HAEMOLYTIC ACTIVITY AND STREPTOMYCIN SUSCEPTIBILITY PROFILE OF BACTERIAL ISOLATES ASSOCIATED WITH NASAL SECRETION

ABSTRACT

Human nasal cavity host major human pathogens which exhibit different haemolytic activities. This study was conducted to determine haemolytic activity and streptomycin susceptibility profile of bacterial isolates associated with nasal secretion. A total of 186 nasal samples (swabs) were collected from both male and female in different age groups in Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria. The samples were analysed using culture techniques, Gram staining, biochemical test and antimicirobial susceptibility testing. Findings from the study revealed that 163 bacteria belonging to different genera and species were isolated. The bacterial isolates included Bacillus (36.20%), coagulase negative Staphylococcus (29.20%), Streptotococcus other than Streptococcus agalactiae (18.40%), coagulase positive Staphylococcus (14.11%) and Streptococcus agalactiae (1.84%). However, bacterial isolates found on blood agar cultured plates showed different haemolytic patterns including alpha, beta and gamma haemolysis. Both Bacillus spp and Streptococcus agalactiae showed beta- haemolysis while coagulase negative Staphylococcus revealed gamma haemolysis. When tested to Streptomycin, Streptococcus organisms showed highest susceptibility followed by Bacillus and the least was coagulase negative Staphylococcus.

TABLE OF CONTENTS

Title page                                                                                                                                i

Certification                                                                                                                           ii

Dedications                                                                                                                             iii

Acknowledgements                                                                                                                iv

Table of contents                                                                                                                    v

List of tables                                                                                                                           vii

Abstract                                                                                                                                  viii

CHAPTER ONE

: INTRODUCTION                                                                               1

•                Background Information                                                                                1

1.2       Objectives of the Study                                                                                              5

1.2.1    Other Specific Objectives                                                                                          5

CHAPTER TWO: LITERATURE REVIEW

2.1       Human Nasal Bacteria                                                                                                6

2.2       Constituents of Human Nasal Secretions and Their Functions                                     7

2.3       Associations of Nasal Bacterial Microbiota and Respiratory Tract Infections        8

2.4       Pathogenic Bacteria Isolates of Human Nasal Discharge                                          11

2.5       Streptomycin                                                                                                              13

2.6       Mechanism of Action of streptomycin                                                                       14

2.7       Haemolysis of Streptococci and the various types                                                     15

2.8       Antimicrobial Resistance in Beta-Haemolytic Streptococci                                     16

CHAPTER THREE: MATERIALS AND METHODS

3.1.      Study Area                                                                                                                  18

3.2.      Population for the study                                                                                             18

3.3       Materials                                                                                                                     18

3.4       Nasal Swab Collection and Handling                                                                         19

3.5       Bacterial Isolation and Identification.                                                                        19

3.6       Biochemical Test                                                                                                        20

3.6.1    Catalase Test                                                                                                               20

3.6.2    Coagulase Test                                                                                                           20

3.6.3    Gelatin Liquefaction Test                                                                                           21

3.6.4    CAMP Test                                                                                                                 21

3.6.5    Indole Test                                                                                                                  21

3.6.6    Methyl Red Test.                                                                                                        22

3.6.7    Citrate Utilization Test                                                                                               22

3.7       Gram Staining                                                                                                            22

3.8       Streptomycin Susceptibility Testing                                                                          23

CHAPTER FOUR: RESULTS                                                                                            24-31

CHAPTER FIVE: DISCUSSIONS

5.1       Discussion                                                                                                                            32

5.2       Conclusion                                                                                                                  33

5.3       Recommendations                                                                                                      33

REFERENCES

APPENDIX

LIST OF TABLES

Table                                                                                             Page

4.1       Distribution of samples according to age                                                                   26

4.2       Distribution of samples according to sex                                                                   27

4.3       Occurrence of bacteria isolates from Nasal secretion                                                28

4.4       Bacterial characteristics on blood agar                                                                       29

4.5       Biochemical characteristics and Cell morphology                                                     30

4.6       Antimicrobial susceptibility test showing zone of inhibition measured in (mm)       31

CHAPTER ONE

1.0    INTRODUCTION

•         Background Information

Nose (Nasal) infections are among the most widespread and serious infections that compel an individual to seek medical attention. It represents some of the most common bacterial disease affecting people of all ages. The infections are among the leading causes of morbidity and mortality in critically ill patients (Witsell et al., 2001).  Nose and other mucosal flora are the frequent sites of infection, because they come in direct contact with the physical environment and are exposed to air borne microorganisms. Disease of nose and mucosal discharges affect the functioning of adults as well as children, with significant impairment of the daily life of affected patients (Witsell et al., 2001). It has been envisaged that with increase in global population, infection remain the most important causes of disease with upper respiratory infections causing hearing loss and learning disability particularly in children (Albert, 1999; Chibuike, et al., 2013; Obiajurn and Chukuezi, 2013). Tinnitus which is now known to be the most common childhood infections lead to annual death of over 50,000 children under 5 years (Rover et al., 2006) in other cases nasal condition may be distressing, as in the case of nasal myiasis (Kuruvilla et al., 2006). Bacterial species such as Staphylococcus aureus, Streptococcus spp, Proteus spp., Haemophilus and coliforms were found to be responsible for most cases of infections. According to Bailey and Scott (1992) and Ikeh et al. (1993), Actinomyces israeli, Mycoplasma pneumoniae, Mycobacterium tuberculosis and Corynebacterium diptheriae are the cause of vary prevalence of nasal infections and disease. Also, Kumar et al. (2013) reported that Pseudomonas, S. aureus, Proteus and Klebsiella are the common bacteria that cause nasal infection in Japura India. This is in addition to a study done on ear, nose and throat infection in Benin City, Nigeria by Osozuwe et al. (2011). The bacteria found in the study; Pseudomonas aeruginosa, S. aureus, Klebsiella spp., S. pneumoniae, E. coli and Citrobacter frenndi. Based on their study, P. aeruginosa was the most prevalent etiologic agent infections in the study. However, E-Mahmoud et al. (2010) reported Streptococcus pyogens, S. pneumoniae, S. aureus, Klebsiella pneumoniae, H. influenzae, Proteus mirabilis and P. aeruginosa, were the bacteria isolated in the ear, nose and throat (ENT) among patients visiting different hospitals in Yola city Nigeria. Based on this study S. pneumoniae was the most prevalent bacteria isolated in both ENT infections. Signs and Symptoms of this infection include, mild to severe pains, fever, and headache (Azeez, 2000), running or stuffy nose, other symptoms and complication include difficulty in swallowing food, meningitis etc. A number of factors have been implicated by previous workers to cause ENT infections and diseases. These included poor habits of cleanness and hard blowing of the nose as well as excessive sniffing which drives infected mucus into the middle ear leading to otitis media in adults (Schnert, 1996). The human ear, nose and throat (ENT) are closely related and inter connected parts of the body. Infections, diseases and health problems related to the ENT are therefore jointly studied and managed like the most other part of the human body. The nose is found to be colonized by a wide range of microorganisms, some of which are more or less harmless under normal condition (Chibuike et al., 2013).

The most important reason for the use of antimicrobial agents is to cure or prevent infectious diseases by using the best available agents. The study of antimicrobial susceptibility patterns of common pathogens in nasal secretions and the periodic review of such data is very essential in modern health care and the data provide a pre-emptive therapy either on the receipt of culture reports or a guide for overall course of treatment where therapy might be wholly empirical without laboratory diagnosis. This is fast becoming the norms in many developing countries as a result of dwindling resources (WHO, 2001). The benefits of the individual who deserves treatment must be weighed against the risk of emergence of resistant micro-organisms to the public. The choice of antimicrobial chemotherapy is initially dependent on clinical diagnosis. However, for many infections, establishing a clinical diagnosis implies determining possible microbiological causes which requires laboratory information from samples collected, preferably before antibiotic therapy is begun. Laboratory isolation and susceptibility testing of organisms make diagnosis to be established and also make drug selection more rational. Microbial flora are those micro-organisms that make their home in some parts of the human body. These micro-organisms of which majority are bacteria comprise of the microbiota also termed normal flora (Allen et al., 2014). The microbial flora consists of the normal and transient flora. The microbial flora of man has physiological peculiarities that enhance their survival in their natural habitats on mucosal surface, and in competition with other bacteria. In the human body, the term normal flora implies that the micro-organisms are harmless, and in most parts they do not cause diseases and are even beneficial some are opportunities pathogens; that is, they may cause infections if tissue injury occurs at specific sites or if the resistance of the body to infection is decreased. The normal flora present colonization of the body area by virulent strains of other micro-organisms and readily re-establishes when it is disturbed while the transient flora inhabits the mucous membrane briefly and are mainly itself from exogenous environment sources (Teo et al., 2015) Nasal secretion can vary in significance, from being innocuous to being indicative of a serious problem. Microbial flora can be responsible for endogenous or exogenous infections and the presence of a foreign body in the nose is a relatively uncommon occurrence. Some of the species of Streptococci and Staphylococci are among the most important pathogens of man and children are the more susceptible to the infection caused by them. A low incidence of common microbial flora of bacteria, fungi, viral and protozoa origin has been observed and the recent study revealed a carrier rate of 14.1% for beta-haemolytic Streptococci. The bacteria flora of the nasal cavity has been studied extensively and exhaustively for the definition of the composition of the normal flora and for the identification of nasal carriers of certain bacterial species such as S. pyogenes or S. aureus for the purpose of epidemiology. The major components of the normal flora of the nasals cavity are coagulase negative Staphylococci (which was reported to be present in widely varied percentage. Ranging from 12 to 81%), S. aureus (6 to 34%) and many aerobic species, such as Streptococci of the viridans group, Meningococci, enteric bacteria and Moraxella species have been isolated occasionally, (Musser and Shelburne, 2009). There are many different types of nasal secretion, it can be serous (clear, watery), mucoid (yellow and mucous- like), purulent (green-yellow, thick, looks like pus) or sanguineous (bloody). Nasal secretion can be unilateral (only ever from one nostril) or bilateral (from both nostrils) which helps identify the source of the secretion. Secretion that is unilateral typically comes from the nasal passage, the sinus while bilateral secretion can arise from the pharynx, or the lower respiratory tract (trachea and lungs) secretion can also be constant or intermittent. Nasal secretion can be acute in onset within hours to a couple of days) or chronic (lasting more than 2-3 days). Nasal discharge may be the only clinical symptom or there may be other clinical symptoms as ocular discharge, enlarged lymph nodes (which is non-specific and present with many types of nasal discharge), fever, cough, abnormal noise when breathing or exercising, lethargy or weight loss. Sometimes, nasal secretion can have a foul odour, which can be specific to certain types of bacterial infections, tissues damage or sinus infections. Antimicrobial resistance is a natural biological phenomenon. It is a predictable outcome of antimicrobial use, the rapid which resistance emerges and its extent are proportional to the intensity of antimicrobial use (Lindsey, 2001). Resistance emerges in population with a high frequency of infection, due to either underlying patient status or interventions compromising host defenses, resulting in high rate or antimicrobial use. The introduction of every antimicrobial agent into clinical practices have been followed by the detection in the laboratory of strains of micro-organism which have resistant traits. Such resistance may either be a characteristic associated with the entire species or emerge in strains of normal susceptible species through mutation or genes transfer (Cheesbrough, 2006). Resistant genes encode various mechanisms, which allows micro-organisms to resist inhibitory effects of specific antimicrobials. These mechanisms offer resistance to other antimicrobials of the same class and sometimes to difference classes of antimicrobial. A nasal swab can be evaluated at the laboratory for some of the more common viruses and bacteria that cause upper respiratory tract infections. Nasal secretion can be benign due to wind or dust and irritation can be due to allergies, it can be indicative of an upper respiratory tract viral infection, early stages of a more serious infection

1.2      Objectives of the Study

The main objective of this study is to evaluate the haemolytic activity and streptomycin susceptibility profile of bacterial isolates associated with nasal secretion.

1.2.1   Specific Objectives

 Specific objectives of this study include;-

•     To determine the prevalence of bacterial pathogens that are commonly associated with nasal secretion.

•     To determine the haemolytic activity of the bacteria associated with nasal secretions.

•     To ascertain the susceptibility of the organisms to streptomycin.

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