For Publication 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 et.al, 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 et.al 2004) Diabetics have a 15% higher risk for amputation than the general population (Synder 2005) due to chronic ulcers. Diabetes causes neuropathy,
4

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 et.al 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

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 et.al, 1998, Lydon et.al, 1989, Mousa 1987, Summannen et.al, 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 et.al 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 et.al 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, et.al, 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,
6

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 et.al, 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. 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, thereby causing series of local and systemic infection from the host tissue (Bowler et.al 2001). 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). Wound infection is therefore the invasion and multiplication of an infectious agent within the body tissues causing signs and symptoms of disease. Wound infection is associated with high rate of morbidity; prolong period of hospital stay, thereby increasing the cost of healthcare delivery for those affected. Owing to the associated resistance, it is also more resource demanding in terms of wound management (Bowler et.al, 2001). Wound is the leading cause of nosocomial infection in the US (Bowler et.al, 2001, Zoutman, McDonald, and Vethanayagan, 1998). It could affect people of any gender and age. Infections associated with wound could be either hospital acquired or community acquired. Wound infection usually results in an increase hospital stay. In the US, a study of post-surgical wound infections following head and neck surgery shows an average
7

increase in the days of hospitalization from 14days to 24days, after it has been infected (Bowler, et.al 2001, Johnson and Yu, 1991). A similar study on post-surgical wound infection of 108 patients in US shows an increase in treatment cost to $3,937 per infected patient (Zoutman, et al., 1998).

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, et.al, 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

Beta-hemolytic

Peptostreptococcus prevotii Streptococcus spp. (fecal) Peptostreptococcus indolicus, Streptococcus spp, (viridans) Peptostreptococcus sp, Corynebacterium xerosis,

Streptococcus intermedius, Corynebacterium sp, Clostridium perfringens, Bacillus sp. Clostridium clostridioforme, Clostridium cadaveris, Escherichia coli, Clostridium

baratii, Escherichia hermanii, Clostridium septicum, Serratia liquefaciens, Clostridium histolyticum, 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 asaccharolytica, Gram-negative pigmented bacillus Fusobacterium necrophorum,

Veillonella spp (Bowler et.al,2001, Brook, 1989, Johnson, et.al,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, et.al, 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, et.al, 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 et.al, 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 et.al 2001). Brook et al (1987) reported that 74% of 39 human and animal bites are infected with poly microbial aerobic and anaerobic microorganisms.

10

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, et al., 1998) and the inability of the polymorphonuclear cell to prevent the invasion of microbial pathogens effectively (Armstrong, et al,. 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 et.al, 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, et.al, 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

11

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 et.al (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, et.al, 1967, Robson, 1968, Robson and Hegger, 1969, and, Robson and Hegger, 1979). Levine, et.al, (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.

12

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 et.al, 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. The manifestation of chronic wounds classically include Delayed (or stalled) healing, Periwound 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, y y y y y 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 patients general condition

In Immunocompromised patients, however there may be modifications to the symptoms described above. For instance in diabetic patient, pain may not be a prominent symptom for foot ulcer, similarly in arterial ulcer, a previously dried wound may become wet if infected (Keryln, 2011). Wound infection may result in serious clinical condition like sepsis and septic shock once a systemic infection has occurred. This may manifest with any of these symptoms; pyrexia or hypothermia, tachycardia, tachypnoea, raised or
13

depressed white blood cell count, and multiple organ dysfunction, hypotension 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). Other 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 et.al, 2001).

14

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 nonselective 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, et.al, 1995, Hansson, et.al 1995, Rudensky et.al, 1992, Perry et.al, 1992, Sapisco et.al, 1984). Based on this fact, 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, et.al, 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

15

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 et.al, 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 et.al, 1969). This procedure has also given significant information on the number of microbes from a wound swab of a surgical wound (Levine et.al, 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, et.al, 1998) in studying rapid diagnostic tests for intra-amniotic infection. With the exception of Gram-

16

positive 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, et.al, 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, et.al, 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, et.al 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 et.al, 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

17

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 et.al, 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, et.al. 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

18

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 Coamoxiclav, cefuroxime, metronidazole Coamoxiclav Coamoxiclav, 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, y Optimization of management of co morbidities, e.g. optimizing glycemic control in diabetic patients and enhance tissue perfusion/oxygenation. y y y 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
19

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. Dakins 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 et.al, 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 et.al, 2001).

20

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 et.al, 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 et.al,1984,Mulder and Walker, 1989, Mulder et.al, 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, et.al, 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, et.al, 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 et.al, 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

21

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, et.al, 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. 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

22

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)

2.7.1.1 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, et.al, 1993, Hodges, 2004)

23

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

24

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

25

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, et.al, 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

26

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 et.al, 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 et.al, 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, et.al, 2009) Biochemical basis of resistance
27

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 -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 a. 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

28

and Cephalosporine rendering the drug inactive. It is responsible for the resistance in staphylococci to Benzyl Penicillin. The presence of - 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 antibiotics. 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

29

infection. The problem of resistance in Africa arises from the uncontrollable use of antibiotics and inappropriate advert and wrong prescriptions. Nicolle et.al. (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 et.al, (2009) 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 et.al (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,et.al., 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 et.al (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 et.al (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).

30

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 rsistance 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
31

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. 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

3.2.2.1 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. Lugols 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
32

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.

3.2.2.2 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.

3.2.2.3 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. 3.2.2.4 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)

33

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.

3.2.2.5 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. 3.2.2.6 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.

3.2.2.7 Growth in 15% sodium chloride This is used to distinguish between staphylococci from other Genera in the family Micrococcacaea

34

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 37OC 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 et.al (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

35

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 -lactamase Iodometrics (cell suspension) method was employed in the detection of -lactamase as described by Sykes (1978)
36

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 1.8109, with McFarland standards (Kolmer et.al 1951) which had been found suitable in standardizing bacterial inoculums density to specific concentrations (Bauer et.al 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.

37

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 et.al, (2010); with slight modification. Twenty Staphylococcus aureus, isolates from wound and HVS swab were grown for 24hrs at 37 C in nutrient broth containing sodium dodecyl sulphate at different concentration of 500g/ml, 250g/ml, 200g/ml, 100g/ml, 50g/ml 25g/ml and 12.5g/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 1g/ml
o

38

to 0.125g/ml for the ampiclox and 1g/ml to 0.25g/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.

39

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.
40

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 500g/ml, 250g/ml. Some of the isolates grow in nutrient broth after treatment with a plasmid curing agents in all of these concentrations. Table 4.7- 4.10 shows the plasmid curing response of bacteria isolates after treatment with mutagen.

TABLE 4.1 Biochemical characterizations of bacterial isolates

STRAIN NO WS 1 WS2 WS3 WS4 WS5 WS6 WS7 WS8 WS9 WS10 WS11 WS12 WS13 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + _ _ + + + + + + + + + + + + + + + + + + + + + + + + + + GLU MLT MAN SUC THG MNS HEM DNASE CAG

41

WS14 WS15 HVS1 HVS2 HVS3 HVS4 HVS5 HVS6 HVS7 HVS8 HVS9 HVS10 HVS11 HVS12 HVS12 HVS13 HVS14 HVS15

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

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

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

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

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

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

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

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

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

Key:
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

42

Table4.2. Antibiotic susceptibility pattern of swab STRAIN NO WS1 WS2 WS3 WS4 WS5 WS6 WS7 WS8 WS9 WS10 WS11 WS12 WS13 WS14 WS15 BLP + + + + + + + + + + + + GENT COT AUG AMX

Staph aureus isolates from wound and hvs

TET

CXC PEF

CEF

CHL

ERY

(10g) (25g) (30g) (25g) (30g) (5g) 21[S] 19[S] R 20[S] 20[S] 17[S] R 24[S] 12[R] R 21[S] 17[S] 12[R] 24[S] 21[S] 16[S] S R 12R R 19[S] R R R R 16[S] 19[S] R R 16[S] R R R R R R R 14[I] R R R R R 14[I] R R R R R R R R 12R R R R R R 12R R R R R R R R R R R R R R R R R
43

(30g) () 22[S] 20[S] 20[S] S R R 10[R] 10[R] R 10[R] R 13[I] R R R R 13[I] R R R R S R R R R R R R 16[S] 17[S] R R 16[S] R R

R R R R R R R R R R R R R R S

22[S] R 21[S] 21[S] 21[S] 21[S] 16[S] R S S R S S 17[S]

22[S] 21[S] 16[S] R S S S S S R

HVS1 HVS2 HVS3 HVS4 HVS5 HVS6 HVS7 HVS8 HVS9 HVS10 HVS11

+ + + + + + + +

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

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

R R R R R R R R R R R

R R R R R R R R R R R

R R R R R R R R R R R

R R R R R R R R R R R

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

R R R R R R R R R R R

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

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

HVS12 HVS13 HVS14 HVS15

+ _ + +

18[S] 19[S] 20[S] 12[R]

R 16[S] S R

R R R R

R R R R

R R R R

R R R R

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

R R R R R

R 16[I]

44

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

Co-trimoxazole (25g)

14(46.7%)

0(0%)

16(53.7%)

Augmentin (30g)

0(0%)

2(6.7%)

28(93.3%)

Amoxicillin (25g)

0(0%)

0(0%)

30(100%)

Tetracycline (30g)

0(0%)

0(0%)

30(100%)

Cloxacillin (5g)

0(0%)

0(0%

30(100%)

Pefloxacin

26(86.7%)

0(0%)

4(13.3%)

Ceftriaxone

17(56.7%)

0(0%)

13(43.3%)

45

Chloramphenicol (30g) Erythromycin (30g)

0(0%) 0(0%)

1(3.3%) 6(20%)

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

46

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%) Resistance 8(34.8%) Sensitivity 7(100.0%) NBLP n=7(23.3%) Intermediat e 0(0.0%) Resistance 0(0.0%)

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

0(0.0%)

13(56.5%)

5(71.4%)

0(0.0%)

2(28.6%)

2(8.7%)

21(91.3%)

0(0.0%)

0(0.0%)

7(100.0%)

Amoxicilin (25g) Tetracycline (30g)

0(0.0%)

0(0.0%)

23(100.0%)

0(0.0%)

0(0.0%)

7(100.0%)

0(0.0%)

0(0.0%)

23(100.0%)

0(0.0%)

0(0.0%)

7(100.0%)

Cloxacillin (5g) Pefloxacin

0(0.0%) 21(91.3%)

0(0.0%) 0(0.00%)

23(100.0%) 2(8.7%)

0(0.0%) 5(71.4%)

0(0.0%) 0(0.00%)

7(100.0%) 2(28.6%)

Ceftriazone Chloramphenicol (30g) Erythromycin (5g)

13(56.5%)

0(0.00%)

10(44.5%)

4(57.1%)

0(0.00%)

3(36.9%)

0(0.0%)

0(0.0%)

23(100%)

0(0.0%)

1(14.3%)

6(85.7%)

3(10.0%)

3(10.0%)

17(80.0%)

0(0.0%)

0(0.0%)

7(100.0%)

47

Table4.6: Response of Staph.aureus isolates to Mutagen on Drug free nutrient agar plate Growth in drug free nutrient agar after exposture to Isolates mutagen 500g/ml WS1 WS2 WS3 WS4 WS5 WS6 WS7 WS8 WS9 WS10 HVS1 HVS2 HVS3 HVS4 HVS5 HVS6 HVS7 HVS8 HVS9 HVS10 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 500g/ml N.G N.G + + + + N.G + + + N.G N.G + + N.G N.G N.G N.G + + 250g/ml N.G N.G + + + + + + + + N.G N.G + + N.G N.G N.G N.G + +

48

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

Amoxycillin N.B 0.5g/ml +ve -ve -ve -ve -ve -ve -ve -ve -ve -ve 0.25g/ml +ve -ve -ve -ve -ve -ve -ve -ve -ve -ve +ve = Mic robi al gro wth on brot h in the pres ence 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 g/ml

Bacterial isolates
1g/ml WS3 +ve 0.5g/ml +ve

Cloxacillin
0.25g/ml +ve 0.625g/ml +ve

49

WS5 WS6 WS8 WS9 WS10 HVS3 HVS4 HVS9

-ve -ve -ve -ve -ve -ve -ve -ve

-ve -ve -ve -ve -ve -ve -ve -ve

-ve -ve -ve -ve -ve -ve -ve -ve

-ve -ve -ve -ve -ve -ve -ve -ve

HVS15

-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

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 250g/ml

Amoxycillin Bacterial isolates 1g/ml WS3 WS5 +ve -ve

50

0.5g/ml +ve -ve

0.25g/ml +ve -ve

WS6 WS8 WS9 WS10 HVS3 HVS4 HVS9 HVS15

-ve +ve -ve -ve -ve -ve -ve -ve

-ve +ve -ve -ve -ve -ve -ve -ve

-ve N.B +ve -ve -ve -ve -ve -ve -ve +ve = Micr obial grow th 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 g/ml

Bacterial isolates
1g/ml WS3 WS5 WS6 WS8 WS9 +ve -ve -ve +ve -ve 0.5g/ml +ve -ve -ve +ve -ve

Cloxacillin
0.25g/ml +ve -ve -ve +ve -ve 0.625g/ml +ve -ve -ve +ve -ve

51

WS10 HVS3 HVS4 HVS9

-ve -ve -ve -ve

-ve -ve -ve -ve

-ve -ve -ve -ve

-ve -ve -ve -ve

HVS15

-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

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
52

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 et.al (2010) identified Staphylococcus aureus as the commonest aetiological agent for Reproductive tract infection in a study carried out in Benin. The work of Akerele et.al, (2002) reveals Staphylococcus aureus as one of the Predominant organism isolated from asymptomatic genital infection. Okesola et.al, (2008) reported Staphylococcus as the predominant bacteria isolated from non-surgical wound infection. This is in agreement with the observation of bowler et.al (2001), it is also similar to the finding of Emele et al, 1999; Basak et al 1992 and the report of Shittu et.al, 2002. Tranet et.al 1998 and Mashita et.al 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 et.al 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 et.al, 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
53

Cloxacillin by Staph aureus isolated from implant surgery wound infections. The outcome of this study is also similar to the finding of Smith et.al (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 et.al (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 et.al, (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 Adejuwon et.al (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
54

Amadi et.al (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 used 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 to quinolone resistant (Smith, 2004) The high rate of resistant by the staphylococci species to Augmentin is almost similar to the report of Bayat et.al (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, et.al 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 et.al, 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
55

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, et.al, 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 enzyme which is borne on plasmid (ref). The work of Akinjogunla et.al 2010, and Akortha et.al, 2010 reveals the presence of plasmid has been attributed to the presence of a beta lactamase

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 et.al 2009). In line with this the outcome of this study presents plasmid curing as an epidemiological tool in relation to the findings of Akinjogunla et.al 2009. 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 250g/ml and 500g/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

56

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 v7aginitis caused by the Grampositive Staph.aureus owing to the massive antibiotic resistance.

REFERENCES
Adejuwon1 A.O., Ogunkanmbi, D., M. A., BisiJohnson, B.O., Fadeyi, O.A Agboola and A.O., Adejuwon, 2011. Staphylococcus aureus isolated from septic caesaerean wound at Ile Ife

Nigeria: Antibiotics susceptibility patterns; International Journal of Medicine and Medical Sciences Vol. 3(5), pp. 149-154, Ahmed, E.A., Ahmed., E.M., Yousif, M.A and Arabi, Y.E. 1996. The relationship between bacterial beta-lactmase production and antibiotic resistance in Kartoum. African journal of Medicine and Medical Sciences 25: 133-136 Ajuwape, A.T.P and Aregbesola, E.A 2001. Biochemical characterization of Staphylococci isolated from rabbits. Israel Journal of Veterinary medicine. 56.2:1-5 Akerele, J and Ahonkai, I.A. 2000. Urinary pathogen and antibacterial susceptibility. A retrospective study of private diagnostic laboratory in Benin City Nigeria. Journal of Medical Laboratory Science 9:47-48

57

Akinjogunla, O. J.1., Adegoke, A. A., Mboto, C. I., Chukwudebelu, I. C. and Udokang, I. P 2009. Bacteriology of automobile accident wounds infection International Journal of Medicine and Medical Sciences Vol.1(2) pp. 023-027, Akortha, E.E and Ibadin, O.K. 2008. Incidence and antibiotics susceptibility pattern of Staphylococcus aureus amongst patient with urinary tract infection in Benin City, Nigeria. African Journal of Biotechnology Akram, M., Shaid, M. and Khan, A.U. 2007. Etiology and antibiotic resistance patterns of communityacquired urinary tract infections in JNMC Hospital Aligarh, India. Annals of Clinical Microbiology and Antimicrobials.6:4 Alos, J.I. and Chacon, J. 1988. Bacteriologia de las infecciones urinaries, extra hospitalaries. Medical Clinic 90:395 Amadi, E.S., Ikeagwu, I.J., Iroha, I.R. Antibiotic sensitivity pattern of Staphyloccoccus aureus in Abakiliki, Nigeria. Pak J Med Sci. 2008; 24(2):231-235

Armstrong, D. G., and Athanasiou, K. A.1998. The edge effect: how and why wounds grow in size and depth. Clin. Podiatr. Med. Surg. 15:105108. Armstrong, D. G., Liswood, P. J. and Todd W. F. 1995. Prevalence of mixed infections in the diabetic pedal wound. A retrospective review of 112 infections. J. Am. Podiatr. Med. Asoc. 85:533537 Basak S., Dutta S.K., Gupta S., Ganguly A.C, De R. 1992. Bacteriology of wound infections. Evaluation by surface swab and quantitative full thickness of wound biopsy culture. Journal of Indian Medical Association. 90: 33-34. Bayat, M, Zia, M, Haghi, M, Hemmatyar, G and Toghyani, M, (2011). Antibiotic resistance pattern of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa isolated from burnt patients in Urmia, Iran. African Journal of Microbiology Research Vol. 5(9) pp. 996-1000 Bello, C.S.S and Quantani, A. 2005. Pitfalls in the routine diagnosis of Staphylococcus aureus. African Journal of Biotechnology 4. 1:83-86 Bendy, R. H., Nuccio, P. A. Wolfe, E Collins, B. Tamburro, C. Glass, W. and Martin, C. M. 1964. Relationship of quantitative wound bacterial counts to healing of decubiti. Effect of topical gentamicin. Antimicrob. Agents Chemother. 4:147155 Bornstein J, Lakovsky Y, Lavi I, Bar-Am A, Abramovici H, 2001. The classic approach to diagnosis of vulvovaginitis: a critical analysis. Infect Dis Obstet Gynecol, 9:105-111.
58

Boulton, A. J., P. Meneses, and. Ennis, W. J., 1999. Diabetic foot ulcers: a framework for prevention and care. Wound Rep. Regen. 7:716. Bowler, PG (1999). The anaerobic and aerobic microbiology of wound: a review wound 10:170-178 Bowler, PG and Davies BJ (2001). The microbiology of acute and chronic wound; wounds 11:72-79 Bowler, PG and Davies, BJ, (1999); The microbiology of infected and non-infected leg ulcers. International Journal of Dermatology 38, 5738 Bowler, PG, Duerden, B I, and Armstrong, DG (2001). Wound microbiology and associated approaches to wound management. Clinical Microbiology Reviews 14, 24469. Breidenbach, W. C., and Trager. S. 1995. Quantitative culture technique and infection in complex wounds of the extremities closed with free flaps. Plast. Reconstr. Surg. 95:860865.

Brendan Healy, Andrew Freedman, (2006) ABC of wound healing. BMJ. Vol32 332:83841

Brian B.O., Tina L.F., Jeanne M.M., and David NF (2008). Diversity of Human Vaginal Bacterial. Anatomy

Textbook.
Brook, I. 1987. Microbiology of human and animal bite wounds in children. Pediatr. Infect. Dis. J. 6:29-32

Brook, I and Frazier FH (1990). Aerobic and Anaerobic microbiology of venous ulcer: International Journal of Dermatology 37: 2974-2976 Brook, I. (1998). Aerobic and anaerobic microbiology of infections after trauma in children: J. Accid. Emerg. Med. 15:162167 Brook, I. 1989. A 12 year study of the aerobic and anaerobic bacteria in intra-abdominal and post surgical abdominal wound infections. Surg. Gynecol.169:387392 Centers for Disease Control and Prevention: 1998 sexually transmitted diseases treatment guidelines. MMWR Morb Mortal Wkly Rep 1998; 47:1111. Chen, K.T., Huard, R C. Della-Latta, P.and Saiman, L, 2006. Prevalence of Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus in Pregnant Women; OBSTETRICS & GYNECOLOGY, VOL. 108, NO. 3, PART 1 Cherry, G. W., Ryan, T and McGibbon, D.1984. Trial of a new dressing in venous leg ulcers. Practitioner 228:11751178.
59

Cherry, GW (1989). Dissolution of wound coagulum and promotion of granulation tissue under duoderm. Wounds 1:95106. Cohen CR, Duerr A, Pruithithada N, et al., 1995. Bacterial vaginosis and HIV seroprevalence among female commercial sex workers in Chiang Mai, Thailand. AIDS 1995; 9:10937. Communities and Associations with Clinically Defined Bacterial Vaginosis. Journal of applied and environmental microbiology, p.48984909 Vol. 74, No. 15 doi:10.1128/AEM.02884-07 Cook, RL, Redondo-lopez, V, Schmitt, C, Meriwether, C and. Sobel, JD 1992. Clinical, Microbiological, and Biochemical Factors in Recurrent Bacterial Vaginosis. Journal of clinical microbiology, p. 870-877 0095-1137/92/040870-08$02.00/0 Vol. 30, No. 4 Cooper, R., Kingsley, A. and White, R. 2003. Wound Infection and Microbiology, Medical Communications (UK) Ltd for Johnson & Johnson Medical. Dancer S. J., Noble W.C., 1991. Nasal, axillary, and perineal carriage of Staphylococcus aureus among women: identification of strains producing epidermolytic toxin. J Clin Pathol; 44:6814. Davis J.P., Chesney P. J., Wand P. J, LaVenture M., 1980. Toxic-shock syndrome: epidemiologic features, recurrence, risk factors, and prevention. N Engl J Med 1980; 303:142935. Diamantopoulos, E. J., Haritos, D. fandi, Y. Grigoriadou, M. Margariti, G. Paniara, O. and Raptis, S. A. 1998. Management and outcome of severe diabetic foot infections. Exp. Clin. Endocrinol. Diabetes 106:346 352. Donders, G.G.G, Bosmans, E, Dekkersmaecker, A, Verecken, A, Bulck, BV and Spitz, B. 2000. Pathogenesis of abnormal vaginal bacterial flora. Am J Obstet Gynecol 182, 872878. Duerden, B. I. 1994. Virulence factors in anaerobes. Clin. Infect. Dis. 18:S253S259. Edoh D, Alomatu B., 2007. Comparison of antibiotic resistance patterns between laboratories in Accra east Ghana. AJST. 8(1):1-7 Edoh, D., and Mensah, A, 2005. Incidence of antibiotic resistant microbes in Accra west, Ghana. African Journal of Science and Technology (AJST) Science and Engineering Series Vol. 8, No. 2, pp. 103 - 109 Edward J.V., Howley, Cohen I.K., 2004. In vitro inhibition of human neutrophil elastase by oleic acid albumin formulation from devitalized cotton wound dressing; Intl journal of pharmacy 284,1121, 11-12 doi: 10.1016/j.pharm 2004 .06 .003 PMID 154554291 Edwards L. The diagnosis and treatment of infectious vaginitis. Dermatol Ther. 2004; 17(1):102-10.2.

60

Ehinmidu, J.O. 2003. Antibiotic susceptibility pattern of urine isolates in Zaria , Nigeria. Tropical Journal of Pharmaceutical Research 2.223-228 Emele, F.E., Izomoh, M.I, Alufohai, E. 1999. Microorganisms associated with wound infections in Ekpoma, Nigeria. West African Journal of Medicine. 18(2): 97-100. Eschenbach, D. A., Critchlow, C. W, Watkins, H., et al. (1983). A dose-duration study of metronidazole for the treatment of nonspecific vaginosis. Scand. J. Infect. Dis. 40(Suppl):73-80 Ferris D.G, Francis S.L., Dickman E.D, Miler-Miles K, Waller J.L, McClendon N, 2006. Variability of vaginal pH determination by patients and clinicians. J Am Board Fam Med. Jul-Aug 2006;19(4):368-73. [Medline] Finegold, S. M. 1989. Classification and taxonomy of anaerobes, p. 24. In S. M. Finegoldand George (ed.), W. L. Anaerobic infections in humans. Academic Press, Inc., San Diego, Calif. Foley, A.A, Nathan, C, Donovan O, and Simon, D, (1991). Eradication of methicillin resistant staph aureus vaginitis with Mupirocin. DICP vol 25:12:1331-1333 Gales, A. C., Jones, R. N., Pfaller, M. A, Gordon, K. A. and Sader, H. S. 2000. Two year assessment of pathogen frequency and antimicrobial resistance pattern among organisms isolated from skin and soft tissue infections in Latin American hospitals: results from the sentry antimicrobial surveillance programme, 1997-98, SENTRY study group. Int. J. Infect. Dis.: 4: 75 84. Goldenberg R, Klebanoff M, Nugent R, Krohn M, Hillier S, Andrews W; for the Vaginal Infections and Prematurity Study Group. Bacterial colonization of the vagina during pregnancy in four ethnic groups. Am J Obstet Gynecol 1996;174:161821. Graner, J. L. 1997., Livingston and the maggot therapy of wounds. Mil. Med. 162:296300. Gupta, K, Stapleton, AE and Hooton, TM (1998). Inverse association of Hydrogen peroxide -producing lactobacilli and vaginal E. coli colonization in women with recurrent urinary tract infections. J Infect Dis 178, 446450. Haghi, M, Maadi, H, Delshad, R, Mohammen AMN, and Saman, SG (2010). Antibiotic resistant pattern of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa from burnt patient. International journal of academic research vol. 2. no.6. part II Hardy, D.J., Legeai, R.J and O Callaghan, R.J. 1980. Kleibseilla neonatal infections: mechanism of broadening aminoglycosides resistance. Antimicrobial Agents Chemotherapy 542-548
Hay, (2006) National Guideline for the Management of Bacterial Vaginosis (2006) Clinical Effectiveness Group British Association for Sexual Health and HIV 61

Hegger, J. P., Robson, M. C and Doran, E. T. 1969. Quantitative assessment of bacterial contamination of open wounds by a slide technique. Trans. R. Soc. Trop. Med. Hyg. 63:532534 Hillier SL, Nugent RP, Eschenbach DA, et al: Association between bacterial vaginosis and preterm delivery of a low birth-weight infant. N Engl J Med 1995; 333:17371741. Hodges, N. 2004. Fundamental features of microbiology, Hugo and Russells Pharmaceutical microbiology. Eds. S.P Denyer, N.A Hodges, and S.P. Gorman. 7th ed. London: Blackwell Sciences. Holmes, et.al, (1984) and Eschenbach et.al (2000) Screening, Diagnosis and Management of Bacterial Vaginosis; Issues of Managing STD in family setting Hooper, D.C 2001. Emerging mechanisms of flouoroquinolone resistance. Emerging Infectious Diseases 7. 2.337-341 Hovik P. 1983. Nonspecific vaginitis in an outpatient clinic: Comparison of three dosage regimens of metronidazole. Scandinavian Journal of Infectious Diseases Suppl 40: 107110. Huebner, J., and Goldman, D. A., 1999. Coagulase negative Staphylococci role as pathogens. Annual Review of Medicine 50:223-236 Hussey, M. J., Levy, E. S. Pombar, X. Meyer, P and Strassner, H. T. 1998. Evaluating rapid diagnostic tests of intra-amniotic infection: Gram stain, amniotic fluid glucose level, and amniotic fluid to serum glucose level ratio. Am. J. Obstet. Gynecol. 179:650656.

Hutchinson, J. J., and Lawrence, J. C., 1991. Wound infection under occlusive dressings. J. Hosp. Infect. 17:8394 Idowu, A.O and Odelola, H.A. 2007. Prevalence of some uropathogenic bacterial isolates and their susceptibility to some quinolones, African Journal of Biomedical Research 10:269-273 Idowu, AO, Oluremi, BB and Seidu, NI, (2011). Incidence and susceptibility pattern of clinical isolates from pus producing infection to antibiotics and Carica papaya seed extract. African Journal of Biotechnology Vol. 10(9), pp. 1700-1704 Jacoby, G.A. 1994. Genetics of extended- spectrum beta-lactamases. European Journal of Clinical Microbiology Infectious Diseases23 (supplement 1): 2-11 Jerve F et al. 1984. Metronidazole in the treatment of non-specific vaginitis (NSV). British Journal of Venereal Diseases 60: 171174.

62

Johnson, M. J. Thatcher, E.and Cox, M. E. 1995. Techniques for controlling variability in Gram staining of obligate anaerobes. J. Clin. Microbiol. 33:755758. Johnson, M. J., Thatcher, E. and Cox, M. E. 1995. Techniques for controlling variability in Gram staining of obligate anaerobes. J. Clin. Microbiol. 33:755758 Jones and Ballet (2010) Open wound; Jones and Ballet Publication Katherine, T, Richard, Huard, Phyllis, Della-latta and Saiman, L (2006). Prevalence of MethicillinSensitive to Methicillin-Resistant Staphylococcus aureus in pregnant women; The American College of Obstetricians and Gynecologists. Kerlyn, C. Controversies in wound infection. International wound infection institute conference Cape town 2011 Kloos W. E, Schleifer K.H., 1975. Simplified scheme for routine identification of human Staphylococcus species. J Clin Microbiol 1: 82-88. Kloss, W.E., and Schleifer, K. H., 1975. Simplified scheme for routine identification of human Staphylococci species. Journal of Cliniocal Microbiology 1: 82-88 Kloss, W.E., Bannerman, T.L., 1994, Update on clinical significance of coagulase negative Staphylococci. Clinical Microbiology Review 7:117-140 Kownhar, H, Muthu, E Vignesh, S. R. Sekar1, R., Velu, V. and Anand Ra, U: 2008. High isolation rate of Staphylococcus aureus from surgical site infections in an Indian hospital; Journal of Antimicrobial Chemotherapy\doi:10.1093/jac/dkm519 Krizek, T. J.,Robson, M. C. and Kho, E. 1967. Bacterial growth and skin graft survival. Surg. Forum 18:518519 Laing, P. 1994. Diabetic foot ulcers. Am. J. Surg. 167:31S36S.

Lambert, P. 2004. Mechanisms of action of antibiotics and synthetic anti-infective agents. Hugo and Russells Pharmaceutical microbiology. Eds. S.P Denyer, N.A Hodges, and S.P. Gorman. 7th ed. London: Blackwell Sciences. 202-219 Larsen B and Monif G.R. (2001): Understanding the bacterial flora of the female genital tract. Clin Infect Dis; 32:e6977. Larsen B, Galask R.P, (1980). Vaginal microbial flora: practical and theoretic relevance. Obstet Gynecol; 55 suppl: 100S13S.

63

Larsen B, Galask R.P, (1982). Vaginal microbial flora: composition and influences of host physiology. Ann Intern Med; 96: 92630. Larsson PG, Forsum U 2005. Bacterial vaginosis--a disturbed bacterial flora and treatment enigma. APMIS, 113:305-316. Lawrence, J. C. 1994. Dressings and wound infection. Am. J. Surg. 167: 21S24S

Levine, N. S., Lindberg, R. B. Mason, A. D and. Pruitt, B. A. 1976. The quantitative swab culture and smear: a quick simple method for determining the number of viable bacteria on open wounds. J. Trauma 16:8994.

Linhares I.M., Giraldo2, P.C. and Baracat, E. C (2010). New findings about vaginal bacterial flora. Rev Assoc Med Bras 2010; 56(3): 370-4 Livingston, S. K. 1936. The therapeutic active principle of maggots. J. Bone Joint Surg. 18:751756.

Livingston, S. K. 1938. Clinical results following the use of a surgical jelly containing the maggot active principle. Am. J. Surg. 41:4950. Louie, T. J., Bartlett, J. G., Tally, F. P, and Gorbach, S. L, (1976). Aerobic and anaerobic bacteria in diabetic foot ulcers. Ann. Intern. Med. 85:461463. Lydon, M. J., Hutchinson, J. J, Rippon, M, Johnson, E, de Sousa, N, Scudder, C Ryan, T. J, and Cherry, G. W (1989). Dissolution of wound coagulum and promotion of granulation tissue under Duoderm. Wounds 1:95106. Mangram, A. J., Horan, T. C. Pearson, M. L. Silver, L. C. and Jarvis, W. R. 1999. Guideline for prevention of surgical site infection. Am. J. Infect. Control 27:97134. Marrazzo, JM, Koutsky, LA, Eschenbach, DA, Agnew, K., Stine, K. and Hillier, S. L. (2002). Characterization of vaginal flora and bacterial vaginosis in women who have sex with women. J Infect Dis 185, 13071313. Martin, L. Barbra A. Richardson, Patrick M. Nyange, Lavreys, L. Sharon L. Bhavna H., Bwayo, J and Kreiss, J. 1999. Vaginal

Kishorchandra, C Mandaliya, J. O. Achola, N

Lactobacilli, Microbial Flora, and Risk of Human Immunodeficiency Virus Type and Sexually Mashita, K., Shinagawa, N., Sato T., Hirata, K., Katsuramaki, T., Mukaiya M and Yura, J. 2000. Bacteria isolated from surgical infections and their susceptibilities to antimicrobial agents.
64

Special references to bacteria isolated between April 1997 and March 1998. Japanese Journal of Microbiology. 53 (80): 533-565 Mbata, T. 2007. Prevalence and antibiogram of urinary tract infection among prison inmate in Nigeria. The internet journal of microbiology3:2 Mc Targgart, I.A., and Elliot, T.S., 1989. Is resistance to novobiocin a test for confirmation of identification to Staphylococcus saprophyticus? Journal of Medical Microbiology 30:253-266 Meislin, H. W. McGehee, M. D. and Rosen, P. 1978. Management and microbiology of cutaneous abscesses. J. Am. Coll. Emergency Physicians7:186191 Melissa, CS, (2006). Vaginal infections. emedicinehealth.com Michael, S, Baggish, M, Mickey, M and Karam, M (2010). Anatomy of the vagina: Anatomy textbook. Mitsuda T, Fujita, A.K, Yokota, S, (1996). Demonstration of mother-to-infant transmission of Staphylococcus aureus by pulsed-field gel electrophoresis. Eur J Pediatr; 155:1949. Monica C (2002). District laboratory practice in tropical countries Part 2 (2nd edition). The Cambridge University Press, The Pitt Building,Trumpinton Street, Cambridge, UK., pp. 141142. Moreo, K (2005). Understanding chronic wound and overcoming challenges of effective case management for patient with chronic wounds: case manager 16(2): 62-3, 67, doi: 10:10161/J.case manager. 2005.01.014. PMID15818347 Morykwas, M. J., and. Argenta, L. C. 1997. Nonsurgical modalities to enhance healing and care of soft tissue wounds. J. South. Orthop. Assoc. 6:279288. Mousa, HA, (1987). Aerobic and Anaerobic fungal wound infection: Journal of Hospital infection 37:317-323 Mulder, G. D., and Walker, A. 1989. Preliminary observations on clotting under three hydrocolloid dressings. J. R. Soc. Med. 82:739740. Mulder, G., Jones, R Cederholm-Williams, S., Cherry, G., and Ryan, T. 1993. Fibrin cuff lysis in chronic venous ulcers treated with a hydrocolloid dressing. Int. J. Dermatol. 32:304306. Mumtaz, S., Akhtar, N. and Hayat, A. (2002). Antibiogram of aerobic pyogenic isolates from wounds and abscesses of patients at Rawalpindi, Pakistan. J. Med. Res.; 41 (1): 16-8. an extract from

65

Mustoe T, (2005). Dermal ulcer healing; Advances in understanding tissue repair and ulcer healing: Molecular mechanisms, therapeutic target and future directions. Paris, France Euro conference Match 17-25 2005 Mustoe, T (2004). Understanding chronic wound a unifying hypothesis on their pathogenesis and implication for therapy; Am .J. Surgery, 187 (5A); 655-705 doi: 10.1016/ J.casemgr, 2005 PMID 15147994
Nagaraja, P. Antibiotic Resistance of Gardnerella vaginalis in recurrent Bacteria vaginosis (2008). India Journal of medical microbiology 26 (2) pg 155-157

Nichols, R. L. 1998. Postoperative infections in the age of drug-resistant Gram-positive bacteria. Am. J. Med. 104:11S16S. Nichols, R. L. 1998. Postoperative infections in the age of drug-resistant Gram-positive bacteria. Am. J. Med. 104:11S16S. Nicolle, L.E., Strubaaugh, L.J. and Garribaldi, R.A 1996. Performance standards for antimicrobial susceptibility testing, tenth informational supplement , vol 18, no 1 National Committee for Central Laboaratory Standards, Wayne.Pa. Nwaneze, P.I., Nwaru, I.M., Oranusi, S., Dinmkpa, U., Okwu, M.U., Babatunde, B.B., Anake, T.A., Jatto., and Asagwara, C.E. 2007. Prevalence and antimicrobial susceptibility pattern. Scientific Research and Essay2.4:112-116//www.academicjournal.org//SRE Odugbemi, T.O., and Coker, A.O., 1987. Prevalence of hospital-acquired infection: cure and control. Journal of infectious Diseases 4:6:15 Ogunshe, A. Bakare, R. and Fasina, N (2009). Microbial Pathogens Implicated in Reproductive Health Infections in a Special Treatment Clinic in Ibadan, Nigeria. Journal of Family and Reproductive Health Vol. 3, No. 1, Okesola, AO and Oni, AA, (2009). Antimicrobial Resistance among common bacterial pathogens in South Western Nigeria. Journal of 330, Olarinde, J.D. 2010 (unpublished). Screening of Staphylococcus Spp from urinary tract infection specimens and antibiotic susceptibility pattern: A Thesis submitted to the Department of Pharmaceutical Microbiology for the award of Master degree in Pharmaceutical microbiology.pg57-62 American-Eurasian J. Agric. & Environ. Sci., 5 (3): 327-

66

Omoreghie, R., Erebor, J.O., Isibor, J.O and Ogefere , H.O. 2008. Observed changes in the prevalence of uropathogens in Benin City, Nigeria. Journal of Medical Laboratory Science 29-31 Onche and Adedeji, (2004).Microbiology of post-operative wound infection in implant surgery Nigerian Journal of Surgical Research Vol. 6, No. 1 - 2: 37 40 Otunoye, N.M., Odunukwe, N.N., Idugbe, E.O., Imosemi, O.D., Smith, S.I., Chigbo, R.T, Bamidele, M., Oparaugo, C.T., Mafe, A.D. and Musa, A.Z (2004). Aetiological agents of vaginitis in Nigerian women. Br J Biomed pg 175-178 Pappasian, C. J., and. Kragel, P. J. 1997. The microbiology laboratorys role in life-threatening infections. Crit. Care Nurs. Q. 20:4459. Parsonnet J, Hansmann M.A., Delaney M.L., Modern P.A., Dubois A.M., Wieland-Alter, W, et al, (2005). Prevalence of toxic shock syndrome toxin 1 producing Staphylococcus aureus and the presence of antibodies to this superantigen in menstruating women. J Clin Microbiol; 43:4628 34. Peel AL (1992). Definition of infection Pg 82-87; EW Taylor led infection modulation in surgical practice, Oxford university press Pelczar, M. J., Chain, E.C.S and Krieg, N.R., 1993. Microbiology. 5th ed. New York: Perry, C. R., Pearson, R. L, and Miller, G. A. 1991. Accuracy of cultures of material from swabbing of the superficial aspect of the wound and needle biopsy in the preoperative assessment of osteomyelitis. J. Bone Joint Surg. 73A:745749. Phillips, D., and Davey, C. 1997. Wound cleansing versus wound disinfection: a challenging dilemma. Perspectives 21:1516. Policar MS: Genital tract infections: how best to treat trichomoniasis, bacterial vaginosis, and candida infection. Consultant 1996; 36:17691774. Popescu, A., and Doyle, R. J. 1996. The Gram stain after more than a century. Biotechnol. Histochem. 71:145151. Raahave, D., Friis-Moller, A Bjerre-Jespen, K. Thiis-Knudsen, J and Rasmussen, L. B.1986. The infective dose of.aerobic and anaerobic bacteria in postoperative wound sepsis. Arch. Surg. 121:924929 Redondo-Lopez, V. Cook, RL and Sobel, JD. (1990). Emerging role of lactobacilli in the control and maintenance of the vaginal bacterial microflora. Rev Infect Dis 12, 856872. Robson, M. C., and Heggers, J. P. 1969. Bacterial quantification of open wounds. Mil. Med. 134:1924.
67

Robson, M. C., and. Heggers, J. P. 1970. Delayed wound closures based on bacterial counts. J. Surg. Oncol. 2:379383 Robson, MC (1997). Wound infection, a failure of wound healing caused by an imbalance of bacteria: Surgical clinical. North America 77; 637-650 Rudensky, B., M. Lipschits, M. Isaacsohn, and M. Sonnenblick. 1992. Infected pressure sores: comparison of methods for bacterial identification. South. Med. J. 85:901903. 212. Russell, A.D. 1994. Types of antibiotics and synthetic antimicrobial agents.Hugo and Russells Pharmaceutical microbiology. Eds. W.B. Hugo and A.D. Russel.5th ed. London: Blackwell Sciences.99-133

Russell, 2004. Types of antibiotics and synthetic antimicrobial agents. Hugo and Russells Pharmaceutical microbiology. Eds. S.P Denyer, N.A Hodges, and S.P. Gorman. 7th ed. London: Blackwell Sciences. 152-185

Sapico, F. L. Witte, J. L. Canawati, H. N. Montgomerie, J. Z. and Bessman, A. N. 1984. The infected foot of the diabetic patient: quantitative microbiology and analysis of clinical features. Schaaf V.M, Perez-Stable E.J, Borchardt K. The limited value of symptoms and signs in the diagnosis of vaginal infections. Arch Intern Med 1990, 150:1929-1933. Schaberg, D. R, Culver, D.H and Gaynes, R.P. 1991. Major trends in the microbial aetiology of Nosocomial infection. American Journal of Medicines 91.3B: 72-75 Schwiertz A, Taras D, Rusch K, Rusch V, 2006. Throwing the dice for the diagnosis of vaginal complaints?. Ann Clin Microbiol Antimicrob. Feb 17 2006;5:4. [Medline]. Sewankambo N, Gray R. H., Wawer M. J, Paxton L., McNaim D, Wabwire-Mangen F, et al. HIV-1 infection associated with abnormal vaginal flora morphology and bacterial vaginosis. Lancet 1997;350:546550. Sha BE, Chen HY, Wang QJ, Zariffard MR, Cohen MH, and Spear GT (2005). Utility of Amsel criteria, Nugent score, and quantitative PCR for Gardnerella vaginalis, Mycoplasma hominis, and Lactobacillus spp. for diagnosis of bacterial vaginosis in human immunodeficiency virus infected women. J Clin Microbiol , 43:4607-4612.

68

Shands KN, Schmid GP, Dan BB, et al. Toxic-shock syndrome in menstruating women:association with tampon use and Staphylococcus aureus and clinical features in 52 cases. N Engl J Med 1980; 303:143642 Sharma VK, Khadka, PB, Joshi A, Sharma R (2006). Common pathogens isolated in diabetic foot infection in Bir Hospital, Kathmandu University Medical Journal), Vol. 4, No. 3, Issue 15, 295301 Shittu A.O, Kolawole d.o. and Oyedepo E.A.R(2002) A study of wound infections in two health institutions in Ile-ife, nigeria. African journal of biomedical research. Pakistan journal of Biological sciences Smith, A. 2004. Bacterial resistance to antibiotics. Hugo and Russells Pharmaceutical Microbiology. Eds. S.P Denyer, N.A Hodges, and S.P. Gorman. 7th ed. Oxford: Blackwell Sciences. Sobel, J.D. (1999). Is there a protective role for vaginal flora? Curr Infect Dis Rep 1, 379383. Steed, D. L., Donohoe, D. Webster, M. W. and Lindsley, L. 1996. Effect of extensive debridement and treatment on the healing of diabetic foot ulcers. Diabetic Ulcer Study Group. J. Am. Coll. Surg. 183:6164. Summanen, P.H.D, Talan, C, Strong, M, Teagre, R, Bennion, JE, Thompson, Vaisanen, Moran, G, Winer, M and Finegold, SM (1995). Bacteriology of the skin and soft tissue infection; comparison of individual with no history of intravenous drug use: clin.infect DIS 20:S279-S282 Supp, DM, Boyce, ST (2005), emedicine surgical treatment and principle: Clinical, Dermatology 23(4):403-12 doi 10.1016/J.clinical dermatology 2004 PMID 16023936. Swedberg J et al. 1985. Comparison of single-dose vs. one-week course of metronidazole for symptomatic bacterial vaginosis. JAMA 254: 10461049. Sweet RL: Role of bacterial vaginosis in pelvic inflammatory disease. Clin Infect Dis 1995; 20(suppl 2):S271S275. Syder RJ (2005). Treatment of Non healing wound with Allograft; Clinical Dermatol 23(4): 388-95(doi 10 10161) Journal of clinical dermatology 2004 PMID16023934 Taylor JE (2005). Extent of iron pick up in defuroxamine coupled with polyurethane material for therapy of chronic wound; Biomatter 26(30) 6024033 Thomas, S., and Jones, M., 1998. The use of larval therapy in wound management. J. Wound Care 7:521524.

69

Thomas, S., Andrews, A., and Jones, M., 1999. Maggots are useful in treating infected or necrotic lesion: Thomson, P. D., and Smith, D. J. 1994. What is infection? Am. J. Surg. 167:7S11S. Todd, J.K. Staphylococcal infection. Pediatric in review Vol.26 No.12 December 2005 Tranet, T.S, Jamulitrat, S., Chongsuvivatvong, V., Geater, A. 1998. Postoperative hospital-acquired infection in Hungvuong Obstetric and Gynaecological Hospital, Vietnam. Journal of Hospital Infection. 40: 141-147 Transmitted Disease Acquisition. JID 1999 Vaginitis, Annex teen clinic, www.annexteenclinic.org

van De Wijgert, JH, Mason, PR , Gwanzura, L, Mbizvo, M T, Chirenje, ZM, Iliff, V, Shiboski, S, and Padian, NS (2000). Intravaginal practices, vaginal flora disturbances, and acquisition of sexually transmitted diseases in Zimbabwean women. J Infect Dis 181, 587594. Velander, PE, Theopold, C, Ghaerrardyn, R, Bleiziffer, O, Yao, F and Erickson, E (2004). Autologous Cultured Keratinocytes, suspension accelerates re-epithelization in diabetics pig: Journal of American college of surgery 199(3 supplementary 1).58 Vickers, A.A., Chaopra, I and O neil, J.A. 2007. Intrinsic novobiocin resistance in Staphylococcus saprophyticus. Antimicrobial Agents and Chemotherapy 51,12: 4484-4485. Vowden, K. R., and Vowden, P. 1999. Wound debridement. 1. Non-sharp techniques. J. Wound Care 8:237240. Wellstood, S. 1992. Gram-Positive Cocci. Encylopedia of Microbiology. London Academic Press. 319349 Wilkinson D, Connolly A, Harrison A, Lurie M, Karim SSA (1997): Sexually transmitted disease syndromes in rural South Africa: results from health facility surveillance. Sex Transm Dis 1998; 25:203. Williams, R. L., and Armstrong, D. G., 1998. Wound healing: new modalities for a new millennium. Clin. Podiatr. Med. Surg. 15:151154. Xu, Z. Li, L., Shirtliff, M.E., Alam, M.J., Yamasaki, S. and Shi, L. 2009. Occurrence and characteristics of class 1 and 2 integron in Pseudomonas aeruginosa in southern China. Journal of Clinical Microbiology 47.1:230-234

70

Yudin, M H, Money, DM (2008). Screening and Management of Bacterial Vaginosis in Pregnancy Society of Obstetricians and Gynecologists of Canada Guidelines

PRACTICE

GUIDELINE
ZAFAR, A., ANWAR, N AND, EJAZ, H, (2007). Bacteriology of infected wounds - a study conducted at children hospital Lahore; E:/Biomedica Vol.23 Jul. Dec./Bio-8 Zhou, X, Stephenson, J, Bent, Maria, G, Schneider, Catherine, C, and Davis (2004). Characterization of Vagina Microbial communities in adult healthy women using cultivation independent method. Microbiology, 150, 25652573 DOI 10.1099/mic.0.26905-0 Zoutman, DS, McDonald, and Vethanayagan D (1999). Total and attributable costs of surgical-wound

infections at a Canadian tertiary-care center; Infect. Control Hosp. Epidemiol. 19:254259

71

72