INCIDENCE OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS ISOLATED FROM NOSTRILS OF SCHOOL PUPILS IN IKWUANO

ABSTRACT

This study was carried out to determine the incidence of Methicillin Resistant Staphylococcus aureus (MRSA) nasal carriage among school pupils of Ikwuano . A total of 100 randomly selected pupils of Divine Vessel of Mercy Academy Nursery/Primary School Oboro Umuariaga and Amawom Community Nursery/Primary School Oboro Amawom both in Ikwuano Local Government of Abia state younger than 12 years were included in the study. Nasal swabs specimens collected were cultured on Mannitol salt agar.  S. aureus isolates were identified by mannitol fermentation, coagulase positivity and catalase positivity. Antimicrobial susceptibility test was performed on Meuller-Hinton Agar (MHA) by modified Kirby-Bauer disc diffusion method. Out of the 100 nasal swabs collected, S. aureus was isolated in 60 (60%). Among the isolates, 63.3% (n=38) were from male children whereas 36.7% (n=22) were from female. There was no significant sex difference in colonization of S. aureus between male and female pupils. Out of 60 isolates, 60% (n=39) were MRSA. MRSA isolates showed high rate of resistance towards antibiotics Imipenem (100%) followed by Cefotaxime (90%), Cefoxitin (65%), Azithromycin (55%), Cefuroxime (50%), Ceftriaxone (41.67%), Gentamycin (18.33%), Ciprofloxacin (18.33%),  and Lefofloxacin (18.33%).This study showed a high incidence of MRSA carriage in school pupils of Ikwuano indicating the spread of MRSA in the community.

TABLE OF CONTENTS

Title Page                                                                                                                                 i

Certification                                                                                                                           ii

Dedication                                                                                                                              iii

Acknowledgements                                                                                                                iv

Table of Contents                                                                                                                   v

List of Tables                                                                                                                          vii

Abstract                                                                                                                                  viii

CHAPTER ONE: INTRODUCTION                                                                                 1

1.1       Virulence Factors of S. aureus                                                                                   3         

1.2       MRSA                                                                                                                         3

1.3       Vancomycin Resistant S. aureus                                                                                6

1.4       Modes of Transmission                                                                                              7

1.5       MRSA Colonization                                                                                                   8

1.6       MRSA Infection                                                                                                         8

1.6.1    Bacteremia                                                                                                                 9

1.6.2    Endocarditis                                                                                                                9

1.6.3    Metastatic Infections                                                                                                  9

1.6.4    Sepsis                                                                                                                          10

1.6.5    Toxic Shock Syndrome                                                                                              10

1.7       Aims and Objective                                                                                                    10       

CHAPTER TWO: LITERATURE REVIEW                                                                   11

2.1       Epidemiology of MRSA                                                                                             12

2.2       Prevalence of MRSA amongst School Children                                                        12

2.3       Antimicrobial Evaluation of MRSA Nasal Carriage amongst Healthy Students         13

2.4       Public Health Burden of MRSA in Africa                                                                  13

CHAPTER THREE: MATERIALS AND METHODS                                                    15

3.1       Study Area                                                                                                                  15

3.2       Collection of Samples                                                                                                15       

3.3       Preparation of Culture Media                                                                                     15

3.4       Inoculation and Isolation                                                                                            15

3.5       Purification of Isolates                                                                                               16

3.6       Identification of the Isolates                                                                                       16

3.7       Gram Staining                                                                                                             16

3.8       Biochemical Test                                                                                                        17

3.8.1    Catalase Test                                                                                                               17

3.8.2    Coagulase Test                                                                                                           17

3.9       Antibiotic Susceptibility Testing                                                                                17

CHAPTER FOUR: RESULTS                                                                                            18

CHAPTER FIVE: DISCUSSION AND CONCLUSION                                                  22

5.1       Discussion                                                                                                                   22

5.2       Conclusion                                                                                                                  23

            References                                                                                                                  24

LIST OF TABLES

CHAPTER ONE

1.0       INTRODUCTION

Staphylococcus. aureus causes a variety of suppurative infections and toxinoses in humans. It causes superficial skin lesions such as boils, styes and furuncules; more serious infections such as pneumonia, mastitis, phlebitis, meningitis, and urinary tract infections; and deep-seated infections, such as osteomyelitis and endocarditis. S. aureus is a major cause of hospital acquired (nosocomial) infection of surgical wounds and infections associated with indwelling medical devices. S. aureus causes food poisoning by releasing enterotoxins into food, and toxic shock syndrome by release of super-antigens into the blood stream (Edward et al., 2013).

Although methicillin-resistant S. aureus (MRSA) has been entrenched in hospital settings for several decades, methicillin-resistant S. aureus MRSA strains have recently emerged outside the hospital becoming known as community associated- methicillin-resistant S. aureus ( (CA-MRSA) or superbug strains of the organism, which now account for the majority of staphylococcal infections seen in the clinic (Emaneini et al., 2016).

The primary reservoir of staphylococci is the nares, with colonization also occurring in the axillae, vulva, pharynx, and other skin surfaces. Nasal carriage in patient admitted to the hospital is common because close contact among patients and hospital personnel is not unusual; transfer of organisms often takes place. Increased colonization in patients and hospital workers frequently occurs in hospitals. Both hospital and community-acquired infections caused by drug resistant S. aureus have increased in the past 20 years (Espinosa-Gongora et al., 2015).

S.aureus is both a human commensal and a frequent cause of clinically important infections. It is frequently found on the human respiratory tract and on the skin. Strains that are associated with disease often result in infections by producing potent protein toxins, and expressing cell-surface proteins that bind and inactivate antibodies. The emergence of antibiotic-resistant forms of pathogenic S. aureus (e.g. MRSA)) is a worldwide problem in clinical medicine. S. aureus screening, today, is mainly done to identify methicillin-resistant S. aureus (MRSA) carriers. The prevalence of methicillin-resistant S. aureus (MRSA) is still quite low in some parts of the world, such as Northern European countries, but there is a worldwide increase in the number of infections caused by methicillin-resistant S. aureus (MRSA) (Emaneini et al., 2016).

Almost 25% of the Health Care Workers are stable nasal carriers, and 30% to 50% of them also possess the bacteria on their hands. Health Care Workers that carry S. aureus in their nares can occasionally cause outbreaks of surgical-site infections (Agumas et al., 2013). Most of the invasive S. aureus infections are assumed to arise from nasal carriage (Calfee et al., 2014).

Staphylococcus aureus is one of the most important pathogens worldwide and has emerged as a prominent organism infecting critically ill persons; the impact of S. aureus infection on human health has dramatically increased as a result of its remarkable ability to become resistant to antimicrobials. Because of its primary habitat is moist squamous epithelium of the anterior nares, most invasive S. aureus infections are assumed to arise from nasal carriage (Annati et al., 2013). The difference between methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible Staphylococcus aureus is resistance to ß-lactam antibiotics; this is often associated with resistance to multiple other antibiotics, which limits the therapeutic options (Castro et al., 2016).

National estimates in the United States from 2000-2002 suggested that the prevalence of S. aureus and methicilin resistant S. aureus (MRSA) colonization ratios were 31.6% and 0.84%, respectively. And about 7% or more of patients admitted to the hospital are colonized with MRSA. Although asymptomatic nasal colonization with S. aureus is common, it appears to be an important factor in the development of most infections due to this organism (Ahanjan et al., 2014).

S. aureus is the most clinically significant species of staphylococci; S. aureus characteristics gave the reason for their pathogenicity; which takes many forms. They grow comparatively well under conditions of high osmotic pressure and low moisture, which partially explains why they can grow and survive in nasal secretions and on the skin. S. aureus has been recognized as an important cause of disease around the world and it has become a major pathogen associated with both hospital and community acquired infections (De-Boeck et al., 2015).

1.1       VIRULENCE FACTORS OF S. aureus

S. aureus expresses many potential virulence factors: 1- surface proteins that promote colonization of host tissues; 2- invasins that promote bacterial spread in tissues (leukocidin, kinases, hyaluronidase); 3- surface factors that inhibit phagocytic engulfment (capsule, Protein A); 4- biochemical properties that enhance their survival in phagocytes (carotenoids, catalase production); 5- immunological disguises (Protein A, coagulase, clotting factor); 6- membrane-damaging toxins that lyse eukaryotic cell membranes (hemolysins, leukotoxin, leukocidin); 7- exotoxins that damage host tissues or otherwise provoke symptoms of disease, and 8- inherent and acquired resistance to antimicrobial agents (Gould, 2010)

1.2       MRSA

Two kinds of methicillin-resistant Staphylo coccus aureus have been described: Hospital- associated methicillin-resistant Staphylococcus aureus (HA-MRSA) and Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA). Naturally occurring strains of methicillin-resistant Staphylococcus aureus were first reported from England in 1961, shortly after the introduction of semi synthetic penicillins. Within ten years, methicillin-resistant Staphylococcus aureus was reported in the United States, with 22 such strains isolated from 18 patients at Boston City Hospitals. Data gathered between July 2004 and December 2005 by the Active Bacterial Core Surveillance Network (the laboratory surveillance component of the Emerging Infections Program of the US Centers for Disease Control and Prevention (CDC) showed an estimated rate of invasive methicillin-resistant Staphylococcus aureus infection (bloodstream or other sterile sites) of 31.8 cases per 100,000 population.                                                                

Hospital associated methicillin-resistant Staphylococcus aureus and Community associated methicillin-resistant Staphylococcus aureus isolates have been found to be distinct microbiologically, implying that Community associated methicillin-resistant Staphylococcus aureus did not originate from Hospital associated isolates that escaped from the hospital setting; rather, Community associated methicillin-resistant Staphylococcus aureus seems to have emerged de novo from established Community associated methicillin-resistant Staphylococcus isolates. A typing scheme established at the CDC showed that the majority of Community associated methicillin-resistant Staphylococcus infections are caused by 2 pulsed-field gel electrophoresis types (USA300 and USA400), whereas the predominant genotypes endemic in hospitals are USA100 and USA200 (Honda et al., 2010).

Additionally, the infections caused by Hospital associated methicillin-resistant Staphylococcus aureus and Community associated methicillin-resistant Staphylococcus are generally different; the Community associated pathogen is most frequently associated with skin and soft tissue (abscesses, boils, and folliculitis), whereas HA pathogen is more likely to infect the respiratory tract, blood stream, urinary tract, and surgical sites. Community associated MRSA is more frequently susceptible to non B-lactam antibiotics (e.g. clindamycin, trimethoprim-sulfamethoxazole, and tetracycline), and also tends to be more aggressive (Khanal et al., 2015)

In another study to determine nasal carriage of methicillin-resistant Staphylococcus and its antibiotic susceptibility pattern in adult hospitalized patients and medical staff in some hospitals in Cameroon the prevalence of nasal carriage of MRSA in medical staff was 41.3% and 32% for in-patients. The carriage rates of MRSA at the regional hospital, Limbe, Yaoundé University Teaching Hospital and Laquintinie Hospital, Douala were 38%, 37.1% and 32.1% respectively. Those who carried methicillin-resistant Staphylococcus were 34.2% and 35% for males and females respectively. It was noted that most MRSA strains were highly sensitive to vancomycin and teicoplanin in patients; while in medical personnel, most strains were sensitive to clindamycin. In the medical staff, the highest rate of resistance was recorded with penicillin G, trimethoprim/sulfamethoxazole and amoxicillin/clavulanic acid; while in the in-patients the highest rate of resistance was with gentamicin and erythromycin (Kumar et al., 2011).

According to a review that looked at Staphylococcus isolates from Denmark and UK between

1957 and 1960, all early methicillin-resistant Staphylococcus strains isolated resembled a large group of the early methicillin-resistant Staphylococcus blood isolates in phenotypic and genetic properties, including phage group, antibiotype (resistance to penicillin, streptomycin, and tetracycline), pulsed-field gel electrophoresis pattern, and spaA type and multilocus sequence type. This strongly suggested that the early methicillin-resistant Staphylococcus examined here represented the progeny of a strain that served as one of the first S. aureus recipients of the methicillin-resistance determinant in Europe.

1.3       VANCOMYCIN RESISTANT S. aureus

Vancomycin used to be an effective antistaphylococcal agent. Not any more, according to the current in vitro and clinical data. The concentration required to inhibit the growth of S.aureus is progressively increasing. Current evidence provides little hope that increasing the dose or using it in combination with another antistaphylococcal agent will improve its efficacy. These strategies, however, require further randomized clinical trials to either reject or validate them

(Lari et al., 2011).

Vancomycin-intermediate S. aureus (VISA) refers to S.aureus that might still respond to large doses of vancomycin. It is also termed glycopeptide-intermediate staphylococcus aureus (GISA), implying resistance to all glycopeptide antibiotics. Vancomycin-resistant S. aureus (VRSA) on the other hand refers to strains of S.aureus that have become resistant to vancomycin. These are extremely rare, though people with the following conditions are more likely to get VISA/VRSA: Underlying medical conditions (such as diabetes or renal disease), previous infections with methicillin-resistant S. aureus (MRSA), recent hospitalizations, use of catheters (e.g. IV lines), recent use of vancomycin or other antibiotics. Detection of VISA is difficult in the laboratory, and special inquiries about susceptibility testing methods may be needed (Mamishi et al., 2012).

Reduced vancomycin susceptibility can occur in S. aureus irrespective of background methicillin susceptibility and that development of intermediate vancomycin susceptibility in MSSA may result in increased tolerance to several classes of anti-staphylococcal agents.The historical U.S. Vancomycin-resistant Staphylococcus case count and geographical information found 13 cases isolated in different states (Center for Disease Control and Prevention). The sources included plantar ulcers, toe wound, urine from a nephrostomy tube, and vaginal swab. Their underlying medical conditions included diabetes, obesity, vascular disease, multiple sclerosis, and hypertension and end stage renal disease.

1.4       MODES OF TRANSMISSION

There are many ways associated with the MRSA transmission; some of these are associated with surrounding persons and others with the surrounding environment. Some modes of transmission are:

1- Person-to-person contact, for example, via transiently colonized hands of staff.

2- Fomites such as bed linens or environmental surfaces are not thought to play a major role in transmission except in special populations, such as patients in burn units or intensive care units (Halablab et al., 2010). Contamination of healthcare workers clothing including white coats, may be a vector for MRSA transmission. A study done on healthcare workers found that about 23% of healthcare workers white coats were contaminated with S. aureus of which 18% of them were MRSA. In a study carried out at University College Hospital Medical School in London, all medical students coats were bacteriologically contaminated, the most organisms were Staphylococcus sp including S. aureus. (Navidinia 2015).

3- Hands of staff appear to be the most likely mode of transmission of MRSA from patient to patient.

4- Droplet-borne transmission is less common, but may be important in patients with tracheotomies who are not able to control their secretions (Ohadian et al., 2015).

1.5       MRSA COLONIZATION

Colonization is the presence, growth and multiplication of the organism in one or more body sites without observable clinical symptoms or immune reaction; colonized patients were considered as a chief source of S. aureus in hospital; approximately 10% to 40% of people on admission have nasal carriage of S. aureus. A ''carrier'' refers to an individual who is colonized with MRSA (Rahimi-Alang et al., 2011). There are three patterns of carriage.

1- Persistent carriers: individuals always carry one type of strains, and those formed about 20% of the carriers and were more common in children than adults.

2- Intermittent carriers: this pattern shows a large proportion of the population (60%) and the strains change with varying frequency.

3- Non carriers: people who almost never carry S. aureus and those are minorities of people (20%) (Saadat et al., 2014).

1.6       MRSA INFECTION

Invasion and multiplication of MRSA in a body site are accompanied by clinical signs and symptoms of infection (e.g., fever, lesions, wound drainage) or increased white blood cell count. Infections caused by MSSA and MRSA are growing concern, particularly among patients in intensive care and surgical units, immunocompromised patients, and elderly patients in hospitals and nursing homes (Shahsavan et al., 2012).

S. aureus infection is a major cause of skin, soft tissue, respiratory, bone, joint, and endovascular disorders. The majority of these infections occur in persons with multiple risk factors for infection. The major diseases caused by S. aureus are:

1.6.1    Bacteremia

The overall rate of mortality from staphylococcal bacteremia, ranges from 11 to 43%. Factors associated with increased mortality include an age of more than 50 years, non-removable foci of infection, and serious underlying cardiac, neurologic, or respiratory disease. The frequency of complications from staphylococcal bacteremia is high, ranging from 11 to 53%. As many as 31% of patients with bacteremia, who do not have evidence of endocarditis, do have evidence of metastatic infection. An increasing percentage of bacteremic infections are related to catheterization (Halablab et al., 2010).

1.6.2    Endocarditis

The incidence of S. aureus endocarditis has increased and accounts for 25 to 35% of cases, it occurs in intravenous drug users, elderly patients, patients with prosthetic valves, and hospitalized patients. S. aureus endocarditis is characterized by a rapid onset, high fever, frequent involvement of normal cardiac valves, and the absence of physical stigmata of the disease on initial presentation.

1.6.3    Metastatic Infections

S. aureus has a tendency to spread to particular sites, including the bones, joints, kidneys, and lungs. Suppurative collections at these sites serve as potential foci for recurrent infections. Patients with persistent fever despite appropriate therapy should be examined for the presence of suppurative collections

1.6.4    Sepsis

A minority of bacteremia or local infections progress to sepsis. Risk factors for sepsis include advanced age, immunosuppression, chemotherapy, and invasive procedures. S. aureus is one of the most common gram-positive pathogens in cases of sepsis.

1.6.5    Toxic Shock Syndrome

The disease is characterized by a fulminant onset, often in previously healthy persons. The diagnosis is based on clinical findings that include high fever, erythematous rash with subsequent desquamation, hypotension, and multiorgan damage (Shakya et al., 2010)

1.7       AIMS AND OBJECTIVES

The aims and objectives of this project are:

1. To isolate and characterize Staphylococcus aureus from the nostrils of school pupils in Ikwuano.

2. To determine the antibiotic susceptibility profile of the isolates

3. To determine MRSA carriage rate among school pupils in Ikwuano.

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