PRODUCTION AND EVALUATION OF ANTIBIOTIC SUSCEPTIBILITY TESTING DISCS

Abstract

Susceptibility of organisms to antibiotics needs to be monitored because of rising antibiotic resistance. For this purpose, disc diffusion technique was employed in this study. In this study, low cost antibiotic discs (BMDR antibiotic discs) were prepared by impregnation of antibiotic solution onto What man filter paper grade TSfo.1. After the preparation of the disc, they were standardized by comparing their efficacy with the commercially available Abtek discs. Both Categories of discs were tested repeatedly against typed strains of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 as well clinical isolates of same organisms in the vitro antimicrobial susceptibility testing. The zones of inhibition in the range of 9mm - 27mm produced by the respective antibiotics on the BMDR discs compared favorably with that of the standard discs presenting zone sizes within the range of 13mm to 23mm for the clinical isolates, it was also observed from this study that in one of the isolates which exhibited complete resistance to all the antibiotics on the Abtek disc, a similar trend was visible on the BMDR antibiotic discs. Both test organisms (Escherichia coli and Staphylococcus aureus) showed 100% resistance to ampicillin on both disc types. In as much, this study demonstrate that, local and foreign discs appear to be producing distinct zones of inhibition, further studies are needed on periodic basis to substantiate the result observed in this study, which was on a small scale.

TABLE OF CONTENTS

Cover page                                                                                                              i

Title Page                                                                                                                ii

Declaration                                                                                                             iii

Certification                                                                                                            iv

Dedication                                                                                                              v

Acknowledgment                                                                                                    vi

Table of contents                                                                                                    vii

Abstract                                                                                                                  viii

CHAPTER ONE                                                                                                 

1.0 INTRODUCTION                                                                                           1

1.1 Aims and objectives                                                                                         5

CHAPTER TWO                                                                                                 5

2.0 Literature review                                                                                               6

2.1 Historical perspective                                                                                        6

2.2 Antibiotics                                                                                                        7

2.2.1Prudent use of antibiotics                                                                               8

2.3 Antimicrobial susceptibility testing techniques                                                9

2.4 principle of antimicrobial susceptibility testing                                               13

2.5 Antimicrobial resistance                                                                                   14

2.6 Standardization of the antimicrobial susceptibility testing methodology        17

2.7  Limitations of antimicrobial susceptibility tests                                              18

2.8 Factors influencing antimicrobial susceptibility testing                                   18

CHAPTER THREE

3.0 MATERIALS AND METHODS                                                                    21

3.1 Media preparation                                                                                            21

3.2 Preparation of filter paper discs                                                                       21

3.3 Preparation of antibiotic stock solution                                                           21

3.4 Test organisms                                                                                                 22

3.5 Gram staining                                                                                                   22

3.6 Biochemical tests                                                                                             22

3.7 Antimicrobial susceptibility testing                                                                 23

CHAPTER FOUR

4.0 Result                                                                                                               25

CHAPTER FIVE

5.0   DISCUSSION                                                                                               34

5.1 Conclusion                                                                                                       36   

REFERENCES

CHAPTER ONE

1.0     INTRODUCTION 

Antimicrobial agents include naturally occurring antibiotics, synthetic derivatives of naturally occurring antibiotics (semi-synthetic antibiotics) and chemical antimicrobial compounds (chemotherapeutic agents). Generally, however, the term ‘antibiotic’ is used to describe antimicrobial agents (usually antibacterial) that can be used to treat infection.

There are a large number of antimicrobial agents available for treating diseases caused by microorganisms. Such drugs are now an essential part of modern medical practice. The antimicrobial agents used in medical practice are aimed at eliminating the infecting microorganisms or at preventing the establishment of an infection (Sudha et al., 2001). To be of therapeutic use, an antimicrobial agent must exhibit selective toxicity; it must exhibit greater toxicity to the infecting pathogens than to the host organism (Chiang et al., 2009). A drug that kills the patient is of no use in treating infectious diseases, whether or not it also kills the pathogens. As a rule, antimicrobial agents are of most use in medicine when the mode of action of the antimicrobial chemicals involves biochemical features of the invading pathogens not possessed by normal host cells (Kiem and Schentag, 2006).

Antibiotics represent a major class of antimicrobial agents. By their very nature, antibiotics must exhibit selective toxicity because they are produced by one microorganism and exert varying degrees of toxicity against others. The discovery and use of antibiotics have revolutionized medical practice in the twentieth century. The formal definition of an antibiotic distinguishes biochemicals that are produced by microorganisms from organic chemicals that are synthesized in the laboratory. This distinction is no longer meaningful because organic chemists can synthesize the biochemical structures of many naturally occurring antibiotics. Additionally, many antibiotics in current medical use are chemically modified forms of microbial biosynthetic products.

Isolating any significant microorganism from a microbiological specimen requires careful evaluation by the clinician and prompt action is usually necessary. If the results of clinical microbiological analyses are to contribute in a meaningful way to the diagnosis and management of patients with infection, then the quality of such tests should be guaranteed. The periodic evaluation of all laboratory procedures is important to ascertain if such procedures continue to conform to standards and are meeting their desired goals (King and Brown, 2001). The lack of uniform standardization and interpretative criteria causes concern, but there are indications that routine susceptibility testing data are suitable for surveillance even if obtained with different methods (Livermore et al, 2000). In the microbiology laboratory, the identified infectious organisms are usually tested for their degree of resistance to various anti-infective substances in order to prevent the administration of ineffective treatments. The treating physician is usually informed of the test results with a report in which the activity of individual drugs against the isolated organism is categorized by one of the three terms "susceptible", "intermediate," and "resistant." This information can be used to optimize treatment for the individual patient, while, in the aggregate, data of this type can be used to form a picture of the degree of resistance to each drug in the population at large. The latter is, in turn, an important criterion in the selection of antibiotics for the initial ("empirical") treatment of infectious diseases (Kyabaggu et al., 2005).

Because susceptibility can vary even within a species (with some strains being more resistant than others), antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial infection in vivo.

Antimicrobial resistance (AMR) has emerged as a significant threat to global health security (Bate et al., 2013). The problem is so serious that it threatens the achievements of modern medicine; and a post-antibiotic era in which common infections and minor injuries can kill is a very real possibility for this century. AMR develops when a microorganism (bacteria, virus, parasite and fungus) no longer responds to a drug to which it was originally sensitive. Drugs for treating infections lose their effect because the microbes change; either they mutate or acquire genetic information from other microbes to develop resistance. The phenomenon is accelerated by use, and especially misuse, of antimicrobial medicines whereby resistant strains survive and aggregate. The problem can be further amplified when antimicrobial agents of substandard or falsified quality are procured and used by patients (Bate et al., 2013). The situation translates into standard treatments no longer working infections are harder or impossible to control; the risk of the spread of infection to others is increased; illness and hospital stays are prolonged, with huge added economic and social costs.

The development of bacterial antimicrobial resistance is neither an unexpected nor a new phenomenon. It is, however, an increasingly troublesome situation because of the frequency with which new emerging resistance phenotypes are occurring among many bacterial pathogens and even commensal organisms. Resistance has been observed to essentially all of the antimicrobial agents currently approved for use in human and veterinary clinical medicine (Walker, 2007). This, combined with the variety of antimicrobial agents currently available, makes the selection of an appropriate agent an increasingly more challenging task. This situation has made clinicians more dependent on data from in-vitro antimicrobial susceptibility testing, and highlights the importance of the diagnostic laboratory in clinical practice (Kiem and Schentag, 2006).

Resistance to antimicrobial agents (AMR) has resulted in increased morbidity and mortality from treatment failures and increased health care costs (Joseph et al., 2006). Although defining the precise public health risk and estimating the increased costs is not a simple undertaking, there is little doubt that emerging antibiotic resistance is a serious global problem. Multidrug-resistant pathogens travel not only locally but also globally, and newly introduced pathogens are spreading rapidly in susceptible hosts (Sudha et al., 2001). Antibiotic resistance patterns may vary locally and regionally, so surveillance data need to be collected from selected sentinel sources. Patterns can change rapidly and they need to be monitored closely because of their implications for public health and as an indicator of appropriate or inappropriate antibiotic usage by physicians in that area.

Antimicrobial susceptibility testing (AST) is performed on bacteria that are isolated from clinical specimens to determine if the bacterial etiology of concern can be killed or inhibited by antimicrobial drugs that are potential choices for therapy, at the concentrations of the drugs that are attainable at the site of infection using the dosing regimen indicated in the drug product’s labeling. The results of in vitro antibiotic susceptibility testing (AST) guide clinicians in the appropriate selection of initial empiric regimens and, drugs used for individual patients in specific situations. The selection of an antibiotic panel for susceptibility testing is based on the commonly observed susceptibility patterns, and is revised periodically (White et al., 2001).

A number of antimicrobial susceptibility testing (AST) methods are available to determine bacterial susceptibility to antimicrobials. The selection of a method is based on many factors such as practicality, flexibility, automation, cost, reproducibility, accuracy, and individual preference (Kerr, 2005).

Not all antimicrobials, at the concentration required to be effective are completely non-toxic to human cells. Most, however, show sufficient selective toxicity to be of value in the treatment of microbial diseases. Standardisation and harmonisation of AST methodologies, used in epidemiological surveillance of antimicrobial drug resistance, are critical if data are to be compared among national or international surveillance/monitoring programmes (Shrivatsava et al., 2009). It is essential that AST methods provide reproducible results in day-to-day laboratory use and that the data be comparable with those results obtained by an acknowledged ‘gold standard’ reference method. Laboratory antimicrobial susceptibility testing can be performed using: A dilution technique and disc diffusion technique dilution technique. Standardization minimizes the impact of these variables so that results will actually measure the organism’s expression of resistance (Brown and MacGowan, 2010). The need to standardize AST therefore became very necessary.

The potency and the accuracy of the antimicrobial content of the discs must be ensured. Antimicrobial discs need to be manufactured within strict control limits and handled correctly within the laboratory, otherwise, they cannot meet the quality and performance standards required. In the developed countries, it is believed that these conditions are adequately met but however, in the developing countries, this may not usually be the case.

1.1 Aims and Objectives

1.      To prepare and standardize low cost antibiotics discs and evaluate their susceptibility pattern to isolates from clinical samples.

2.      To compare the efficacy of the locally prepared disc with commercial antibiotic discs.

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