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Microbial prevalence and antimicrobial susceptibility of wound isolates from tertiary institution

CHAPTER ONE

INTRODUCTION

1.0 Background of the study

A wound is a type of injury which happens relatively quickly in which

skin is torn, cut, or punctured (an open wound), or where blunt force trauma

causes a contusion (a closed wound). In pathology, it specifically refers to a

sharp injury which damages the dermis of the skin. Intact skin is the perfect

defence to bacterial invasion, but damage to the skin allows bacteria, fungi and

yeasts to enter (Young, 2012). More than 200 different species of bacteria

normally live on the skin1 and an open wound provides a moist, warm and

nutritious environment perfect for microbial colonisation and proliferation.

Bacteria colonise all chronic wounds and low levels of bacteria can benefit the

wound by increasing the amount of neutrophils, monocytes and macrophages

in the wound, thus improving levels of prostaglandin E2 and the formation of

collagen (Edwards, & Harding, 2004). When one or more microorganisms

multiply in the wound, local and systemic responses occur in the host, which

can lead to infection and a subsequent delay in healing (Angelet al.,2011).

Maintaining the bacteria at a level at which the host is in control is an

important part of avoiding wound infection (Cutting, 2010).


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Regardless of large amounts of bacteria, many wounds continue to heal

well. The ability of the patients immune system to deal with bacteria (host

response) and the type and amount of bacteria involved determines whether

clinical problems will occur (Young, 2012). Chronic wounds are open for

extended periods of time and the patients usually have underlying disease

processes, which leads to heavy colonisation with bacteria and/or fungi (Young,

2012). When chronic wounds are poorly perfused they are more susceptible to

infection, as blood delivers oxygen, nutrients and immune cells, thus providing

little opportunity for microorganisms to colonise and proliferate (Bowler et al.,

2001). Devitalised tissue, combined with fluid and nutrients from wound

exudate provide an ideal setting for bacterial proliferation (Cutting 2010). The

host response can often be improved by correcting or improving the underlying

diseases (Young, 2012).

1.2.0Aim and Objectives

Aim

This study is aimed at determining the Microbial prevalence and

antimicrobial susceptibility of wound isolates from tertiary institution.

The specific objectives were to:


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1.Isolate, characterize and identify the morphological biochemical test of

wound collected from tertiary institution

2.To identify types of Microbial prevalence over wound

3.To determine antimicrobial susceptibility of wound isolates from tertiary

institution

CHAPTER TWO

LITERATURE REVIEW

Overview of Wounds

A wound is a breakdown in the protective function of the skin; the loss of

continuity of epithelium, with or without loss of underlying connective tissue

(Bowler et al., 2001). Wounds can be accidental, pathological or post-operative.

An infection of this breach in continuity constitutes wound infection. Wound

infection is thus the presence of pus in a lesion as well as the general or local

features of sepsis such as pyrexia, pain and induration. Infection is believed to

occur when virulence factors expressed by one or more microorganisms in a

wound out-compete the host natural immune system (Bowler et al., 2001).

Wound infection is important in the morbidity and mortality of patients

irrespective of the cause of the wound. It is also important because it can delay


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healing and cause wound breakdown (Alexander, 1994). This is also associated

with longer hospital stay and increased cost of healthcare (Sule et al., 2002).

Wound infections are also significant in that they are the most common

nosocomial infection (Dionigi et al., 2001).

Studies on wound infection have largely focused on surgical site infections

(Sands et al., 1996; Garner, 1986 and Gaynes etal., 2001). This might be

because other types of wound infection are not problematic in the developed

world where most of these studies have been done. However, in developing and

resource-poor countries, other types of wound infection in addition to surgical

site infection are still important causes of morbidity and mortality (Mehta et al.,

2007; Anguzu & Olila, 2007; Fadeyi et al., 2008). Where studies have been

done on wound infections generally, regional and local variations have been

observed in terms of the causative micro-organisms (Sule et al., 2002; Wariso &

Nwachukwu, 2003; Egbe et al., 2011). This means that physicians need to

know the prevalent organisms and the resistance patterns existing in their

localities.

Classification

According to level of contamination a wound can be classified as


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Clean wound, a wound made under sterile conditions where there are no

organisms present in the wound and the wound is likely to heal without

complications (Kidd et al., 2000).

Contaminated wound, where the wound is as a result of accidental

injury where there are pathogenic organisms and foreign bodies in the

wound.

Infected wound, where the wound has pathogenic organisms present and

multiplying showing clinical signs of infection, where it looks yellow,

oozing pus, having pain and redness (Kidd et al., 2000).

Colonized wound, where the wound is a chronic one and there are a

number of organisms present and very difficult to heal as in a bedsore

(Kidd et al., 2000).

Abrasion

In dermatology, an abrasion is a wound caused by superficial damage to

the skin, no deeper than the epidermis. It is less severe than a laceration, and

bleeding, if present, is minimal. Mild abrasions, also known as grazes or

scrapes, do not scar or bleed, but deep abrasions may lead to the formation of

scar tissue. A more traumatic abrasion that removes all layers of skin is called

an avulsion (Kidd et al., 2000).


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Abrasion injuries most commonly occur when exposed skin comes into

moving contact with a rough surface, causing a grinding or rubbing away of

the upper layers of the epidermis (Kidd et al., 2000).

The abrasion should be cleaned and any debris removed. A topical

antibiotic (such as Neosporin or bacitracin) should be applied to prevent

infection and to keep the wound moist (Kidd et al., 2000). Dressing the wound

is optional but helps to keep the wound from drying out which interferes with

healing. If the abrasion is painful, a topical analgesic (such as lidocaine or

benzocaine) can be applied, but for large abrasions. A systemic analgesic may

be necessary. Avoid exposing abraded skin to the sun as permanent

hyperpigmentation can develop (Kidd et al., 2000).


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Fig 2.1: Abrasion on the palm of a right hand, shortly after falling

Sources: Kidd et al., 2000

Chronic wound

A chronic wound is awoundthat does not heal in an orderly set of stages and

in a predictable amount of time the way most wounds do; wounds that do not

heal within three months are often considered chronic (Robert, 2005). Chronic

wounds seem to be detained in one or more of thephases of wound healing.

For example, chronic wounds often remain in theinflammatorystage for too

long (Robert, 2005; Taylor et al., 2005). In acute wounds, there is a precise

balance between production and degradation ofmoleculessuch ascollagen; in

chronic wounds this balance is lost and degradation plays too large a role

(Edwards et al., 2004; Schnfelder et al., 2005).

Chronic wounds may never heal or may take years to do so. These wounds

cause patients severe emotional and physicalstressand create a significant

financial burden on patients and the whole healthcare system (Augustin &

Maier, 2003).

Acute and chronic wounds are at opposite ends of a spectrum of wound healing

types that progress toward being healed at different rates (Kathleen, 2005).

Signs and Symptoms
https://en.wikipedia.org/wiki/Woundhttps://en.wikipedia.org/wiki/Wound_healinghttps://en.wikipedia.org/wiki/Inflammationhttps://en.wikipedia.org/wiki/Moleculehttps://en.wikipedia.org/wiki/Collagenhttps://en.wikipedia.org/wiki/Stress_(medicine)https://en.wikipedia.org/wiki/Wound_healinghttps://en.wikipedia.org/wiki/Inflammationhttps://en.wikipedia.org/wiki/Moleculehttps://en.wikipedia.org/wiki/Collagenhttps://en.wikipedia.org/wiki/Stress_(medicine)https://en.wikipedia.org/wiki/Wound


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Chronic wound patients often report pain as dominant in their lives (Krasner,

1998). It is recommended that healthcare providers handle the pain related to

chronic wounds as one of the main priorities in chronic wound management

(together with addressing the cause). Six out of ten venous leg ulcer patients

experience pain with their ulcer (Hofman, 1997), and similar trends are

observed for other chronic wounds.

Persistent pain (at night, at rest, and with activity) is the main problem for

patients with chronic ulcers (Catherine, 2006). Frustrations regarding

ineffective analgesics and plans of care that they were unable to adhere to were

also identified.

Cause

In addition to poor circulation, neuropathy, and difficulty moving, factors that

contribute to chronic wounds include systemic illnesses, age, and repeated

trauma. Comorbid ailments that may contribute to the formation of chronic

wounds include vasculitis (an inflammation of blood vessels), immune

suppression, pyoderma gangrenosum, and diseases that cause ischemia

(Robert, 2005). Immune suppression can be caused by illnesses or medical

drugs used over a long period, for example steroids (Robert, 2005). Emotional

stress can also negatively affect the healing of a wound, possibly by raising


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blood pressure and levels of cortisol, which lowers immunity (Augustin & Maier,

2003). What appears to be a chronic wound may also be a malignancy; for

example, cancerous tissue can grow until blood cannot reach the cells and the

tissue becomes an ulcer (Hofman et al., 1997). Cancer, especially squamous

cell carcinoma, may also form as the result of chronic wounds, probably due to

repetitive tissue damage that stimulates rapid cell proliferation (Hofman et al.,

1997). Another factor that may contribute to chronic wounds is old age

(Thomas, 2004). The skin of older people is more easily damaged, and older

cells do not proliferate as fast and may not have an adequate response to stress

in terms of gene upregulation of stress-related proteins (Thomas, 2004). In

older cells, stress response genes are overexpressed when the cell is not

stressed, but when it is, the expression of these proteins is not upregulated by

as much as in younger cells (Thomas, 2004). Comorbid factors that can lead to

ischemia are especially likely to contribute to chronic wounds. Such factors

include chronic fibrosis, edema, sickle cell disease, and peripheral artery

disease such as by atherosclerosis (Robert, 2005). Repeated physical trauma

plays a role in chronic wound formation by continually initiating the

inflammatory cascade. The trauma may occur by accident, for example when a

leg is repeatedly bumped against a wheelchair rest, or it may be due to

intentional acts. Heroin users who lose venous access may resort to 'skin


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popping', or injecting the drug subcutaneously, which is highly damaging to

tissue and frequently leads to chronic ulcers (Williams & Southern, 2005).

Children who are repeatedly seen for a wound that does not heal are sometimes

found to be victims of a parent with Munchausen syndrome by proxy, a disease

in which the abuser may repeatedly inflict harm on the child in order to receive

attention (Vennemann et al., 2006).

Pathophysiology

Chronic wounds may affect only the epidermis and dermis, or they may affect

tissues all the way to the fascia (Crovetti et al., 2004). They may be formed

originally by the same things that cause acute ones, such as surgery or

accidental trauma, or they may form as the result of systemic infection,

vascular, immune, or nerve insufficiency, or comorbidities such as neoplasias

or metabolic disorders (Crovetti et al., 2004). The reason a wound becomes

chronic is that the bodys ability to deal with the damage is overwhelmed by

factors such as repeated trauma, continued pressure, ischemia, or illness

(Crovetti et al., 2004). Though much progress has been accomplished in the

study of chronic wounds lately, advances in the study of their healing have

lagged behind expectations. This is partly because animal studies are difficult

because animals do not get chronic wounds, since they usually have loose skin


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that quickly contracts, and they normally do not get old enough or have

contributing diseases such as neuropathy or chronic debilitating illnesses

(Thomas, 2004). Nonetheless, current researchers now understand some of the

major factors that lead to chronic wounds, among which are ischemia,

reperfusion injury, and bacterial colonization (Thomas, 2004).

Ischemia

Ischemia is an important factor in the formation and persistence of wounds,

especially when it occurs repetitively (as it usually does) or when combined

with a patients old age (Thomas, 2004). Ischemia causes tissue to become

inflamed and cells to release factors that attract neutrophils such as

interleukins, chemokines, leukotrienes, and complement factors (Thomas,

2004). While they fight pathogens, neutrophils also release inflammatory

cytokines and enzymes that damage cells (Thomas, 2004; Robert, 2005). One of

their important jobs is to produce Reactive Oxygen Species (ROS) to kill

bacteria, for which they use an enzyme called myeloperoxidase (Thomas, 2004).

The enzymes and ROS produced by neutrophils and other leukocytes damage

cells and prevent cell proliferation and wound closure by damaging DNA, lipids,

proteins (Alleva et al., 2005), the extracellular matrix (ECM), and cytokines that

speed healing (Thomas, 2004). Neutrophils remain in chronic wounds for longer


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than they do in acute wounds, and contribute to the fact that chronic wounds

have higher levels of inflammatory cytokines and ROS (Taylor et al., 2005).

Since wound fluid from chronic wounds has an excess of proteases and ROS,

the fluid itself can inhibit healing by inhibiting cell growth and breaking down

growth factors and proteins in the ECM. This impaired healing response is

considered uncoordinated (Krishnaswamy et al., 2014). However, soluble

mediators of the immune system (growth factors), cell-based therapies and

therapeutic chemicals can propagate coordinated healing (Lasagni et al., 2010).

It has been suggested that the three fundamental factors underlying chronic

wound pathogenesis are cellular and systemic changes of aging, repeated bouts

of ischemia-reperfusion injury, and bacterial colonization with resulting

inflammatory host response (Mustoe, 2004).

Bacterial Colonization

Since more oxygen in the wound environment allows white blood cells to

produce ROS to kill bacteria, patients with inadequate tissue oxygenation, for

example those who suffered hypothermia during surgery, are at higher risk for

infection (Thomas, 2004). The hosts immune response to the presence of

bacteria prolongs inflammation, delays healing, and damages tissue (Thomas,

2004). Infection can lead not only to chronic wounds but also to gangrene, loss


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of the infected limb, and death of the patient. More recently, an interplay

between bacterial colonization and increases in reactive oxygen species leading

to formation and production of biofilms has been shown to the generate chronic

wounds (Dhall, 2014). Like ischemia, bacterial colonization and infection

damage tissue by causing a greater number of neutrophils to enter the wound

site (Robert, 2005). In patients with chronic wounds, bacteria with resistances

to antibiotics may have time to develop (Halcon & Milkus, 2004). In addition,

patients that carry drug resistant bacterial strains such as methicillin-

resistant Staphylococcus aureus (MRSA) have more chronic wounds (Halcon &

Milkus, 2004).

Treatment

Though treatment of the different chronic wound types varies slightly,

appropriate treatment seeks to address the problems at the root of chronic

wounds, including ischemia, bacterial load, and imbalance of proteases

(Thomas, 2004). Various methods exist to ameliorate these problems, including

antibiotic and antibacterial use, debridement, irrigation, vacuum-assisted

closure, warming, oxygenation, moist wound healing, removing mechanical


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stress, and adding cells or other materials to secrete or enhance levels of

healing factors (Velander et al., 2004).

Preventing and Treating Infection

To lower the bacterial count in wounds, therapists may use topical antibiotics,

which kill bacteria and can also help by keeping the wound environment moist

(Brem, 2004; Patel, 2000), which is important for speeding the healing of

chronic wounds (Taylor et al., 2005; Thomas et al., 2005). Some researchers

have experimented with the use of tea tree oil, an antibacterial agent which

also has anti-inflammatory effects (Halcon & Milkus, 2004). Disinfectants are

contraindicated because they damage tissues and delay wound contraction

(Patel et al., 2000). Further, they are rendered ineffective by organic matter in

wounds like blood and exudate and are thus not useful in open wounds.[32]

A greater amount of exudate and necrotic tissue in a wound increases

likelihood of infection by serving as a medium for bacterial growth away from

the hosts defenses (Thomas, 2004). Since bacteria thrive on dead tissue,

wounds are often surgically debrided to remove the devitalized tissue (Patel et

al., 2000). Debridement and drainage of wound fluid are an especially

important part of the treatment for diabetic ulcers, which may create the need

for amputation if infection gets out of control. Mechanical removal of bacteria


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and devitalized tissue is also the idea behind wound irrigation, which is

accomplished using pulsed lavage (Thomas, 2004). Removing necrotic or

devitalzed tissue is also the aim of maggot therapy, the intentional introduction

by a health care practitioner of live, disinfected maggots into non-healing

wounds. Maggots dissolve only necrotic, infected tissue; disinfect the wound by

killing bacteria; and stimulate wound healing. Maggot therapy has been shown

to accelerate debridement of necrotic wounds and reduce the bacterial load of

the wound, leading to earlier healing, reduced wound odor and less pain. The

combination and interactions of these actions make maggots an extremely

potent tool in chronic wound care.

Negative pressure wound therapy (NPWT) is a treatment that improves ischemic

tissues and removes wound fluid used by bacteria (Thomas, 2004; Kathleen,

2005). This therapy, also known as vacuum-assisted closure, reduces swelling

in tissues, which brings more blood and nutrients to the area, as does the

negative pressure itself (Kathleen, 2005). The treatment also decompresses

tissues and alters the shape of cells, causes them to express different mRNAs

and to proliferate and produce ECM molecules (Kathleen, 2005; Robert, 2005).

Treating Trauma and Painful Wounds


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Persistent chronic pain associated with non-healing wounds is caused by

tissue (nociceptive) or nerve (neuropathic) damage and is influenced by

dressing changes and chronic inflammation. Chronic wounds take a long time

to heal and patients can suffer from chronic wounds for many years.[33]

Chronic wound healing may be compromised by coexisting underlying

conditions, such as venous valve backflow, peripheral vascular disease,

uncontrolled edema and diabetes mellitus. If wound pain is not assessed and

documented it may be ignored and/or not addressed properly. It is important to

remember that increased wound pain may be an indicator of wound

complications that need treatment, and therefore practitioners must constantly

reassess the wound as well as the associated pain.

Optimal management of wounds requires holistic assessment.

Documentation of the patients pain experience is critical and may range from

the use of a patient diary, (which should be patient driven), to recording pain

entirely by the healthcare professional or caregiver (Osterbrink, 2003). Effective

communication between the patient and the healthcare team is fundamental to

this holistic approach. The more frequently healthcare professionals measure

pain, the greater the likelihood of introducing or changing pain management

practices.


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At present there are few local options for the treatment of persistent pain,

whilst managing the exudate levels present in many chronic wounds. Important

properties of such local options are that they provide an optimal wound healing

environment, while providing a constant local low dose release of ibuprofen

during war time.

If local treatment does not provide adequate pain reduction, it may be

necessary for patients with chronic painful wounds to be prescribed additional

systemic treatment for the physical component of their pain. Clinicians should

consult with their prescribing colleagues referring to the WHO pain relief

ladder of systemic treatment options for guidance. For every pharmacological

intervention there are possible benefits and adverse events that the prescribing

clinician will need to consider in conjunction with the wound care treatment

team.

Diagnosis of Wounds

Diagnostic and therapeutic devices have gone through a great development

since the invention of Ignc Semmelweis published in 1846. Paradoxically

opportunities of healthcare represent the most important factor of recent

nosocomial infections (Katalin, 2010).


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Diagnosis of wound infection can be a daunting task in resource-poor settings.

There is often a lack of adequate diagnostic equipment or requisite personnel

(Hart and Kariuki, 1998). Thus, a diagnostic dilemma confronts physicians in

the absence of local epidemiological data on wound infections which could aid

empiric treatment. This dilemma coupled with the fact that there are no

established evidence-based clinical practice guidelines for wound infections,

makes management of wound infections difficult in resource-poor settings like

the Niger Delta region of Nigeria.

Our study was designed to establish baseline indices of wound infection at the

tertiary institution, Oghara Teaching Hospital, Oghara, by looking at the

prevalent micro-organisms involved in wound infections, associated factors and

drug resistance patterns.

Pus

Suppuration, the formation of pus, is a common sequel of acute inflammation.

Pus consists of living, dead and disintegrated neutrophils, living and dead

microorganisms and the debris of tissue cells, all suspended in the

inflammatory exudates. An abscess is a localized or discrete focus of pus.

However, pus may occur diffusely in loose tissues or body cavities.


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Bacterial infection is the usual cause of suppuration and such bacteria are

said to be pyogenic (pus forming) and include Staphylococcus aureus,

Streptococcus pyogenes,Pseudomonas aeruginosa,Proteusspecies,Escherichia

coli,Klebsiella species,Clostridium perfringes, Bacteroides among others.

Pyogenic infections are either polymicrobial or monomicrobial and they maybe

endogenous or exogenous. Pyogenic infections occur in abscesses, chronic

wounds from diabetic patients, decubitus ulcer or bed sores, burns wound

infections, post-operative wound infections, cellulitis, bites, suppurative

lymphadenitis, exudates from body cavities and pyomyositis.

Various studies across the globe have been consistent enough to show a

predictable bacterial profile in pyogenic wound infections. This makes an

important observation for a clinician who intends to start empirical treatment

to his patients, while laboratory cultures reports are awaited.

A study on aerobic bacterial profile and antimicrobial susceptibility pattern of

pus isolates in a South Indian tertiary care hospital revealedStaphylococcus

aureus(24.29%) was the most common isolates, followed byPseudomonas

aeruginosa(21.49%),Escherichia coli(14.02%),Klebsiella pneumonia(12.15%),

Streptococcus pyogenes (11.23%),Staphylococcus epidermidis (9.35%) and

Proteusspecies (7.47%) (Raoet al.,2014). Another study on isolation of


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different types of bacteria from pus revealed alsoStaphylococcus aureusto be

the predominant microorganism (40%) followed byKlebsiellaspecies (33%),

Pseudomonasspecies (18%),

Escherichia coli(16%), and

Proteusspecies (7%)

(Verma 2012).

A study done in a University teaching hospital in Nigeria, revealed

Staphylococcus aureus(42.3%),Pseudomonas aeruginosa(32.9%),Escherichia

coli(12.8%) andProteus mirabilis(12.8%) are associated with surgical wound

infections (Nwachukwuet al.,2009). These findings agree with those reported

in Kenya on surgical site infections, thatStaphylococcus aureuswas the most

prevalent bacterial isolate (Dindaet al.,2013). These findings also agree with a

study done in Uganda that identifiedStaphylococcus aureusas the commonest

causative agent of septic post-operative wounds (Anguzuet al.,2007).

A study done on the bacteriology of surgical site infections in Karachi , revealed

the most common pathogen isolate wasStaphylococcus aureus (50.32%),

followed by Pseudomonas aeruginosa (16.33%),Escherichia coli(14.37%),

Klebsiella pneumonia (11.76%), Streptococcus pyogenes (1.30%), and

miscellaneous gram negative rods (5.88%) includingAcinetobacter baumannii,

Proteus mirabilisandCitrobacter diversus(Mahmood 2010). A cross-sectional

study designed to determine the distribution of the bacterial pathogens and


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their antimicrobial susceptibility from suspected cases of post-operative wound

infections, also revealedStaphylococcus aureus(63%) was the most frequently

isolated pathogenic bacteria, followed byEscherichia coli(12%),Pseudomonas

species (9.5%),Klebsiellaspecies (5%),Proteusspecies (3.5%) and coagulase

negativeStaphylococcusspecies (3.5%) (Shriyanet al.,2010).

Various studies across the globe have been consistent enough to show a

predictable bacterial profile in pyogenic wound infections. This makes an

important observation for a clinician who intends to start empirical treatment

to his patients, while laboratory cultures reports are awaited.

A study on aerobic bacterial profile and antimicrobial susceptibility pattern of

pus isolates in a South Indian tertiary care hospital revealedStaphylococcus

aureus(24.29%) was the most common isolates, followed byPseudomonas

aeruginosa(21.49%),Escherichia coli(14.02%),Klebsiella pneumonia(12.15%),

Streptococcus pyogenes (11.23%),Staphylococcus epidermidis (9.35%) and

Proteusspecies (7.47%) (Raoet al.,2014). Another study on isolation of

different types of bacteria from pus revealed alsoStaphylococcus aureusto be

the predominant microorganism (40%) followed byKlebsiellaspecies (33%),

Pseudomonasspecies (18%),Escherichia coli(16%), andProteusspecies (7%)

(Verma 2012).


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A study done in a University teaching hospital in Nigeria, revealed

Staphylococcus aureus(42.3%),Pseudomonas aeruginosa(32.9%),Escherichia

coli(12.8%) andProteus mirabilis(12.8%) are associated with surgical wound

infections (Nwachukwuet al.,2009). These findings agree with those reported

in Kenya on surgical site infections, thatStaphylococcus aureuswas the most

prevalent bacterial isolate (Dindaet al.,2013). These findings also agree with a

study done in Uganda that identifiedStaphylococcus aureusas the commonest

causative agent of septic post-operative wounds (Anguzuet al.,2007).

A study done on the bacteriology of surgical site infections in Karachi , revealed

the most common pathogen isolate wasStaphylococcus aureus (50.32%),

followed by Pseudomonas aeruginosa (16.33%),Escherichia coli(14.37%),

Klebsiella pneumonia (11.76%), Streptococcus pyogenes (1.30%), and

miscellaneous gram negative rods (5.88%) includingAcinetobacter baumannii,

Proteus mirabilisandCitrobacter diversus(Mahmood 2010). A cross-sectional

study designed to determine the distribution of the bacterial pathogens and

their antimicrobial susceptibility from suspected cases of post-operative wound

infections, also revealedStaphylococcus aureus(63%) was the most frequently

isolated pathogenic bacteria, followed byEscherichia coli(12%),Pseudomonas

species (9.5%),Klebsiellaspecies (5%),Proteusspecies (3.5%) and coagulase

negativeStaphylococcusspecies (3.5%) (Shriyanet al.,2010).


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A study on microbiological profile of diabetic foot ulcers and its antibiotic

susceptibility pattern in a teaching hospital in Gujarat, revealed that

Pseudomonas aeruginosa(27%) was the most common isolate causing diabetic

foot infections followed byKlebsiellaspecies (22%),Escherichia coli(19%),

Staphylococcus aureus (17%), Proteus species (7%), Enterococci (3%),

Acinetobacter(2%), CoNS (2%) andProvidencia(1%) (Mehtaet al.,2014). The

predominance of gram negative bacilli in diabetic pus has also been reported in

another study (Sivakumariet al.,2009). However,Staphylococcalspecies was

the primary pathogen in most of wound infections of diabetic patients (Daniel

et al.,2013).

A study done in a tertiary hospital, Pakistan on burn wounds, revealed

Staphylococcus aureus(57.98%) to be the most causative organism in burn

wound infections followed byPseudomonas aeruginosa(19.33%),Klebsiella

pneumonia(8.4%),Proteusspecies (4.2%),Staphylococcus epidermidis(3.36%),

Escherichia coliandEnterobacter(2.52%) each,CitrobacterandSerratia(0.84%)

each (Ahmedet al.,2013). Though a study done in Ibadan, Nigeria on burn

wound infections revealedKlebsiellaspecies to be the most commonly isolated

pathogen, constituting 34.4%, closely followed byPseudomonas aeruginosa

(29.0%) andStaphylococcus aureus(26.8%) (Kehindeet al.,2004).


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In a two year period study done on bacterial profile of burn wounds infections

at a burn unit Nishtar hospital Multan, the frequency of gram negative

organisms was found to be high withPseudomonas aeruginosa(54.4%) being

the most common isolate, followed byStaphylococcus aureus(22%),Klebsiella

species (8.88%),Staphylococcus epidermidis (5.79%),Acinetobacterspecies

(4.63%),Proteusspecies (2.70%) andEscherichia coli(1.54%) (Shahzadet al.,

2012).

A three year review of bacteriological profile and antibiogram on burn wounds

isolates in Van,Turkey revealed the most frequent bacterial isolate was

Acinetobacter baumannii(23.6%), followed by coagulase negativeStaphylococci

(13.6%),Pseudomonas aeruginosa (12%),Staphylococcus aureus (11.2%),

Escherichia coli(10%),Enterococcusspecies (8.8%) andKlebsiella pneumonia

(7.2%) (Bayram et al.,2013). Even though gram negative bacteria are being

increased significantly but stillStaphylococcus aureusis being continued as a

major etiological agent of pyogenic infections.

Antimicrobial resistance

The prevalence of antimicrobial resistance varies greatly between and within

countries and different pathogens. Also antimicrobial resistance patterns of

bacteria isolates keep changing and evolving with time and place.


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Data from the past several years show an increasing resistance to ampicillin,

penicillin and amoxicillin which were considered first line drugs for treatment

of pyogenic infections ( Anguzuet al.,2007, Shriyanet al.,2010, Binduet al.,

2014).

A study on prevalence and antimicrobial susceptibility of bacteria isolated from

skin and wound infections revealed gram positive cocci were highly sensitive to

vancomycin, teicoplanin, linezolid and chloramphenicol and gram negative

bacilli showed high degree of sensitivity to imipenem, piperacillin/tazobactam

and aminoglycosides. The least sensitivity was exhibited for penicillin,

ampicillin, tetracycline, cotrimoxazole and cephalosporins (Kaupet al.,2014).

Gram positive isolates in pus were most susceptible to vancomycin,

levofloxacin, oxacillicin and clindamycin whereas among the gram negative

isolates in pus, the most susceptible drugs were piperacillin/tazobactam,

levofloxacin, imipenem, aztreonam and amikacin (Raoet al.,2014).

Raoet al., 2013, reported that out of 144 aerobic isolates from pus samples in

post-operative wound infections 94.4% were sensitive to imipenem, 75.5% to

amikacin, 27% to ciprofloxacin, 22.2% to gentamicin, 21.5% to cotrimoxazole,

12.5% to cefotaxime, 9.7% to ceftazidime and 6.25% to amoxicillin/clavulanic

acid. All isolates were resistant to ampicillin. 33% ofStaphylococcus aureus


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were sensitive to methicillin and among the CoNS, 58.3% were sensitive

methicillin. All gram positive cocci isolated were sensitive to vancomycin and all

gram negative isolates were sensitive to imipenem (Raoet al., 2013).

S.aureusisolates showed the highest resistance to penicillin (100%), ampicillin

(95.5%), ceftriaxone (81.8%), vancomycin (65.2%) while the least resistance was

exhibited to amoxicillin/clavulanic acid (30.3%).Klebsiellaspp were resistant to

gentamicin (100%), chloramphenicol(87.5%), ceftriaxone (87.5%) and

ciprofloxacin (62.5%).E.colispp were resistant to ampicillin (100%), gentamicin

(46.7%), chloramphenicol(40%), ceftriaxone (40%) and ciprofloxacin (40%).

Proteus spp were resistant to ampicillin(100%), chloramphenicol(66.7%),

gentamicin (33.3%) and ceftriaxone (33.3%).Pseudomonasspp were resistant

to gentamicin (50%), chloramphenicol (100%), amoxicillin/clavulanic acid

(100%), ampicillin (100%) and ceftriaxone (100%). Allproteus and

pseudomonasisolates were susceptible to ciprofloxacin. Isolates of CoNS

showed 100% resistance to vancomycin, ceftriaxone, ampicillin and penicillin

but sensitive to chloramphenicol. Single and multiple antimicrobial resistances

were observed in 6.8% and 93.2% of the isolates, respectively. No bacterial

isolates was found to be sensitive to all antibiotics tested ( Dessalegnet al.,

2014). Aminoglycosides and quinolones were found to be the most susceptible

drugs in aerobic bacterial isolates from wound infections (Al-azawi, 2013,


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Anguzu et al.,2007). Sensitivity ofS.aureus isolates from burn wound

infections at a hospital in Ethiopia were 93.9% vancomycin, 90.9%

clindamycin, 86.4% kanamycin and 86.4% erythromycin. Resistance of

S.aureusisolates above 50% rates was observed in penicillin, methicillin,

polymyxin B and chloramphenicol 95.5%, 77.3%, 68.2% and 51.5%

respectively (Tigistet al., 2012).

Acinetobacterisolates showed almost complete resistance to cephalosporins

(cephalexin 98.7%, cefuroxime 98.2%, cefotaxime 93.2%, ceftriaxone 93.3%,

ceftazidime 87.5%, cefaclor 97.4%), piperacillin ( 94.7%), gentamicin (81.3%),

while lower rates of resistance were shown in amikacin 68.3% and ciprofloxacin

69.7%. The most effective antimicrobial drug was doxycycline with the lowest

resistance rate of 22.1% (Elmanama 2006).

Azithromycin , gatifloxacin, amikacin, ampi/subbuctam and ciprofloxacin were

found to be highly susceptible to gram negative organisms in pus while

amikacin, azithromycin, ciprofloxacin, clindamycin, cloxacillin,

chloramphenicol, moxifloxacin, linezolid and gatifloxacin were highly sensitive

for gram positive organisms in pus (Vermaet al.,2012, Verma 2012). However,

most of gram negative isolates in diabetic foot ulcers were resistant to

amikacin, piperacillin/tazobactam, gentamicin, ampicillin-sulbactam and

gatifloxacin. The gram negative bacilli were highly sensitive to imipenem and


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polymyxin. 69.4% of GNB were ESBL producer. Gram positive isolates were

found to be susceptible to vancomycin, linezolid, ampicillin/sulbactam,

tetracycline and neomycin. 60% ofStaphylococcus aureuswere methicillin

resistant and were sensitive to vancomycin and linezolid (Mehtaet al.,2014).

Gram negative organisms were highly resistant to ampicillin and ceftriaxone (

lactam antibiotics). Ciprofloxacin was highly active against all gram negative

organisms and also gram positive cocci (Nwachukwu et al.,2009). 100%

vancomycin resistanceStaphylococcus aureuswas isolated from wounds of

diabetic patients (Danielet al.,2013). In that studyStaphylococcus aureusonly

showed sensitivity to gentamycin and tetracycline.

CHAPTER THREE

MATERIALS AND METHODS

Sample size

A total of 80 wound swabs submitted at the general culture bench from in-

patients in different wards of the hospital, 36 of which were male and 34

female. Inclusion criterion was patients with purulent wounds.

Clinical specimens


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Specimens were collected aseptically with sterile cotton wool swabs from post

operative wound infections. Pus samples /wound swabs were collected with

aseptic precautions and were transported to the laboratory without delay.

Blood agar, MacConkey agar and Nutrient agar were used for isolation and

study of cultural characters. The plates were incubated at 37C for 24 hours in

an incubator. Isolated colonies were subjected to Gram staining and

biochemical tests for identification. Biochemical tests are performed by API20E

and Vitek2 systems. Most resistant isolate is further identified by 16S rRNA

sequencing.

Culture of specimen

The specimens were inoculated on blood, chocolate and MacConkey agar plates

(Oxoid, Basingstoke, U.K). The plates were incubated aerobically at 37 0C for

24 to 48 hours. Pure colonies were kept in nutrient agar slants. The nutrient

agar slants were incubated at 37oC for 18 24 h before storage in the

refrigerator at 4C pending biochemical analysis.

Identification of bacterial pathogens

Pure cultures were characterized using morphological appearances on selective

and differential media. Motility test and biochemical tests such as catalase,


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coagulase, oxidase, Voges Proskauer, hydrogen sulphide production, urease,

methyl red, indole, citrate and sugar utilization tests were carried out according

to standard techniques.

Antibiotics

A total of eight (8) antibiotics, which represent the most commonly prescribed

antibiotics for treatment of wound infections in the study area, were used in

the study. Oxoid antibiotic discs used were amoxicillin (AMX, 10 g),

ceftriaxone (CRO, 30 g), ceftazidime (CAZ, 30 g), ciprofloxacin (CIP, 10 g)

and gentamicin (CN, 30 g).

Antibiotic susceptibility testing

Antimicrobial susceptibility test were carried out on isolated and identified

colonies of Gram-negative bacteria using commercially prepared antibiotic disk

(Span diagnostics) on Nutrient agar plates by the disk diffusion method,

according to the Central Laboratory Standards Institute (CLSI) guidelines.

Antibiotics used in our study were Gentamycin (GEN), Angmenycin (AUG),

Cefitriazonc (CTR), Erythromycin (ERY), Cefixime (CEF), Imepenem (IMP),

Meroparem (MEM) and Lavofloxacin (LEV).


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CHAPTER FOUR

RESULT PRESENTATION

During the study, 80 wounds swabs were collected and cultured on Mac-

conkey, Blood and Chocolate Agar, this was after they were incubated at 37o

for 24hrs, thus in the wound sample fifty (50) out of eighty (80) samples have

growth which amounted to 62.5% growth rate. The bacterial isolate recorded in

this study areKlebsiellapnemoniae, Psuedomonas aeruginosa,Escherichia

coli,Staphylococcusaureus,ProteusvulgarisandProteusMirabilisas shown in

table 4.1. Gram negative bacilli were responsible for 70% of wound

infections.Staphylococcus aureuswas the only gram positive organism

isolated.Staphylococcus aureuswas the most prevalent pathogen detected in

the swabs, whilePseudomonasaeruginosa,Proteusmirabilisandvulgariswas

the least detected isolate.

Based on cultural, morphological and biochemical characteristics of the

organisms isolated, a total of six (6) bacterial species isolates were identified in

the 50 wounds swabs samples studied.Escherichia coli(20%),ProteusMirabilis

(10%),ProteusVulgaris(10%),Pseudomonasaeruginosa(10%), ,Klebsiella

pneumonia(20%) andStaphylococcusaureus(30%). Thus, table 2 above shows


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that staphyloccous aureus is more prevalent in the studied wound swab and

this is closely followed byEscherichia coliandKlebsiellapneumonia.

The bacterial isolates exhibited a high resistance to the antibiotics tested,

with the organismsKlebsiellapneumoniaandStaphylococcusaureusresistant

to all of the tested antibiotics in the same vein majority of the organisms

isolated with the exception of two (2) strains ofProteusmirabiliswere all

resistant to cefixime (Table3).Pseudomonas aeruginosaexhibited a very high

resistance to the tested antibiotics with few sensitivity observed in meropenem,

imepenem, levofloxacin and cefixime, the lowest recorded resistance being

E.coliwas relatively susceptible to gentamycin, meropenem, imepenem,

levofloxacin, and Augmentin.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3705182/table/T5/http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3705182/table/T5/


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4.1 Microscopic and Morphological characteristics of the isolates

Table 4.1.Identification chart for bacteria isolates

Cultural

xteristics

Morp. Gram

stain

Catalase Oxidase Citrate indole Motility H2S Urease Lactose Coaulase

test

!erobic

test

"athoen

identified

Creamy round

mucoid

irreular colony

#od $ % & % & $ & % % % + Kleb

Pneumon

Cream flat

colony 'ith

undulatin ede

#od $ $ % % $ % $ $ $ $ % Pseudom

aerogino

"in( colony

'ith oriod

shape

#od $ % $ $ % % $ % % % + Escheric

.coli

Cream mucoid

colony 'ith

irreular ede

#od $ % & & & % % % $ % + Proteus

Mirabilis

)hite and flat

colonies

#od $ % $ $ % % % % $ $ + Proteus

Vulgaris

Creamy on

blood aar

Cocci

in

cluste

rs

% % $ $ % % $ % % % +

Staphylo

s aureus

*ey $+ neati,e to the test-%+ "ositi,e


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Table 4. 2: "re,alence of indi,idual bacterial isolates from 'ound infection

Oranism o /0

Staphylococcus aureus 12/30

Escherichia coli 1/50

Proteus mirabilis 2/10

Proteus vulgaris 2/16

Klebsiella pneumonia 1/50

Pseudomonas aeruginosa 2/10

Total 2/1


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Table 4.3: Antibiotic sensitivity/resistance of the isolated organism

Isolates CTR GEN MEM IMP CEF ERY LEV AUG

Pseudomonas

aeruginosa

R R S S R R S S

Klebsiella

Pneumonia

R R R R R R R R

Proteus

vulgaris

R R R R S S S S

Proteus

mirabilis

S S R R S S S S

Staph. aureus R R R R R R R R

E coli R S S S R R S S

Key:

GEN = Gentamycin

AUG = Augmentin

MEM = Meropenem

IMP = Imepenem

CEF = Cefixime

ERY = Erythromycin

LEV = Levofloxacin

CTR = Cefixime

CHAPTER FIVE


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DISCUSSION AND CONCLUSION

Discussion of result

Epidemiological surveillance of antimicrobial resistance is indispensable

for empirical treatment infections, implementing control measures, and

preventing the spread of antimicrobial resistant microorganisms (Goosens and

Sprenger, 1998). Also Bacterial contamination of wounds is a serious problem

in the hospital, where the site of a sterile operation can become contaminated

and subsequently infected.

Our study demonstrated a high prevalence (62.5%) of pathogenic

bacteria in wounds. This high figure is consistent with that obtained in similar

studies in Nigeria as rightly reported by Wariso and Nwachukwu, (2003) and

Taiwo et al., (2001), but different from another study in East Africa reporting a

prevalence of 70.5% as authored by Mulugeta and Bayeh, (2011). These

differences may be due to study design. The rates might be equally high if only

wounds with a high suspicion of infection are investigated as opposed to all

wounds.

Although there was no association between the type of wound and the

type of micro-organism isolated, it is important to note that all swabs from

traumatic wounds yielded significant bacterial growth and were thus deemed to

indicate infection. However, two previous studies carried out by Otokunefor et


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al., (1990) and Okesola and Kehinde, (2008) associated specific micro-

organisms with particular wound types]. More studies are required to clarify

this observation.

As in previous studies, documented by Taiwo, et al., (2002) and Egbe et

al., (2011); Gram-negative bacteria were the most commonly isolated

pathogens. Our observation ofStaphylococcus aureusas the most common

pathogen in wound infections is in-line with the works of other authority in

Nigeria who reported thatStaphylococcus aureusis the predominant organism

isolated from wound (Egbe et al., 2011).Klebsiella pneumoniaewhich in our

study amounted for 20% of the prevalence rate of organism isolated from

wounds was observed as the most common pathogen in wounds in a study

conducted by Sule et al., (2002). This is evidence of the existence of local and

regional variability and shows that each health facility has to determine the

prevalent micro-organisms and other associated indices. Most of these studies,

including ours, are limited by the fact that anaerobic cultures were not done for

a variety of reasons, the main one being a lack of equipment and funds. Thus,

anaerobic bacteria, which are also important in wound infections, could not be

isolated.

Pathogenic isolates of have relatively high potentials for developing

resistance (Karlowsky etal., 2004). High resistance of organism to


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antimicrobial agents tested was observed in this study withStaphylococcus

aureusandKlebsiellapneumoniaresistant to all the tested antibiotic.

Staphylococcus aureusis a very common cause of infection thus it is not

surprise to have isolated it in wounds.

Staphylococcus aureuswas found to be a frequent isolate in wound sepsis

(Emmerson, 1994). A study by Ndip,et al., (1997) at Ilorin, Nigeria reported

wound infections of 38% as the highest frequency of S. aureus isolates. This

agrees with the result in the present study where S. aureus also had the

highest isolate of 30%.Staphylococcus aureusdevelops resistance very quickly

and successfully to different antimicrobials over a period of time. The highest

frequency of S.aureusoccurred with susceptibility to antimicrobial agent

Levofloxacin.

The high level resistance could be associated with earlier exposure of

these drugs to isolates which may have enhanced development of resistance.

There is high level antibiotic abuse in this environment arising from self-

medication which is often associated with inadequate dosage and failure to

comply to treatment, and availability of antibiotics to consumers across the

counters with or without prescription. It had been observed that the

indiscriminate use of antibiotics without prescriptions in the developing


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countries such as Nigeria where there are no regulatory policies in this respect

has rendered the commonly used antibiotics completely ineffective in the

treatment ofStaphylococcus aureusinfections.

This is similar to what was observed by Aibinuetal.,(2004) who reported

100% resistance of their E. coliisolates to ampicillin and amoxicillin.

Resistance to Augmentin and levofloxaxin observed in this study was similar to

what was observed in South Africa, Israel, (62% - 84%) and Hong Kong,

Philippines (64 - 82%) (Stellinget al., 2005). Densenclosetal. (1988)) reported

53% of theirE. coliisolates were resistant cotrimoxazole and 67% to

tetracycline. Their finding is in harmony with the report of this study, showing

69% and 88% resistance to antibiotics agents. The reason for this high

resistance to commonly used antibiotics may be due to widespread and

indiscriminate use in our environment.

Antibiotic resistance by the isolates to commonly prescribed antibiotics

was high. This high level of resistance is a cause for concern. The absolute

resistance to levofloxacin was not unexpected considering the fact that

levofloxacin is a component of Ampiclox, an antibiotic frequently implicated in

self-medication in Nigeria (Yah et al., 2008). Augmentin, which are among the

least prescribed antibiotics in Nigeria (Yah et al., 2008), are neither widely

abused in this country nor easily affordable by the patients in the Niger Delta


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region. The development of resistance to Augmentin observed in this study is

thus a wake-up call for action on antimicrobial resistance. The poor availability

of antibiotics, as well as their unregulated use and misuse, has been shown to

contribute to increasing antimicrobial resistance in developing countries (Hart

and Kariuki, 1998). The lack of diagnostic facilities in these developing regions

encourages empiric treatment and overtreatment, which contribute to the

increased resistance (Hart and Kariuki, 1998).

Conclusion

Severe antimicrobial resistance in wound infections was observed among

patients in Delta State University Teaching Hospital (DELSUTH), Oghara, Delta

State of Nigeria. There is a need for serious and urgent intervention to stem the

spread and further evolution of this resistance. A rigorous infection control

policy combined with rational drug use play an important role in this fight

against antimicrobial resistance..

Recommendations

Multiple antibiotic resistance in bacterial populations is a great challenge

in the effective management of wound infections. This calls for monitoring and

optimization of antimicrobial use. We suggest a multidisciplinary approach to

wound infection management involving both clinicians and microbiologists.


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Strengthening of laboratory services at local and national levels will ensure

effective surveillance of antimicrobial resistance. We also advocate routine

microbiological surveillance of wounds and testing for antimicrobial

susceptibility before drug use. Thus the inclusion of anaerobic culture in

routine microbiology culture investigations will be of immense contribution

also. Finally, Since antimicrobial resistant patterns are constantly evolving, and

present global public health problem, there is the necessity for constant

antimicrobial sensitivity surveillance. This will help clinicians provide safe and

effective empiric therapies.

ese project

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