediated by a plasmid or a transposon. It is a common phenomenon among different genera of Gram- negative bacteria, transfer of plasmid also occur among Gram positive bacteria. A single plasmid can confer resistant on bacteria to a wide variety of antibiotics simultaneously i.e. cross resistance.

Transformation Transformation is a process by which bacteria pick up pieces of a naked DNA from an environment and add them up to their own chromosome. The process may occur spontaneously in which the bacterial chromosome leak out from the donor cell to the recipient cell or it may occur by an artificial means as a result of extraction by chemical procedures. It may also be due to cellular break after lysis by bacteriophages. Transfer only takes place between competent cells. Transfer occurs only in bacteria and it is commonly found in Gram positives bacteria which are capable of taking up high molecular weight DNA from the aqueous environment. Many bacteria species commonly Gram positive Haemophilus spp Bacillus spp and Escherichia coli are capable of acquiring resistance by this method. Transduction Transduction is a process of genetic transfer in which a small portion of the DNA is transferred from a donor cell to the recipient cell through temperate bacteriophages. The phage which acts as the agent of genetic transfer becomes integrated in the genome of the donor cell, replicating along with the host genome. It also transcribed at the expense of the host genome. Once this has occurred, the phage DNA is passed along to the daughter cell alongside the mother cell. Under stressed condition, either by heat or chemical the phage may be lysed loosing part of its DNA to the host cell or carry part of the host chromosome upon excision. Subsequent, infection of another bacterium by the phage which had just been lysed, from the other host and carrying the additional DNA picked up from the host may influence the introduction of the former DNA segment to its new host. This process is known as transduction. It occurs in bacterial genera such as Salmonella, Shigella, Staphylococcus, Pseudomonas, Vibrio, Proteus, Escherichia, and Bacillus.

Transposition The discovery of transposons (or transposable element) often referred to as jumping gene has provided explanation for frequent occurrence of drug resistance in bacteria. Transposons are units of DNA that move from one molecule of DNA to another, inserting themselves nearly at random (Pelczar,, 1993). Transposons transfer is not restricted like plasmid which is based on close relatedness of host genome. It can insert between one plasmid and another, or between a plasmid and a portion of bacterial chromosome within a bacteria cell without reliance on DNA sequence homology. Complex transposons usually have genes that code for many antibiotics and it is their activities that have resulted in R-Plasmid with resistance markers to the antibiotics (Russell, 2004).

Biotechnological method for gene acquisition Resistance gene can now be introduced into a wild type bacterium for the purpose of experiment by biotechnological methods. The most commonly used method is electroporation, and it is the most generalized way of achieving the introduction of resistant gene into a wild type bacterium, even though transformation, conjugation and transduction may also be applicable (Smith, 2004). Resistant genes are isolated and fragmented using restriction endonucleases. The genes excised are ligated into vectors such as plasmid so that they can be maintained i.e. replicated and expressed. After this process, the recombinant DNA are purified and are introduced into the bacterium to acquire it, by subjecting it to a high electric field so it can be induced to take up the DNA. This method allows the uptake of most sizes of plasmids. It was used recently to determine the importance of the gene gyrB in the resistant of Staph saprophyticus to novobiocin. The gene was introduced into Staph aureus RN4220 and it was found to

confer an approximately 64-fold increase in resistance to novobiocin relative to wild types (Vickers, 2007).

Integrons Integrons are gene capture system found in plasmids, chromosomes and transposons. They recongnise and capture multiple gene cassettes. A gene cassette may encode genes for antibiotic resistance, although most genes in integrons are uncharacterized. Therefore, integrons have been identified as a primary source of resistant genes within microbial population and were suspected to serve as reservoir of antimicrobial resistance gene within microbial population (Xu, 2009). The cassette has a specific recombination sites that confer mobility because it is recognized by recombinase encoded by the integrons that catalyses its integration into specific site within the integrons (Smith, 2004). Four classes of integrons have been identified with only one member of class 3 described and class 4 integrons are limited to Vibrio cholerae (Smith, 2004). The class 1 is found in both Gram-negative and Gram- positive bacteria while the class 2 integrons are frequently associated with the members of the family of Enterobacteriaceae such as Salmonella, Escherichia coli. The same class 2 was recently detected in Pseudomonas aeruginosa in which multi- drug resistant rates of integrons positive and negative strains were reported as 93.2% and 18.2% respectively (Xu,, 2009) Biochemical basis of resistance Changes in target site Most antibiotics exhibit their activities on microorganisms by acting on a particular site on the microbes referred to as the drug target site on the microorganisms. These sites are usually enzymes on the microbes. The inhibitors i.e. the antibiotics usually have a high affinity for the sites on the microbes and can out-compete any other substrate that may also depend on this site. However if this site is modified or altered it may lead to reduced or loss of affinity for the antibiotics and the bacteria with such altered site become

resistant to the inhibitor. This kind of resistance has been found in some organisms that showed resistant to sulphonamides. Sometimes it is the target proteins in the microbial cell that are altered. The altered penicillin binding protein in the cell wall of a particular strain of Staph aureus confer on it resistance to β-lactam and on Streptococcus pneumoniae resistance to penicillin. Alteration of target site had also been identified as the biochemical basis for the resistance in Enterococcus species to Aminoglycosides; in this group of bacteria, the ribosomal binding site has been altered. Reduction in cellular permeability There is a need for antibiotics to overcome certain mechanical barrier presented by the host cell membrane before they can elicit their activities. Mutants in bacteria have been identified to produce protective coat in the bacteria which reduce or inhibit the cellular uptake of antibiotics such as aminoglycosides and beta-lactams, chloramphenicol, bacitracin, and Isoniazid. Decreased uptake of flouroquinolone compound has been found associated with the changes in the porin protein of the bacterial cell membrane. Conversion of active drug into inert products Some bacteria develop resistance to antibiotics by producing enzymes which inactivates the antibiotics of choice either by

Destroying the antibiotics in use. These enzymes act by breaking one or more molecular covalent bonds in the antibiotics molecule which are central to the activities of the antibiotics. This mechanism is typical of Staph aureus which produces beta lactamases that breaks the bond of the beta lactam ring in penicillin and Cephalosporine rendering the drug inactive. It is responsible for the resistance in staphylococci to Benzyl Penicillin. The presence of β- lactamase gene on plasmid which can be exchanged among many bacterial species has confered penicillin resistance on many non- beta- lactamase producing which were initially susceptible to such penicillins. Some Gram- negative bacteria such as Serratia,


Enterobacter, and Pseudomonas have developed resistance to penicillin through this mechanism.
b. Inactivating an active product may be by the inhibition of a vital moiety in the


Most Gram-negative bacteria usually inactivate antibiotics by

producing phosphorylating and adenylating enzymes which destroys the activity of the antibiotics. Resistance to chloramphenicol is observed in bacteria that produce chloramphenicol acetyl transferases, which acts in the presence of acetyl Co A to catalyze the acetylation of 3’-OH group in the chloramphenicol molecule. The compound form from the catalysis is inactive. Increased production of biochemical intermediate Bacteria may also develop resistance to an antibiotic by increasing the production of a metabolite which competes with the active site of the antibiotics. This mechanism has been described associated with Staph aureus resistance to sulphonamide because sulphonamide is an analogue to paraamino-benzoic acid (PABA) in Staph. aureus which develops resistance to sulphonamide by increasing the production of PABA that would competitively displace sulphonamide. This unusual increased production of PABA has been found to be due to mutation in the regulatory gene of phosphate biosynthetic pathway.

2.7.2 Antibiotic resistance among wound pathogens and vaginal pathogens There has been continuous emergence of resistance among wound pathogens either by intrinsic ability of the bacteria to develop resistance or through the acquisition of resistant gene. Resistance of wound pathogens has remained a major challenge to the treatment of wound infection and has limited the drug of choice available for the treatment of wound infection. The problem of resistance in Africa arises from the uncontrollable use of antibiotics and inappropriate advert and wrong prescriptions. Nicolle (1996) observed that most predisposing factors contribute to the increasing frequency of antibiotic-resistant organisms in many homes. Considering this fact it is

essential to review the efficacy of the commonly used antibiotics and the newer ones, especially the fluoroquinolones by the available reports of various studies in different places and most especially Nigeria. This is essential because antibiotic susceptibility vary from regions to regions and among localities. Okesola, (1999) observed a significant level of resistance among wound pathogen to ampicillin, penicillin, cloxacillin, gentamicin, ceftriazone, ceftazidime, but with an acceptable level of sensitivity to pefloxacin and azithromycin. The study of Idowu (2009) shows that the wound pathogens shows resistant to various antibiotics with a considerable level of sensitivity to ofloxacin and gentamicin, 99% of the isolate in the study were sensitive to gentamicin and ofloxacin. These bacteria however show a high level of resistant to other antibiotics used in their study. The antibiotics used in this study include amoxycillin, cotrimoxazole, nitrofurantoin, nalidixic acid, and tetracycline. In Nigeria there are several reports that have shown the effectiveness of newer fluoroquinolones against both Gram positive and Gram negative bacteria isolates (Odelola and Idowu, 2007; Ehinmidu, 2003 Akortha and Ibadin, 2008; Nwaneze,, 2007). However, there have been advices that there is a need for caution in the way these drugs are administered, so that its use will not be abused and hence prevent the emergence of resistance to them. Results have shown emergence of resistance to these agents especially by beta-lactamase producing bacteria which are also resistant to other agents such as sulphonamide, aminoglycosides, tetracycline, chloramphenicol, trimethoprim. zhanel (2005) that had previously reported resistance to ciprofloxacin to be at a rate of 1.2% in 2000 reported an increase resistance to 5.5% in 2005 to the same antibiotics for out-patients. In India, Akram (2007) reported a high resistance rate of 69% against ciprofloxacin. Bacteria isolates gave a resistance of 24.1% against ofloxacin a flouroquinolone (Akortha and Ibadin, 2008). Among the aminoglycosides gentamicin still maintains a good level of activities both on Gram-positive and Gram-negative bacteria including Pseudomonas (Mbata, 2007). This has been attributed to its availability only on parenteral dosage only, thereby giving room to a lower chance of abuse. Penicillin which are often used with aminoglycosides, also have been limited in their use because of beta-lactamase producing bacteria; hence the noticed decrease in sensitivity of

even Gram- positive bacteria to amoxicillin and ampicillin and penicillin with improved stability to beta-lactamase. Okesola (2009) studied the antibiotic resistance among common bacterial pathogen reported that a considerable level of résistance among wound pathogens to various antibiotics used in the study include amoxicillinclavulate, cefuroxime, cefotaxime, chloramphenicol, ciprofloxacin, gentamicin, penicillin, streptomycin, with 72.6% of the isolates showing resistant to amoxicillinclavulate, resistant to cefotaxime, 83.1% to Chloramphenicol, 37.7% to ciprofloxacin, 20% to Gentamicin, 100% resistant to penicillin, 91.8% resistant to streptomycin, 61.6% to cefuroxime. Similarly antibiotic resistance has been described among the common pathogens associated with vaginal related infections particularly among the aerobes and bacteria which are members of the Gram-positive cocci. Ogunshe and Bakare (2009) reported resistance among bacteria which are involved in STI/ vaginal infection to some older antibiotics such as ampicillin, Septrin, chloramphenicol, Gentamicin, and tetracycline. These organisms are members of both Gram-positive and Gram- negative bacteria that can be isolated from the reproductive region. They include Klebsiella, Gardnerella, Streptococcus, E.coli and Staphylococcus. These organisms show varying degree of higher resistance to the above mentioned antibiotics. In addition to this some level of resistance were also found related to the use of fluoroquinolones though they were identified as being very potent. A resistance rate of about 45% was observed in some strain of E.coli Streptococcus and Staphylococcus. From the outcomes of the various finding above it is evident that resistance in microorganism has led to an increased reduction in the activities of antibiotics, which were once the antibiotics of first and second choice in the past. It is therefore essential that physician treats the patient with the appropriate agent as soon as the susceptibility result is out. It is also imperative that there should be re-evaluation of empirical drugs and there should be continued surveillance for emerging resistance among wound pathogens and vaginal pathogens.


2.8.1 Plasmid Curing Plasmids are extra-chromosomal genetic materials that are often present in the bacteria chromosomes as an independent unit capable of self replication without the activity of the bacteria chromosome. They are normally inherited stably in the next generation and can only be lost spontaneously from the host cell by replication error (segregation). Many phenotypic characters are encoded by Plasmid borne genes. These include properties such as resistance to antibiotics and heavy metals. Among all the known plasmids, R- plasmids are significant because they confer resistance on bacteria to various antibiotics, thereby posing a great threat to chemotherapy. R-plasmids may be classified as transferable or non-transferable. Transferable plasmids are detected by conjugation while non transferable plasmids are detected by their spontaneous loss from the host cell owing to some error in replication or segregation (Dorylo and Lorkiewiz; Novick, 1969). Curing may be defined as the elimination of a plasmid from a bacterial culture, it is the best method to substantiate the relationship between a genetic trait and carriage of specific plasmid by the culture as the phenotypic characters which are associated with the plasmid are not expressed but on the re- introduction of the plasmid into the cured strain, the lost phenotype re-appears (Gerbhardt, 1994). In other to cure plasmid both physical and chemical agents are used (Caro,, 1984; Gerbhardt,, 1994). These agents interfere with DNA replication or affect a particular organelle or enzyme of the bacterial cell (Stanisich, 1988). While some compound may cause curing in a rather nonspecific way, they damage and stress the cells whereas some others eliminate the sex factor F. agents that have been reported to for successful isolation of plasmid free cells includes intercalating dyes such as acridine orange or ethidium bromide (Mitsuhashi,, 1961; Watanabe and Fukasawa, 1961), different antibiotics (Hooper,, 1984), Sodium Dodecyl Sulfate (SDS), transition metals (Lakshimi,1988), temperature and plumb gin (Bharathi and Polsa, 1991; Lakshimi and Thomas, 1996). Curing experiments are conducted on trial and error basis, both with respect to the choice of curing agent and the culturing conditions used. Curing by Sodium Dodecyl Sulfate seems to be effective against a wide variety of bacteria. Similarly ethidium bromide is reported to be effective agent against Bacillus, Staphylococcus, Streptococcus, and others

(Clewel,, 1974, Stanisich, 1988). The probability that a curing agent can eliminate an extra-chromosomal element from its host depend on whether the plasmid is bound to the membrane or associated with the folded chromosome, because the former location will favour plasmid curing while the later will not. Multicopy plasmid or those located in both positions have a lower tendency to be lost. The extent of spontaneous loss or segregation is a property of a particular plasmid. The losses are also more frequent when Shigella or Salmonella host strain are used (Mitsuhashi, 1961; Watanabe and Fukasawa, 1961). Susceptibility to curing agents also varies among plasmids. There are some reports which reveals that some plasmid are not cured when treated with acridine orange and ethidium bromide. Twelve R- plasmids of Gram-negative bacteria isolated from Poultry in Karachi were studied for their curing by ethidium bromide. There has been previous report that these plasmids were not cured by acridine orange. Three out of this R- plasmid were cured following treatment

2.9 Justification of the study Staphylococcus aureus has been identified as the most commonly isolated pathogen from wound sepsis or infection and has also been described as one of the pathogens often isolated in clinical cases of bacteria vaginosis. Although the presence of Staphylococcus aureus in vagina discharge had not been fully regarded as a pathogen associated with vaginitis, its presence in vaginal discharge of women with vaginitis may symbolize its significance in this disease condition. Otunoye (2009) identified Staphylococcus species as part of the broad spectrum of pathogens that are involved in vaginitis among women in Nigeria. Staphylococci have been known to exhibit resistance to resistance to a variety of antibiotics particularly the β-lactam antibiotics. In recent time resistance to the new and commonly used antibiotics have been identified in staphylococci species isolated from wound and HVS, Otunoye (2009) recorded 60% resistant of Staphylococci species to ciprofloxacin; an example of flouroquinolone a recent antibiotics. The failure in the treatment of wound infection and vaginitis is due to the

emergence of resistance of the microorganism associated with this infection to previously effective drugs. It is therefore necessary that there is continuous surveillance on the emergence of microbial resistance to newer antibiotics and susceptibility testing for cost effective customization of empiric antibiotic therapy so as to control the increasing resistant to antibiotics among wound pathogens and vaginal pathogens as recommended by Ozunba (2005). Many works had been done on the resistant pattern of staphylococci species isolated from wound sepsis or infection to antibiotics, but there are various variations in the susceptibility pattern of the antibiotic of choice in different environment which thus necessitate the need for a time to time review of the susceptibility pattern of Staphylococci species from wound infection. Similarly much had been done in the study of Gardnerella vaginalis resistance to antibiotics as the predominant causative agent of vaginitis and group b Streptococcus. Nagara, (2005) identifies the resistant of Gardnerella vaginitis to metronidazole which is the common antibiotics often employ in the treatment of bacteria vaginosis, this is similar to the report of Sobel who observed various level of resistant of microbial population isolated from vaginitis resistance to various antibiotics. However not much work has been carried out in respect of staphylococcal species isolated from vaginitis resistant behavior. In view of this, this work is carried out to screening staphylococcus aureus species isolated from wound and HVS swab and hence study the antibiogram of these microorganisms as well as the comparative study of their plasmid curing responses. Previous works have also suggested an epidemiological role for antibiotic resistance curing among bacterial isolates from different clinical hence the need to examine the epidemiological role of antibiotic plasmid curing in Staphylococci aureus from HVS source and the wound source and to establish its epidemiological role in the diseases associated with these routes. This study therefore tries to study the epidemiological role of plasmid curing on the route of infection caused by Staphylococcal aureus.


CHAPTER THREE Material and Methods
3.1Materials 3.1.1 Equipment and glassware During the course of this study the following materials were used among others namely: Portable autoclave: (Gallenkamp, England) was used to sterilize appropriate culture for 15mins media at 121oc Hot air oven: was used for the sterilization of glassware and cotton wool at 160oc

Incubator: (Gallenkamp, England) operated at 37oc and was used to provide the environment for proper growth of bacteria used in the study Refrigerator: (Thermocool 250, England) used for maintaining organisms on agar slant at a temperature of 4oc Light microscope: (Olympus, Japan) was used to view stained bacterial smear on microscope slides. Vortex mixer: (Griffin and George Ltd, Britain). This was used for thorough mixing of overnight broth culture to obtain uniform inocula Weighing balance: (Mettler PC 400) was used to measure chemicals and media powder Water bath: (Electro-thermal, England) was used to boil media powder suspended in water for proper dissolution before sterilization and melt solid media. The glassware used included beakers, conical flasks, measuring cylinder, test tube, Pasteur pipette, glass slides, bijou bottle, Syringe and needle. They were properly washed and rinse several times with distilled water. After washing they were dried and sterilize in hot air at 160oc for 1hr

3.1.2 Culture media The culture media used were nutrient agar Mcconkey agar, Manitol salt agar Mueller Hinton agar, Dnase test agar, peptone water broth, Nutrient broth. Their composition and preparation were stated in the Appendix. 3.1.3 Antibiotics Multiple sensitivity disks from Abtek Biologicals limited were used and they comprised (Amoxycillin), (ceftriazone), (co.trimoxazole), (erythromycin), (pefloxacin), (ciprofloxacin), (gentamicin), (ampiclox), (chloramphenicol), (streptomycin). 3.2 Methods

3.2.1 Collection of isolates Thirty staphylococcal isolates from Wound and high vaginal swab specimen of patients with bacteriologically confirmed wound infection and sexually transmitted infections (15 staphylococcal isolates in each case) were obtained from the Department of Medical Microbiology University College Hospital between July 2010 and April 2011. The isolates were collected from various media which include Blood agar, Chocolate agar, and Sensitivity agar Mcconkey agar and were sub-cultured aseptically into nutrient agar slope. The isolates on receipt were immediately taken into the departmental laboratory for further identification and other necessary test. This was however done only usually after they have been kept in the incubator for 24hrs at 37oc after collection. This collection was done weekly and cultures not worked on were kept in the refrigerator. All isolates identified as Staphylococcus were maintained as stock cultures on slants and stored in the refrigerator at 4oc. Screening on manitol salt agar; Dnase agar and Gram staining were done periodically to guarantee the purity, the preservation of morphology and viability of the organisms. Biochemical identification The methods used for the identification and species differentiation were as described by Cheesbrough (2002) below Gram staining A thin smear of diameter of between 15-20mm of each isolate was made on a clean grease-free slide with wire loop. This was allowed to air dry and then fixed with heat by passing gently over a bursen burner flame. The smear on the slide was flooded with crystal violet for 60s and then rinsed off. Lugol’s iodine was added to fix the primary stain and allowed to stay for 60s after it was rinsed in water. It was then decolorized by adding 95% alcohol only for few seconds, rinsed in water and finally dilute safranin was added and allowed for about 2mins.the slides was rinsed in water and allowed to dry. The back of the slide was cleaned with blotting paper and the stained smear was observed

under the objective lens (×100) light microscope after the application of immersion oil. The result was recorded. Catalase test This test is used to determine the ability of an organism to produce the enzyme Catalase which releases oxygen from hydrogen peroxide. A loopful of bacterial colony was transferred from a nutrient agar culture plate to a drop of hydrogen peroxide on a slide. This was observed for the presence of bubbles of gas. The presence of bubble of gas indicates that the organism is positive for Catalase production. Coagulase test This test shows the presence of coagulase, an enzyme that clots plasma by converting fibrinogen into fibrin. Tube method 0.2ml of human blood plasma was pipette into EDTA anti coagulated tube. To each tube was added 0.8ml of 18-24hr broth culture of each organism. The tubes were incubated at 37oc and observed for clotting after 1hr. the tubes were tilted when checking for clot. If there was no clotting; the tubes were then examined at 30min interval for 6hr and 24hr. Dnase test This test is use to differentiate Staphylococcus aureus from coagulase negative Staphylococci. It detects the ability of an organism to degrade deoxyribonucleic acid (DNA)

A loopful of overnight broth culture of each staph isolates was streaked on Dnase agar plates containing methyl green and incubated at 37oc for up to 48hrs. The colonies on plate were then observed for the presence of clear zones. Haemolytic Pattern Layered blood agars were used for this test. It was prepared by pouring 8ml of nutrient agar into Petri dishes. These were allowed to set before equal volume molten blood agar was used to overlay them. Oxidation –fermentation test Bacteria differ in their ability to utilize carbohydrates under aerobic and anaerobic condition. This ability is used to differentiate organism into those that can oxidize or ferment carbohydrates. This is basically important for distinguishing between Micrococcus and Staphylococcus. One percent peptone water was prepared by dissolving one gram of peptone water broth in 100ml distill water. One percent of the various sugar (glucose, sucrose, and manitol) made by adding 1g of various sugar to the peptone water. To each peptone water, sugar solution was added 0.1g of Nacl (0.1%), followed by the addition of indicator, bromocresol purple. The mixture was dispensed into tube and sterilized by autoclaving at 121oc for 5mins. The tubes were then inoculated with each of the test organisms and incubated at 37oc for 24hrs to 72hrs. In the case of peptone – glucose solutions, there were duplicate tubes for each isolate. After incubation the tubes were observed for acid production indicated by colour change from purple to yellow. Gas production was observed by putting Durham tube in the test tube and checking for gas bubbles in them. Growth in 15% sodium chloride This is used to distinguish between staphylococci from other Genera in the family Micrococcacaea


Each isolate was grown in nutrient broth containing 15% sodium chloride at 37oc for 24hrs Overnight broth culture were then streak on nutrient agar and incubated at 37 OC for 24hrs. Plates were observed for growth and result taken.

3.2.3 Preparation of McFarland standards The McFarland standards were prepared as described by Komer (1951). Clean sterile screw capped universal bottles with numbered 1 to 10. One percent aqueous solution of barium chloride (Bacl2) was dispensed into bottles starting from 0.1ml and ending in 1ml in the 10th bottle. Appropriate volume of aqueous solution of concentrated tetraooxosulphate (VI) acid was added to the 1% aliquot of Barium chloride to make a total volume of 10ml in each bottle. The caps were then tightened and filled with paraffin tape. When needed for use the mixtures were shaken and matched with the bacterial suspension. Corresponding densities were interpreted as number of bacterial cell per ml 1= 3x 108 2 = 6 x 108 3 = 9 x 108 4 = 1.2 x 109 5 = 1.5 x 109 6 = 1.8 x 109 7 = 2.1 x 109 8 = 2.4 x 109

9 = 2.7 x 109

3.2.4 Preparation of iodine reagent Iodine crystal Potassium iodide 0.506g 13.30g

The two substances were dissolved in 25ml distilled water, starting with potassium iodide. The mixture were shaken well and stored in a brown bottle away from light until when the reagent is needed.

3.2.5 Preparation of starch solution The starch solution was prepared fresh as 1% w/v aqueous concentration by dissolving 0.25g of soluble iodine in 25ml of distilled water. The mixture was boiled in electro thermal water bath with intermittent stirring to give a white gelatinous solution. It was allowed to cool before use. 3.2.6 Phosphate buffer 0.1M Solution A: 1.36g of potassium dihydrogen phosphate (KH2PO4) was dissolved in 100ml of distilled water, pH 5.5. Solution B: 1.42g of disodium hydrogen phosphate, anhydrous (Na2HPO4) dissolved in 100ml of distilled water pH 8.8 34ml of solution A was then mixed 66ml of solution B to give phosphate buffer of pH 7.0. The buffer was then dispensed in 10ml amount into clean universal bottle and

sterilized by autoclaving. Thereafter, the buffer was stored away in the refrigerator until when needed.

3.2.7. Detection of β-lactamase Iodometrics (cell suspension) method was employed in the detection of β-lactamase as described by Sykes (1978) Overnight broth culture of each Staph.aureus was subcultured by streaking on nutrient agar plate and incubated at 37oc for 18-24hrs. A cell suspension was prepared in triplicates by emulsifying bacterial colonies with sterile wire loop in 0.5ml of freshly prepared phosphate buffer solution containing penicillin G (10,000 unit or 0.06mg/ml). Its density was determined to be about 1.8×109, with McFarland standards (Kolmer 1951) which had been found suitable in standardizing bacterial inoculums density to specific concentrations (Bauer 1966). The suspensions contained in small sterile tube were homogenized on a vortex mixer briefly. These were then incubated at room temperature 25oc for a minimum of 1hr. After then two drops of freshly prepared 1% aqueous solution was added without shaking the mixture. The mixtures were allowed to stand at room temperature for 10mins. For a colour change from blue-black to colorless. The results were interpreted as negative if there is no colour change within 10mins.

3.2.8 Antimicrobial susceptibility testing All staphylococcal isolates were further tested for their susceptibility pattern according to National Committee for Clinical Laboratory Standard guidelines (NCCLS, 2002 and Cheesbrough 2002). The antibiotic disc diffusion method was used as described by Kirby-Bauer was used


Isolates from pure overnight culture were suspended in sterile normal saline of 5ml. this was adjusted to 0.5mlMcFarland standard (108 cfu/ml) by adding normal saline if turbidity was too much than that of the standard Staph aureus. Seeding method was used for the antibiotic sensitivity susceptibility testing; 0.2ml of bacterial isolate was picked from the normal saline suspension and was transferred to 9.8ml of melted sensitivity agar. The agar medium was poured into a Petri-dish aseptically and was allowed to set. With the lid still in place, the seeded agar plate were left for 5mins to allow any surface moisture to be absorbed before applying the antibiotic disks. The multo-disks were placed firmly on the surface of the dried plate using sterile forceps. The plates were allowed to stand for 30mins before incubating aerobically and at inverted position at 37oc. Zones of growth inhibition were measured after 18-24hrs of incubation. Resistance was determined according to the reference zone diameter interpretation standard of NCCLS (2002) The Multiple Antibiotic Resistance (MAR) index was calculated as follow: MAR index for isolates= {Number of Antibiotics to which isolate is resistant/Number of antibiotic tested}. 3.2.9 Determination of the genetic basis of Resistance

The genetic basis for the resistance strains of Staphylococcus aureus isolated from the two sources was carried out by subjecting them to a plasmid curing test. The plasmid curing was done to determine the location of the resistant marker in the bacteria (whether it is plasmid borne or located on the chromosome. The curing (elimination) of the resistant plasmids of the (Staphylococcus aureus and Coagulase negative Staphylococcus spp.) isolated was done using various concentration of sodium dodecyl sulphate as described by Yah et al (2008); Akortha, (2010); with slight modification. Twenty Staphylococcus aureus, isolates from wound and HVS swab were grown for 24hrs at 37oC in nutrient broth containing sodium dodecyl sulphate at different concentration of 500µg/ml, 250µg/ml, 200µg/ml, 100µg/ml, 50µg/ml 25µg/ml and 12.5µg/ml. After

24hrs, the broth was agitated to homogenize the content and a loopful of bacteria in the broth medium were then sub-cultured onto drug free nutrient agar plates and were incubated for 48hrs at 37oc. Distinct colonies were then picked from plates which shows growth after 48hrs of incubation onto nutrient broth which was incubated overnight. From these broths, double fold dilutions of bacteria were prepared and from this dilution different dilutions were made into which MIC (minimum inhibitory concentration), of two of the drugs (cloxacillin and amoxycillin) to which antibiotics showed 100% resistance were added. The concentrations of these antibiotics were varied from 1µg/ml to 0.125µg/ml for the ampiclox and 1µg/ml to 0.25µg/ml for the amoxycillin. One of the broth dilutions in each concentration act as negative control which contains no bacterial colony and no drug while one of them act as the positive control containing no drug but only bacteria colony. These were incubated at 37oc for 24hrs, after which the activities of the antibiotics were observed. Absence of growth on nutrient broth which is indicated by showing a clear broth without turbidity indicates plasmid cured, thereby signifying plasmid as the agent which confer resistance on bacteria which enable it resist antibiotics has been cured. While the presence of growth on nutrient broth indicates that plasmid is not cured. This was done for twenty Staphylococcal isolates ten each from HVS and Wound swab.


CHAPTER FOUR RESULTS A total of thirty staphylococcal isolates were obtained from patients diagnosed with wound infection and HVS swab were used in this study. Fifteen of these isolates were from wound swabs while the rest were from high vaginal swabs. All the isolates were Gram-positive cocci and were found mostly in cluster of four and more. They were all positive for Catalase. The result for distinguishing staphylococci from micrococci showed that all fermented glucose and grew in broth containing 15% sodium chloride. All isolates grew in manitol salt agar. They were thus confirmed as Staphylococci Further test to identify the isolates to species level revealed that they were all coagulase- positive in human plasma. All the staphylococci were also positive for DNAse activity. The result of oxidation-fermentation is presented in Table 4.1 for all the staphylococcal isolates. All the Staphylococcus isolates fermented manitol aerobically, glucose and 80% fermented sucrose. All the Isolates but two grew in thioglycollate broth, most of them shows a high proportion of haemolytic ability as about 95% have one type of haemolysis or the other on blood agar plate.

The result of antibiotic susceptibility testing showed that there was a high proportion of susceptibility of staphylococcal isolates to Pefloxacin (90%), Gentamicin (67%) and ceftriazone (57%). The isolates were fairly sensitive to co-trimoxazole (54%). They showed a high level of resistance to Amoxicillin (100%) Tetracycline (100%) Cloxacillin (100%) Augmentin (94%) and Chloramphenicol (93%) (Table 4.2 and 4.3) Calculated multiple antibiotic resistant (Table 4.4) showed that all the isolates exhibited multiple antibiotic resistant. Some of the isolates (27%) were resistant to eight of the antibiotics tested. Table 4.5 also shows the pattern of antibiotic sensitivity pattern of beta lactamase producing strains (BLP) and non- beta lactamase producing strains (NBLP). Seventy-six percent of all the strains produced Beta-lactamase, out of which 100% were resistant to tetracycline, amoxicillin, and cloxacillin, and chloramphenicol 91.3% of

the isolates were resistant to Augmentin and, 80%, 56.5%, 44.5%, 34% and 8.7% of the isolates were resistant to erythromycin, cotrimoxazole, gentamicin ceftriazone and pefloxacin respectively. The non-beta lactamase producers also show a high rate of resistant to Augmentin, Amoxicillin, Cloxacillin, Tetracycline, and Erythromycin (100%) 85.7% resistant was observed to chloramphenicol, 36.9% resistant to ceftriaxone, 28.6% resistant to co-trimoxazole and pefloxacin. They were all sensitive to gentamicin. Comparing the resistance rate between the beta lactamase producers and the non beta lactamase producers, beta lactamase producers have a higher percentage of resistance than the non beta lactamase producing strains. Table4.2 and 4.3 can also be used to compare the independent in vitro behavior of staph aureus from Isolates from wound swab exhibited a higher rate of resistance to the antibiotic used as 60% of the isolates were resistant to gentamicin from wound infections against the 20% resistance observed in high vaginal swab isolates, co.trimoxazole, 40% against 36.7% in HVS, pefloxacin, 13.3% in wound against 6.7% HVS and 30% in wound for erythromycin against 20% resistant in HVS. Different patterns of resistant was however noticed in Augmentin where resistant is higher in HVS (100%) than in wound isolates (87.7%) and ceftriaxone where the resistant rate observed is 33.3% in wound against 53.3% in HVS isolate. This observation is similar for chloramphenicol in which resistant observed is 83.7% in wound against 100% in HVS. Table 4.6 shows the response of isolates after treatment with sodium dodecyl sulphate; most of the isolates at concentration of 500µg/ml, 250µg/ml. Some of the isolates grow in nutrient broth after treatment with a plasmid curing agents in all of these concentration. Table 4.7 shows the plasmid curing response of bacteria isolates after treatment with mutagen.

TABLE 4.1 Biochemical characterizations of bacterial isolates



NO WS 1 WS2 WS3 WS4 WS5 WS6 WS7 WS8 WS9 WS10 WS11 WS12 WS13 WS14 WS15 HVS1 HVS2 HVS3 HVS4 HVS5 HVS6 HVS7 HVS8 HVS9 HVS10 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + _ _ + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + + +


+ + + + + +

+ + + + + +

+ + + + + +

+ + + +

+ + + + + +

+ + + + + +

+ + + + + +

+ + + + + +

+ + + + + +

WS1 – WS2 = Wound Swab
HVS1 – HVS2 = High vaginal swab Glu =Glucose MLT = Maltose MAN = Manitol Suc = Sucrose THG = Thioglycollate MNS = Manitol salt HEM = Haemolysis DNASE = Dnase agar CAG = Coagulase


Table4.2. Antibiotic susceptibility pattern of swab

Staph aureus isolates from wound and hvs


ANTIBIOTIC SUSCEPTIBILITY PATTERN OF WOUNDS AND HVS SWAB GENT COT AUG AMX TET BLP (10µg) (25µg) (30µg) (25µg) (30µg) + 21[S] 16[S] R R R + 19[S] S R R R + R R R R R 20[S] 12R R R R + 20[S] R R R R 17[S] 19[S] R R R + R R R R R + 24[S] R 14[I] 12R R + 12[R] R R R R + R R R R R + 21[S] 16[S] R R R 17[S] 19[S] R R R + 12[R] R R R R + 24[S] R 14[I] 12R R

THE STAPHYLOCAOCCAL ISOLATES FROM CXC (5µg) R R R R R R R R R R R R R R PEF 22[S] 20[S] R 22[S] 21[S] 21[S] 16[S] S S R 22[S] 16[S] S S CEF 20[S] S R R 21[S] 21[S] R S S 17[S] 21[S] R S S CHL (30µg) 10[R] 10[R] R 10[R] R 13[I] R R R R 13[I] R R R ERY () S R R R R R R R 16[S] 17[S] R R 16[S] R


+ + + + + + + + + + _ + +

21[S] 20[S] 20[S] 19[S] 13[I] 20[S] 16[S] 14[I] 12[R] 12[R] 18[S] 14[I] 18[S] 19[S] 20[S] 12[R]

16[S] 19[S] S 16[S] S 16[S] R 12[R] 24[S] R R 12[R] R 16[S] S R





S 21[S] 18[S] 22[S] R 20[S] S S S S S S 24[S] 22[S] 21[S] S

R 21[S] R 20[S] R R R R S S R R R 20[S] 21[S] S


R R R R R R R R 14[I] 16[I] 10[R] R R R R 16[I]


Table4.3. Summary of the antibiotic susceptibility pattern of the strain of Staph aureus Categories Antibiotics Gentamicin (10µg) Sensitivity 19(63.3%) Intermediate 3(10%) Resistance 8(27.7%)

Co-trimoxazole (25µg)




Augmentin (30µg)




Amoxicillin (25µg)




Tetracycline (30µg)




Cloxacillin (5µg)









Ceftriaxone Chloramphenicol (30µg) Erythromycin (30µg)

17(56.7%) 0(0%) 0(0%)

0(0%) 1(3.3%) 6(20%)

13(43.3%) 29(96.7%) 24(80%)

N=21 Categories of susceptibility as defined using NCCLS (2002

Table4.4 Multiple antibiotic resistance (MAR) index of the isolated Staph aureus MAR index Frequency of MAR index (n=20) (100%)

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

0(0.00%) 0(0.00%) 0(0.00%) 0(0.00%) 0(0.00%) 4(13.33%) 8(26.67%) 8(26.67%) 8(26.67%) 1(3.33%) 1(3.33%)

NB: Number of Antibiotics to which isolate is resistant Number of Antibiotic used

Table 4.5 Summary of antibiotic susceptibility pattern of beta –lactamase producing (BLP) and non-beta-lactamase producing strain of Staph aureus isolates BLP n=23(76.7%) Intermedia Antibiotics Sensitivity te 3(13.0%) 0(0.0%) Resistance 8(34.8%) 13(56.5%) Sensitivity 7(100.0%) 5(71.4%) NBLP n=7(23.3%) Intermediat e 0(0.0%) 0(0.0%) Resistance 0(0.0%) 2(28.6%)

Gentamicin (10µg) 12(52.2%) Co-trimoxazole (25µg) Augumentin (30µg) 0(0.0%) 9(39.1%)






Amoxicilin (25µg) Tetracycline (30µg) Cloxacillin (5µg) Pefloxacin







0(0.0%) 0(0.0%) 21(91.3%)

0(0.0%) 0(0.0%) 0(0.00%)

23(100.0%) 23(100.0%) 2(8.7%)

0(0.0%) 0(0.0%) 5(71.4%)

0(0.0%) 0(0.0%) 0(0.00%)

7(100.0%) 7(100.0%) 2(28.6%)

Ceftriazone Chloramphenicol (30µg) Erythromycin (5µg)







0(0.0%) 3(10.0%)

0(0.0%) 3(10.0%)

23(100%) 17(80.0%)

0(0.0%) 0(0.0%)

1(14.3%) 0(0.0%)

6(85.7%) 7(100.0%)

Table4.6 : Response of Staph.aureus isolates to Mutagen on Drug free nutrient agar plate


Growth in drug free nutrient agar after exposture to mutagen 500µg/ml 250µg/ml N.G N.G N.G N.G + + + + + + + + N.G + + + + + + + N.G N.G N.G N.G + + + + N.G N.G N.G N.G N.G N.G N.G N.G + + + +

N.B N.G: no growth on drug free nutrient agar + = Growth on Nutrient Agar

Table 4.7: Staph aureus isolates response to varied concentration of Amoxycillin up to their MIC conc. after treatment with sodium dodecyl sulphate at a conc. of 500µg/ml


Bacterial isolates 1µg/ml WS3 WS5 WS6 WS8 WS9 WS10 HVS3 HVS4 HVS9 HVS15 +ve -ve -ve -ve -ve -ve -ve -ve -ve -ve

Amoxycillin 0.5µg/ml +ve -ve -ve -ve -ve -ve -ve -ve -ve -ve 0.25µg/ml +ve -ve -ve -ve -ve -ve -ve -ve -ve -ve


N.B +ve = Microbial growth on broth in the presence of the above named antibiotics after plasmid has been cured -ve = Absence of growth on broth when exposed to antibiotics

Table4.8: Staph aureus isolates response to varied concentration of Cloxacillin after treatment with sodium dodecyl sulphate at a conc. of 500 µg/ml

Bacterial isolates
1µg/ml WS3 WS5 WS6 WS8 WS9 WS10 HVS3 HVS4 HVS9 HVS15 +ve -ve -ve -ve -ve -ve -ve -ve -ve -ve

0.5µg/ml +ve -ve -ve -ve -ve -ve -ve -ve -ve -ve 0.25µg/ml +ve -ve -ve -ve -ve -ve -ve -ve -ve -ve 0.625µg/ml +ve -ve -ve -ve -ve -ve -ve -ve -ve -ve


+ve = Presence of growth on broth in the presence of Cloxacillin -ve = Absence of growth on broth on nutrient broth containing Cloxacillin

Table 4.9: Staph aureus isolates response to varied concentration of Antibiotics up to their MIC conc. after treatment with sodium dodecyl sulphate at a conc. of 250µg/ml Amoxycillin Bacterial isolates 1µg/ml WS3 WS5 WS6 WS8 WS9 WS10 HVS3 HVS4 HVS9 HVS15 +ve -ve -ve +ve -ve -ve -ve -ve -ve -ve 0.5µg/ml +ve -ve -ve +ve -ve -ve -ve -ve -ve -ve 0.25µg/ml +ve -ve -ve +ve -ve -ve -ve -ve -ve -ve


N.B +ve = Microbial growth on broth in the presence of the above named antibiotics after plasmid has been cured -ve = Absence of growth on broth when exposed to antibiotics

Table4.10: Staph aureus isolates response to varied concentration of Antibiotics after treatment with sodium dodecyl sulphate at a conc. Of 250 µg/ml


Bacterial isolates
1µg/ml WS3 WS5 WS6 WS8 WS9 WS10 HVS3 HVS4 HVS9 +ve -ve -ve +ve -ve -ve -ve -ve -ve

0.5µg/ml +ve -ve -ve +ve -ve -ve -ve -ve -ve 0.25µg/ml +ve -ve -ve +ve -ve -ve -ve -ve -ve 0.625µg/ml +ve -ve -ve +ve -ve -ve -ve -ve -ve






N.B +ve = Presence of growth on broth in the presence of Cloxacillin -ve = Absence of growth on broth on nutrient broth containing Cloxacillin

Chapter Five
Discussion and Conclusion


The general microbiological believe in the various techniques used in identifying staphylococcus aureus confirm the fact that they can be used in the rapid differentiation of Staphylococcus from micrococci. Both the tube coagulase method and the DNAse test have shown that all bacteria isolates are Staph aureus. Coagulase test is often used in rapid blood culture to identify Staph. aureus to a greater advantage over DNAse technique which may take a longer period (Ajuwape and Aregbesola 2001, Olarinde, 2010 (not published)). The results of the fermentation test also conform to the outcome of the work of other workers (Ajuwape and Aregbesola, 2001). It however does not agree that all strain must ferment manitol sucrose maltose and mannose. All the isolates except one shows one type of haemolysis or the other similar to the study of Ajuwape and Aregbesola, (2001) and the report of the findings of Olarinde (2010). This study however does not lay emphasis on the type of haemolysis since it is not an important biochemical characteristic in distinguishing coagulase and non coagulase producing Staphylococci species (Bello and Qahtani, 2005).

Staphylococcus aureus is a major pathogen in the genus Staphylococci and has been found to be the major cause of many suppurative and wound infection. Its presence had also been linked to bacteria vaginosis a polymicrobial disease condition which is common among women of all age. Staphylococcus aureus has been described as the leading cause of death in Nosocomial infection associated with wound infection. The ability of this organism to be readily transmitted from one host to another is associated with their ability to cope in the presence of minimal nutrient and tendency to survive in a harsh condition of growth. The result of this study supports the claim of Akerele and Akonkhai (2000) that Staphylococcus aureus as the leading organism being isolated

from cultures. The work of Onche and Adedeji agrees that Staphylococcus aureus is the commonest bacteria isolated from wound infection. Similarly Omoregie (2010) identified Staphylococcus aureus as the commonest aetiological agent for Reproductive tract infection in a study carried out in Benin. The work of Akerele, (2002) reveals Staphylococcus aureus as one of the Predominant organism isolated from asymptomatic genital infection. Okesola,

(2008) reported Staphylococcus as the predominant bacteria isolated from non surgical wound infection. This is in agreement with the observation of bowler (2001), it is also similar to the finding of Emele et al, 1999; Basak et al 1992 and the report of Shittu, 2002. Tranet 1998 and Mashita 2000 reported Staph aureus as the most frequently microorganism from wounds caused by incision to reach pus or fluid collection under the skin surface and from wound types observed in their study. Staph aureus is an opportunistic pathogen and a Normal flora of the skin (Bowler 2001) and of the female perineum and vulva (Davies, 1996) which can colonize wound when there is a breach in the skin to cause infections. It may be spread to the vaginal during sexual intercourse or when the environment of the vagina has an overgrown population of Staphylococcus aureus. Some of the virulence factors produced by some human strain of Staphylococcus aureus include: Panton-Valentine toxin, alpha toxin, protein coagulase and super-antigen which causes toxic shock syndrome (TSS). Staph aureus are known to be well pathogenic than all other species of Staphylococci. Staph aureus however does not spread readily through tissue and are such are carried or transmitted by the blood or within the neutrophil (Pelczar, 1993). The high resistance rate observed in the staph aureus isolated in this study against Cotrimoxazole (53.7%) Erythromycin, (80%), Augmentin (93.3%) Chloramphenicol, (96.7%) Tetracycline, (100%) Amoxicillin, (100%) Tetracycline, (100%) and Cloxacillin (100%) suggests an index that the cheap and commonly available antibiotics may not be useful in the empirical treatment of wound infections and vaginitis except as signified by the result of susceptibility testing. The result of this study with respect to resistance is similar to the observation of the report of Onche and Adedeji (2004) which observed complete resistance to Cloxacillin by Staph aureus isolated from implant surgery wound infections. The outcome of this study is also similar to the finding of Smith (2009) where all isolated species of Staph were susceptible to Gentamicin the outcome of this study reveals 60% susceptibility of the isolated strain to gentamicin, which is only available in parenteral form and may have been the reasons for the reduction in resistance. The high rate of resistance observed in chloramphenicol, and Tetracycline follows the report of Ogunshe and Bakare (2009) in which the Gram-positive bacteria isolates from their study exhibit a high rate of resistant to Chloramphenicol, Tetracycline, Co- trimoxazole (Septrin), Ampicillin and Penicillin. It however contradicts their observation in which there is a highly level of susceptibility to Erythromycin as the outcome of

this study reveals high level resistant to Erythromycin (80%). Similarly, Haghi (2010) in a study in Iran observed 100% Staphylococcus aureus resistant to Amoxicillin, Tetracycline, Chloramphenicol, Augmentin and cotrimoxazole. It however has a slight variation to the outcome of this study as the Staphylococcus aureus strains in this study show moderate level of resistant to Gentamicin unlike what was reported in their study where all Staphylococcus aureus were resistant to Gentamicin. Their study also reveals 100% resistant of isolates to Cotrimoxazole which is different from the outcome of this study where only 53.7% of Staphylococcus aureus isolates were resistant to Co-trimoxazole. The Cephalosporine antibiotic was represented by ceftriazone which is a third generation Cephalosporine. The result of this study reveals moderate level of susceptibility to ceftriazone (40%) and a moderate to high level resistant to ceftriazone which follows the observation of Bayat, (2009) in which 100% resistant was recorded for Staphylococcus aureus. It however contradicts the report of Ogunshe and Bakare (2009) that observed a high rate of susceptibility to ceftriazone and attributed this to the presence of acylamino-side chain. This observation may be due to the wide use of this antibiotic in the empiric treatment of infection before antimicrobial susceptibility testing. The flouroquinolone was represented in this study by pefloxacin, is of no doubt an efficient chemotherapeutic agent against wound infection and vaginal infection. There is a high rate of susceptibility of the Staphylococcus species to it. However there is reduction in the susceptibility pattern compare to what had been previously reported. The study of Adejumo (2011) in Ibadan reported 100% Susceptibility of Staphylococcus aureus isolate to flouroquinolone in a study of wound infection in Ile Ife hospital. The result obtained agrees with the findings of Ikeagwu (2008) where they reported reduction in the susceptibility pattern of Staphylococcus aureus isolated from Hvs wound and semen to Fluoroquinolones (65%). The reducing antimicrobial activities of the fluoroquinolones against Staph aureus can be traced to the indiscriminate use of these antibiotics in recent time despite their relatively high cost although it has been suggested that they should be use as a last line antibiotics due to the side effect profile especially in children. Resistant strains are gradually emerging due to selective pressure from constant use and incoherent prescriptions. This therefore suggests the need for caution before the emergence of a failure therapeutic effect in the treatment of wounds or vaginal

infection after the use of fluoroquinolones, pointing towards limitation in their application in the treatment of wound and vaginal infection. Resistance in bacteria to fluoroquinolones is primarily due to induced changes in the conformational structure, resulting from mutation in the gyrA gene which encodes the A subunit of the DNA gyrase. This mutation prevents fluoroquinolones from binding to the DNA molecule. A mutation which occurs to the gyr1A of the topoisomerase IV in Staph aureus can also cause t quinolone resistant (Smith, 2004) The high rate of resistant by the staphylococci species to Augmentin is almost similar to the report of Bayat (2010), where 100% resistance was recorded associated with the antibiotic sensitivity testing of Staph aureus in a hospital in Iran. This high rate of resistant may be associated with the consistent usage in clinical condition as well as indiscriminate application, owing to the presence of clavulanic acid as an additive which can inhibit beta-lactamases. The continuous use of this agent however might have resulted in the organism devising a mechanism of resisting the activities of clavulanic acid on the beta-lactamases. The expression of beta-lactamases is the most common mechanism of bacterial resistant to betalactam (Ahmed, 1996; Smith, 2004). In this study 80% of the isolate were beta-lactamase positive or shows beta-lactamase production. A similar or closed figure of 78% for the production of beta-lactamase was obtained for Staph aureus isolated from Post-operative wound swab (Ahmed, 1996). MAR index is a tool that reveals the spread of resistant isolates in a given population. A MAR index greater than 0.2 implies the strains of such bacteria originate from an environment where several antibiotics are used (Ehinmidu, 2003). Ehinmidu (2003) found MAR indices to a large number of bacteria selected against ten different antibiotics to be greater than 0.2. His findings is supported by the outcome of this study, as at least 90% of all the isolates have MAR index greater than 0.2. This shows that majority of the isolates have been exposed to various antibiotics. Plasmid is known to enhance the transfer of genetic material including resistant gene between bacterial species and genera (Miranda,, 2004). Plasmids have been implicated as one of the mechanism employed by microorganisms to resist the activities of antimicrobial agents.

Resistance to beta lactam antibiotics

has been attributed to the presence of a beta lactamase

enzyme which is borne on plasmid (ref). The work of Akinjogunla 2010, and Akortha, 2010 reveals the presence of plasmid

as a significant factor in the resistance observed in certain strains of Staphylococci and other bacteria; this is in accordance with the findings of this study where a significant proportion of the bacteria (Staph. aureus) become susceptible after curing had been carried out. Plasmid replication is inhibited by various agents especially acridine (acridine orange) that intercalates between the bases of DNA, without inhibiting the chromosomal DNA replication. Plasmid profile determination has been described as an important process in the epidemiological studies of infection (Akinjogunla 2010). In line with this the outcome of this study presents plasmid curing as an epidemiological tool in relation to the findings of Akinjogunla 2010. In this study, only four out of the ten Staphylococcus aureus isolates from HVS swab were able to survive in the presence of the mutagen while eight out of ten were able to survive the effect of the mutagen (sodium dodecyl sulphate at concentration of 250µg/ml and 500µg/ml.) this observation may be attributed to their varied sources of isolation. Similarly plasmid borne resistance was observed in all Staphylococcus aureus isolates from HVS while two strains of Staph.aureus of wound origin were not cured by the plasmid curing process thus inferring that resistance to antibiotic in them are chromosomal mediated. The outcome of this study however shows that Staph aureus isolates from wound were able to withstand the effect of the mutagen and response better to plasmid curing although a small fraction shows that the resistance exhibited by them were chromosomal mediated and not plasmid borne as they retain their resistance after curing. The Staph.aureus isolates from HVS were unable to cope with the used concentration of the mutagen but those that grew in the presence of the mutagen all become susceptible to the antibiotics to which they were previously resistant. This implies that all Staph.aureus isolates from this region have the resistant they showed associated with plasmid and not on the chromosomes. In conclusion, in this study plasmid had been shown to play a key role in Staph aureus resistance to antibiotics. It can also be deduced that Staph aureus response to plasmid curing process can be

attributed to their sources or site of isolation. Also Staph.aureus was discovered as a possible pathogen that could cause vaginitis and remains the commonly isolated bacteria from wound infections. The drugs that were found effective against the bacteria isolated in this study are Pefloxacin, Ceftriazone, Gentamicin, and Co-trimoxazole. This thus implies that there is limited range of antibiotics for the treatment of wound infection and vaginitis caused by the Grampositive Staph.aureus owing to the massive antibiotic resistance.

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APPENDIX Tube dilution method Preparation of Stock antibiotic suspension for cloxacillin Manufacturer: Elcee Pharmaceutical limited Indicated weight=500mg or 0.5g Weight of vial + powder = (a) = 14.5g Weight of empty vial= (b) =14.0g Weight of powder =(c) = 0.5g Weight of excipient = (c)g – indicated weight = 0.5 – 0.5g = 0.0g 0.02g of powder was dissolved in 200ml of sterile distilled water = 100µg/ml

Amoxycillin Manufacturer: Smithkline Beecham Pharmaceutical Brentford England Indicated weight = 500mg or 0.5g Weight of vial + powder = (a) = 13.8g Weight of empty vial = (b) = 13.3g Weight of powder = (c)g = 0.5g Weight of excipient = (c)g – indicated weight = 0.0mg 0.02g of powder was dissolved in 200ml of sterile distilled water = 100µg/ml


Composition and Preparation of Media Nutrient Agar Peptone Beef extract Sodium extracts Agar No 2 g/L 5.0 3.0 8.0 12.0

Twenty-eight gram was dissolved in 1L of distilled water, allowed to boil to dissolve properly before it was autoclaved at 121oC for 15minutes.

Blood Agar Blood agar was prepared the same way as nutrient agar except that it contained 5% human blood. The blood was added after sterilizing the nutrient agar. Nutrient Broth Peptone Yeast extract Beef extract Sodium chloride pH 7.4±0.2 g/L 5.0 2.0 1.0

Thirteen grams of the powder was dissolved in 1L of distilled water. This was gently boiled to dissolve completely. The broth was dissolved into test tubes and covered with caps and autoclaved at 121oC for 15minutes. Peptone Peptone (bacteriological) pH g/L 1.0 7.0 (approx)

Ten gram of powder was dissolved in 1L of distilled water and sterilized at 121oC for 15minutes.

Mueller- Hinton agar (M-HA) Oxoid Cm337 Beef, dehydrated infusion From casamino acids Casein hydrolysate Starch Agar pH 7.3 ± 0.2 at 25oC


300.0 17.5 1.5 1.5

Thirty-eight grams of the powder was dissolved in 1L of distilled water. This was boil to dissolve the agar and sterilized at 121oC for 15mins. For sensitivity test the agar was supplemented with 2% NaCl.

Thioglycollate broth (Brewer) Beef extract Yeast extract Balanced Peptone No Sodium Chloride Dextrose Sodium thioglycollate Methylene blue Agar No. 2 pH 7.1 ± 0.2 g/L 1.0 20.0 5.0 5.0 1.1 0.002 1.0


Approximately 20g of the powder in 1L of distilled water. This was brought to boil and later sterilized at 121oC for 15mins.

Mannitol salt agar (LABM) Typical Beef extract Balanced peptone No Sodium Chloride D- manitol Agar No Phenol red

g/L 1.0 10.0 75.0 10.0 12.0 0.0025

Approximately 111g of the powder is dissolved in 1L of distilled water, brought to boil and sterilize in the autoclave at121oc for 15mins

DNAse Agar (Oxoid) Tryptose Deoxyribonucleic acid Sodium Chloride Agar pH 7.3±0.2 at 25oC

g/L 70.0 2.0 5.0 12.0

Exactly 39g of the powder was dissolved in 1L of distilled water, This was sterilize in the autoclave at 121oc for 15mins.

Chemicals and reagents Chemicals Ethanol (70%, 95%, 100%) Hydrogen peroxide, H2O2

Liquid paraffin Sodium dodecyl sulphate

Indicator Bromocresol Purple Methyl green Reagents Gram stain Crystal violet Lugol’s iodine Acetone alcohol decolourizer Carbon fuschin/Safranin

Standard solutions Physiological Saline, 0.85%. Exactly 0.85g of sodium chloride was put in 100ml of distilled water. This was autoclaved and stored at room temperature.


McFarland standard solution; 0.5ml of aqueous solution of barium chloride was added to 99.5ml of 1% concentrated sulphuric acid in tubes to obtain 0.5 McFarland Standard which matches a broth culture containing approximately 1.5× 108 cells/ml


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