ABSTRACT
The sensitivity of biofilm forming staphylococcus aureus from hospital environment to different antibiotics which includes: ciprofloxacin, erythromycin, levofloxacin, gentamicin, trimethoprim, ampiclox, rifampicin, amoxycilin, norfloxacin, chloramphenicol, cefuroxime and ceftriaxone were evaluated. Fifty (50) clinical swab sample were collected within the hospital with respect to different sections which includes; Door handles, beddings, surgical table surfaces, surgical equipment, furniture. Biofilm detection test was carried out using Congo Red Agar method, a total of 12 isolates were strong biofilm producers while 6 isolates were weak biofilm producers and 32 isolates were non biofilm producers. In the antimicrobial susceptibility testing Ciprofloxacin gave the highest zone of inhibition, followed erythromycin, rifampicin, levofloxacin, trimethoprim, gentamicin, chloramphenicol, norfloxacin, ceftriaxone, cefuroxime, ampiclox and then amoxycilin give the least zone of inhibition. Ciprofloxacin seems a reliable antibiotic which can be used in combating the menace of biofilm in hospital environment.
TABLE OF CONTENTS
Certification i
Dedication ii
Acknowledgement iii
Table of content iv
List of table vii
List of figures viii
Abstract ix
CHAPTER ONE
INTRODUCTION
1.1 Background of study 1
1.2 Objectives of Study 5
1.3 Problem Statement 6
1.4 Significance of the Study 6
CHAPTER TWO
LITERATURE REVIEW
2.1 Definition of Biofilm 7
2.2 Biofilm formation and Maturation 8
2.2.1 The
Conditioning Layer 9
2.2.2 Reversible
Adhesion 9
2.2.3 Irreversible
Adhesion 10
2.2.4 Population
Growth 11
2.2.5 Final Stages
of Biofilm Development 12
2.3 Factors Favoring Biofilm Formation 13
2.3.1 Substratum
Effect 13
2.3.2 Conditioning
Films 13
2.3.3 Hydrodynamics 14
2.3.4 Horizontal
Gene Transfer 14
2.3.5 Quorum
Sensing 15
2.4 Biofilm Structure and Function 16
2.4.1
Extracellular Polymeric Substances 16
2.4.2 Protein 17
2.4.3 Interaction
of Particles 18
2.5 Biofilm and
Pathogenesis 18
2.5.1 Native Valve
Endocarditis 18
2.5.2 Otitis Media 19
2.5.3 Chronic
Bacterial Prostitis 19
2.5.4 Cyctic
Fibrosis 19
2.5.5
Periodontitis 20
2.6 Environmental Factors Affecting Biofilm
Development 21
2.6.1 Effect of pH 21
2.6.2 Rheological
and Adhesive Properties of Biofilm 21
2.6.3 Effect of
Temperature 22
2.6.4
Antimicrobial Susceptibility Testing 23
CHAPTER THREE
MATERIALS AND METHOD
3.1 Sample
Collection 25
3.2 Preparation of
Media and Sterilization 25
3.3 Sample
Inoculation 26
3.3.1 Isolation of
Bacteria 26
3.4 Identification of the Bacterial Isolate 26
3.4.1 Macroscopic
Examination 26
3.4.2 Microscopic
Examination (Gram Staining) 26
3.5 Biochemical
Test 27
3.5.1 Catalase
Test 27
3.5.2 Coagulase
Test 27
3.5.3 Oxidase Test 27
3.5.4 Methyl Red-
Voges Proskauer test 27
3.6 Detection of the Biofilm using Congo red
agar 28
CHAPTER FOUR
RESULTS
4.1 Results 29
CHAPTER FIVE
DISCUSSION,
CONCLUSION AND RECOMMENDATION
5.1 Discussion 37
5.2 Conclusion 38
5.3 Recommendation 39
LIST OF TABLES
Tables
Title Page
Table
4.1 Shows an assessment of biofilm
formation of S. aureus determined by CRA
method 31
Table
4.2 Antimicrobial susceptibility
testing of biofilm producing S. aureus from door
handles within the hospital environment 32
Table
4.3 Antimicrobial susceptibility
testing of biofilm producing S. aureus from hospital
beddings within the hospital
environment 33
Table
4.4 Antimicrobial susceptibility
testing of biofilm producing S. aureus from surgical
table surfaces within the hospital
environment 34
Table
4.5 Antimicrobial susceptibility
testing of biofilm producing S. aureus from surgical
equipment within the hospital
environment 35
Table
4.6 Antimicrobial susceptibility
testing of biofilm producing S. aureus from furniture
within the hospital environment 36
LIST OF FIGURES
Figure Title Page
Figure 4.1 Formation of biofilm on Congo red agar
showing positive, intermediate and
negative results 30
CHAPTER ONE
INTRODUCTION
1.1
BACKGROUND OF STUDY
Bacteria
generally exist in one of two types of population: planktonic, freely existing
in bulk solution, and sessile, as a unit attached to a surface or within the
confines of a biofilm. Biofilms were observed as early as 1674, when Antoine
van Leuwenhoek used his primitive but effective microscope to describe
aggregates of ‘‘animalcules” that he scraped from human tooth surfaces. Since
then, many advances in technology and laboratory working practices have allowed
more accurate descriptions of biofilms to be made, although even today there is
still ambiguity: A biofilm consists of cells immobilized at a substratum and
frequently embedded in an organic polymer matrix of microbial origin. Biofilms
are a biologically active matrix of cells and extra-cellular substances in
association with a solid surface. Biofilms are sessile microbial communities
growing on surfaces, frequently embedded in a matrix of extracellular polymeric
substances. A biofilm may be described as a microbial derived sessile community
characterized by cells that attach to an interface, embedded in a matrix of Exo
- polysaccharide which demonstrates an altered phenotype. Micro colonies are
discrete matrix enclosed communities of bacterial cells that may include cells
of one or many species. Depending on the species involved, the micro-colony may
be composed of 10–25% cells and 75–90% extracellular polymeric substances (EPS)
matrix. Bacterial cells within the matrix are characterized by their lack of
Brownian motion, and careful structural analysis of many micro-colonies often
reveals a mushroom-like shape. Although descriptions of biofilms have varied
over the years, the fundamental characteristics are frequently maintained. A
biofilm is attached to a substrate and consists of many bacteria co-adhered by
means of physical appendages and extra-cellular polymeric substances. If one of
these ingredients is omitted, a biofilm will not form. However, it should be
noted that without water bacterial motility and nutrient availability is
reduced and osmotic pressures become less viable to most bacteria. For
bacteria, the advantages of biofilm formation are numerous. These advantages
include: protection from antibiotics, disinfectants, and dynamic environments.
Intercellular communications within a biofilm rapidly stimulate the up and down
regulation of gene expression enabling temporal adaptation such as phenotypic
variation and the ability to survive in nutrient deficient conditions. About
99% of the world’s population of bacteria are found in the form of a biofilm at
various stages of growth and the films are as diverse as the bacteria are
numerous.
Over
the past few decades biofilm growth has been observed in many industrial and
domestic domains. Unfortunately, in most cases the growth of biofilms has been
detrimental. Many industries suffer the ill-effects of biofilm growth of one
type or another, which can result in heavy costs in cleaning and maintenance.
Examples of such industries include the maritime, dairy, food, water systems,
oil, paper, opticians, dentistry and hospitals. Perhaps the environment where
people are exposed to biofilms most frequently is the domestic environment.
Product spoilage, reduced production efficiency, corrosion, unpleasant odors
(malodors), unsightliness, infection, pipe blockages and equipment failure are
examples of the detrimental effects of biofilms. For these reasons and the
emergence of restrictive legislation regarding the effects of cleaning agents
on the environment and to user health and safety (Commission Regulation EC No.
1048/ 2005), there is a lot of industrial interest in developing materials and
methods which can remove and actively prevent the formation of biofilms. The
usefulness of biofilms is well known, especially in the field of
bioremediation. The use of organisms to remove contaminants, e.g. metals and
radio nuclides, oil spills, nitrogen compounds and for the purification of
industrial waste water, is now commonplace. Indeed the adhesive characteristics
of natural human flora are now considered as a tool for preventing the adhesion
of pathogenic bacteria to avert infection. However, major problems due to the
inappropriate formation of biofilms exist.
In
the UK, it is estimated that 9 million cases of intestinal disease every year,
much of which originates at home, where human excreta are the primary source of
infection Estimates show that for every case of infectious disease reported to
the Communicable Disease Surveillance Centre (CDSC), 136 unreported cases occur
in the community causing considerable morbidity. Global data on the incidence
of infectious disease combined with concerns about emerging and re-emerging
pathogens has led to a new governmental initiative to improve home hygiene, for
example, the safe removal of bacteria from domestic surfaces. Approximately 16%
of food poisoning outbreaks in England and Wales may be associated with meals
prepared in private houses.
In
the food industry biofilms cause serious engineering problems such as impeding
the flow of heat across a surface, increases in fluid frictional resistance of
surfaces and increases in the corrosion rate of surfaces leading to energy and
production losses. Pathogenic micro flora grown on food surfaces and in
processing environments can cross-contaminate and cause post-processing
contamination. If the microorganisms from food-contact surfaces are not
completely removed, they can lead to mature biofilm formation and so increase
the bio transfer potential. Examples of the food sectors that pay particular
attention to the possibility of cross-contamination are the milk industry and
the slaughter industry.
Hospital-related
infection (nosocomial infection) periodically provokes sensationalist
headlines, for good reason. Surgical instruments and fluid lines, e.g.
scalpels, drips and catheters, are common sources of biofilm growth and
subsequent infection. Biofilm forming Methicillin-resistant Staphylococcus aureus (MRSA) is
particularly important due to its ubiquity in the National Health Service (NHS)
and repeated resistance to all but a few antibiotic programs. Frequent sources
of MRSA are the patients themselves. Dentists have been under scrutiny in
recent years due to some serious breaches of health and safety laws, in
particular the sterility of instruments and Dental Unit Water Lines (DUWL).
Water lines create optimal conditions for biofilm formation due to ideal
surface chemistries, laminar flow and surface area. Potential sources of
infection include mouth sprays with dysfunctional valves and contaminated hand
pieces. The oil industry has cited many problems resulting from biofilm
formation by sulphate-reducing bacteria (SRB). Examples include pipe and rig
corrosion, blockage of filtration equipment and oil spoilage. Contamination by
SRB can result when oil reservoirs are subjected to water flooding for
secondary oil recovery in fields found under the sea bed. Such contamination
may arise from temperature-resistant organisms originating from hydrothermal
vents.
Conversely,
the effects of oil spills can result in shifts in the relative abundance of
microbial flora which impacts fish and invertebrate mortality, growth and
reproduction.
The
implications of biofilm growth are enormous and they pose a potential threat to
everybody and every surface. The sheer varieties of surfaces and environments
that have been occupied by biofilms are almost infinite. It follows that
combinations of the biofilm structural and temporal heterogeneity are just as
numerous. Considering the threat to health and industry that biofilms pose, it
is not difficult to realize the magnitude of the problem. It is thought that
further understanding of the mechanisms used by microorganisms to adhere to
various surfaces, with the use of the techniques currently available to measure
the adhesive strengths of various populations, will provide a basis for the
development of better strategies for cleaning surfaces.
Biofilm growth is governed by a number of
physical, chemical and biological processes. Attachment of a cell to a
substrate is termed adhesion, and cell-to-cell attachment is termed cohesion.
It is the mechanisms behind these forms of attachment, which ultimately
determine the adhesive and cohesive properties a biofilm will exhibit. Fletcher
described the accumulation of microorganisms on a collecting surface as a
process of three stages: adsorption, or the accumulation of an organism on a
collector surface i.e. substrate (deposition); attachment, or the consolidation
of the interface between an organism and a collector, often involving the
formation of polymer bridges between the organism and collector; colonization,
or growth and division of organisms on the collector’s surface.
Although
useful as a snap shot of biofilm growth, this type of profile is limited when
considering the intimate processes of cell–substrate/cell–cell interaction.
Characklis and Marshal later described an eight-step process which included the
formation of an initial conditioning layer, reversible and irreversible
adhesion of bacteria, and the eventual detachment of cells from a mature biofilm
for subsequent colonization.
1.2 OBJECTIVES OF STUDY
·
To isolate and identify S. aureus from surfaces around the
hospital environment to be submitted to Medical Microbiology Laboratory for
identification and characterization.
·
To determine and quantify
biofilm production of the S. aureus by
the Congo red agar method.
·
To determine the
antimicrobial susceptibility pattern of the S.
aureus.
·
To determine the
relationship between biofilm production and multi drug resistance among biofilm
producing organisms.
·
To detect MRSA among the
organisms of S. aureus isolated
from hospital environment.
1.3
LIMITATIONS OF STUDY
One
of the limitations of this study that sought to mar the effective carrying of
the work was that of getting the samples from the respective locations and also
that of the financial aspect and time. The study operated with a small budget
and lasted for only four weeks and a relatively huge sum of money and time were
needed to study the samples.
1.4 SIGNIFICANCE OF THE
STUDY
This
research work would be of great importance to the government and the public in
general giving a sensitization of biofilm forming organisms and an inherent
idea about nosocomial infections which are the major problems facing many
public edifice in the control and better management of biofilm producing
organisms .
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