EFFECT OF BIOCIDES ONBIAL GROWTH RATE (BACTERIA)

ABSTRACT

This study was to determine the effect of biocides on microbial growth rate. A total of Six (6) different household biocides (Dettol, Septol, TCP, Izal, Savlon and Ethenol) were screened for their efficacy to control microbial growth. Environmentally isolated Escherichia coli and Staphylococcus aureus as well as ATCC strains of both organisms (S. aureus, ATCC25923 and ATCC25922) were used in this study as test organisms. To determine the effect of the various biocides on the test organisms at times 6hrs, 12hrs and 16hrs, agar well diffusion technique was employed to evaluate/determine the potency of these biocides on the test organisms. Results from this study revealed that Savlon recorded the highest zones of inhibition against Staphylococcus aureus (26 mm) at 3 hours followed by Dettol (24mm), Septol (23mm), Izal (21mm) and Ethanol (15mm) at 6 hours time interval and the two pathogens (Staphylococcus aureus and Escherichia coli) were sensitive to Savlon and Dettol at different concentrations. Results of this experiment also indicated that different pathogens acquired resistance to disinfectants (TCP and at less extent ethanol), and also suggested that the antibacterial effects of these biocides are not only dependent on the types of disinfectant but also on their concentrations. Resistance against antibiotics by pathogenic bacteria is a major concern in the antimicrobial therapy for both humans and animals. The effectives of biocides are very important to enhance the antimicrobial activity of these disinfectants towards controlling microbial population which includes prevention of diseases transmission and infection.

TABLE OF CONTENTS

Title Page                                                                                                                                  i

Certification                                                                                                                           iii

Dedication                                                                                                                              iv

Acknowledgement                                                                                                                  v

Table of Contents                                                                                                                   vi

List of Tables                                                                                                                          vii

Abstract                                                                                                                                  ix

CHAPTER ONE

Introduction                                                                                                                            1

1.1       Aim and Objectives                                                                                                    3

CHAPTER TWO

Literature Review                                                                                                                   4

2.1       Microbial Resistance to Biocides                                                                               4

2.2       Reduced Microbial Susceptibility to Biocides                                                           5

2.3       Mechanisms by Which Biocide Exert Their Antimicrobial Action                                    6

2.4       Mechanisms Which Reduce Microbial Susceptibility to Biocides                                    8

2.4.1    Intrinsic Properties of Bacteria Conferring Reduced Susceptibility to Biocides      8

2.4.2    Reduced Susceptibility to Biocides Resulting from Phenotypic Changes                      11

2.4.3    Plasmid-Mediated Mechanisms                                                                                 12

2.4.4    Mutational Resistance to Biocides                                                                             13

2.5       Possible Links between Antibiotic Resistance and Reduced

            Susceptibility to Biocides                                                                                           14

2.5.1    Examples of Studies Showing Reduced Susceptibility to Biocides in

            Antibiotic-Resistant Bacteria                                                                                     14

2.5.2    Examples of Studies Showing No Change in Susceptibility to Biocides in

            Antibiotic-Resistant Bacteria                                                                                     15

2.6       Active Substances                                                                                                       16

2.7       Production, Use and Fate of Biocides                                                                        17

2.8       Application of Biocides                                                                                              18

2.8.1    Biocides (Disinfectants) on Medical Devices and Surfaces                                       18

2.8.2    Biocides (Disinfectants and Antiseptics) Used on Skin and Mucosa                                    20

2.9       Biocides in Consumer Products                                                                                  20

2.9.1    General Aspects                                                                                                          20

2.9.2    Cosmetics and Personal Care Products                                                                      21

2.9.3    Household Products                                                                                                    21

CHAPTER THREE

Materials and Methods                                                                                                           23

3.1       Sample Collection                                                                                                      23

3.2       Sterilization of Materials                                                                                            23

3.3       Materials and Media Used                                                                                          23

3.4       Media Preparation                                                                                                      23

3.4.1    Inoculation of Test Organisms                                                                                   24

3.5       Biochemical Test                                                                                                        24

3.5.1    Catalase Test                                                                                                               24

3.5.2    Indole Test                                                                                                                  24

3.5.3    Citrate Utilization Test                                                                                               24

3.5.4    Hydrogen Sulphide (H₂S) Production Test                                                                24

3.5.5    Starch Hydrolysis                                                                                                       25

3.5.6    Motility Test                                                                                                               25

3.5.7    Voges-Proskauer Test                                                                                                 25

3.5.8    Urease Test                                                                                                                 26

3.5.9    Methyl Red Test                                                                                                         26

3.5.10  Carbohydrate Fermentation                                                                                        26

3.5.11  Coagulase Test                                                                                                           27

3.5.12  Oxidase Test                                                                                                               27

3.6       Biocides Testing                                                                                                         27

3.6.1    Determination of Biocidal Activity                                                                            27

3.6.2    Determination of Minimum Inhibitory Concentration and Minimum

            Bacteria Concentration                                                                                               28

CHAPTER FOUR

Results                                                                                                                                    30

4.1       Diameter Zones of Inhibition Produced After Three Hour                                        30

4.2       Diameter Zones of Inhibition Produced After Six Hours of Growth                                    30

4.3       Diameter Zones of Inhibition after Sixteen Hours of Growth                                  31

4.4       Minimum Inhibitory Concentration and Minimum Bactericidal Concentration

            Value of Selected Biocides against the Test Bacteria                                                            31

CHAPTER FIVE

Discussion, Conclusion and Recommendation                                                                      35

5.1       Discussion                                                                                                                   35

5.2       Conclusion                                                                                                                  37

5.3       Recommendation                                                                                                       37

            References

LIST OF TABLES

CHAPTER ONE

1.0      INTRODUCTION

Biocides, in the broadest sense, are substances formulated to be harmful to (or to otherwise control) living organisms (Scenihr, 2009). Many authorities adopt more restrictive definitions, similar to those for a microbicide, for the purposes of considering non-antibiotic antimicrobial agents, albeit still with diverse chemical characteristics and applications. Useful definitions in this mould have been provided by (Tumah, 2009) and (Sheldon, 2005), which the authors of the present review regard as reasonable in scope and practicality. With few exceptions such as iodide salts that show some efficacy in the treatment of certain veterinary fungal and bacterial infections (Fraser et al., 2001) biocides are not sufficiently selective to be used within body tissues, but some may be used as antiseptics on body surfaces. Other common applications include use as disinfectants on equipment and surfaces in many environments including farms and hospitals, as decontaminants on carcass surfaces following slaughter, as sporicidal sterilants for medical equipment and as preservatives in pharmaceuticals, cosmetics and food (Scenihr, 2009).

Antiseptics and Biocides are used extensively in hospitals and other health care centres to control the growth of microbes on both living tissues and inanimate objects. They are essential parts of infection control practices and aid in the prevention of nosocomial infections (Larson et al., 1991). But a common problem is the selection of Biocides and antiseptics because different pathogens vary in their response to different antiseptics or disinfectants (Russell, 1996). Dettol is widely used in homes and healthcare settings for various purposes including disinfection of skin, objects and equipments, as well as environmental surfaces. With prior cleaning before application, the number of microorganisms colonizing the skin and surfaces are greatly reduced (Rutala, 1996). The antimicrobial properties of chloroxylenol, the main chemical constituent of Dettol and other chlorinated phenols have been extensively studied (Hugo and Bloomfield, 2001). The antimicrobial properties of the disinfectant against some pathogenic bacteria have earlier been reported (Mellefont et al., 2003). Moreover, microorganisms are continuously acquiring resistance to new antiseptics and disinfectants; as a result, no single antiseptic or Biocides will be appropriate for all pathogens (Tortora et al., 1998). Therefore, it is necessary to evaluate the effectiveness of an antiseptic or disinfectant against a specific pathogen so that an appropriate agent can be easily selected. It is very important not only choose the appropriate disinfectant if not show that the concentrations, time, temperature and physical action that is used is efficient and profitable for those who use them.

This research demonstrated experimentally the effect of selected biocides on some test organisms. Also obtain security in the process of disinfection using a methodology of a standardized procedure. Entis, (2002), mentioned that the disinfectant function is to destroy microorganisms and prevent the spread of these. A disinfectant is a biocide that destroys the growth of microorganisms on surfaces and inanimate objects (McDonnell and Russell, 2009). The mechanisms of action of biocides, together with the factors that influence its activity, has become a key feature for the best use of biocidal formulations and control the emergence of resistant organisms (Gibson et al., 2009). It is considered that the active ingredients of the biocides are generally products that may contain one or more actives principles.

Determining the biocide efficacy is often carried out in suspension tests. This type of test determines the concentration of disinfectant which shows a definite log reduction in the number of microorganisms at a given time. In practice, meanwhile, the microorganisms are subjected to disinfection of surfaces in food production and that remains after cleaning, are commonly of the surface (Gibson et al., 2009).

Since some microorganisms have gained resistance to biocides due to mutation and resistance, lack of proper quality control in the production of biocides will reduce the efficacy of the biocides on microorganisms, considerations should be placed on concentrations of biocides used to combat microorganisms. Biocide of choice is also to be tackled because pathogens differ in their response to different Biocides.

The study of this work is for people to know the effectiveness of some biocides used against microorganisms (Staphylococcus aureus and Escherichia coli), and also to know the amount of concentration of a biocide that will inhibit the growth of microorganisms. Minimum Inhibitory Concentration was used to determine the lowest concentration of an antimicrobial that inhibit the visible growth of a microorganism after overnight incubation and Minimum Biocidal Concentration which is the lowest concentration of an antibacterial agent required to kill a particular bacterium.

1.1       AIM AND OBJECTIVES

To determine the effect of biocides on microbial growth rate (bacteria), while the specific objects are;

·       To determine the antimicrobial effect of various biocides tested against the selected bacterial strains

·       To determine the minimum biocide concentration on selected pathogens

·       To determine the minimum inhibitory concentration of the various biocides on selected pathogens

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