Chapter
One
Introduction
Micro-organisms play an important role in all our lives and many
are so small they can only be seen under a microscope. Many
micro-organisms are helpful to us whilst others can be harmful;
micro-organisms that harm humans tend to be referred to as germs
( Wilson and Heaney, 1999). These germs can be a bacteria, fungi
and virus. Once inside the home, classroom, air, laboratory, they
can be transferred from person to person or from the source to a
person by direct contact, or through indirect contact via a
surface and back again ( Rusin et al., 1998).These bacteria are
transmitted through a variety of means including direct person to
person spread or contact with body fluids, contact with droplets
or airborne spread by droplet nuclei, and indirect transmission
through hand contact with a contaminated intermediate object
(Barbosa and Levy 2000).Contaminated sites and surfaces in the
home have been classified into 1 of 3 general categories
(reservoirs, reservoir disseminators, and hand/food contact
surfaces) for which the risks of contamination and cross
1
contamination are higher. Bacteria can be introduced into the
home via the human occupants, pets, and foodstuffs. Health care
workers may be exposed to infectious materials including
infectious body fluids, contaminated medical supplies and
equipment, contaminated environmental surfaces, or contaminated
air. Nosocomial transmission of infectious organisms, including
methicillin-resistant Staphyloccus aureus (MRSA), occurs
primarily through the hands of health care personnel, which may
come into contact with colonized patients. Diseases commonly
spread by means of environmental surfaces such as computers,
classroom walls, classroom door handles, laboratory, toilets,
chairs, and so on include the common cold, cold sores,
conjunctivitis, giardiasis, impetigo, meningitis, pin worm
disease, diarrhea and pneumonia, to mention but a few (WHO,
1980). Bacteria such as Escherichia coli, Shigella dysenteriae, Streptococcus
pneumoniae, Klebsiella pneumoniae and Staphylococcus aureus as well as
Corynebacterium diphtheriae cause diarrhoea, dysentery, pneumonia,
food poisoning and intoxication as well as whooping cough
respectively (FAO, 1989; WHO, 1980). Chances of contamination of
environmental surfaces are increased when there are lack of
2
running tap water, boreholes, drainage systems and heaps of
domestic waste especially in a classrooms, laboratory and
metropolis densely populated (Calamari et al., 1994). Human hands
have been implicated as the major transmitter of microorganisms
to environmental surfaces. Curtis et al. (2003) and Lorna et al.
(2005) reported that hands often act as vectors that carry
disease-causing pathogens including bacteria and viruses from
person to person either through direct contact or indirectly via
surfaces. Defective personal hygiene can facilitate the
transmission of some of these pathogenic bacteria found in the
environment to human hands (Mensah et al., 2002).
A true antibiotic is an antimicrobial chemical produced by
microorganisms against other
microorganisms ( Wilson and Heaney, 1999). Mankind has made very
good use of these antimicrobials in its fight against infectious
disease. Many drugs are now completely synthetic or the natural
drug is manipulated to change its structure somewhat, the latter
called semisynthetics.
The risk of infection from pathogenic microorganisms on
environmental surfaces derives not only from their presence but
3
also from their ability to survive on many surfaces. The
persistence of pathogenic microorganisms has been established in
studies of their survival on surfaces in institutional,
commercial, and domestic settings (Weber and Rutala WA, 2001;
Rusin et al., 1998; Buckalew et al., 1996; Wilson and Heaney,
1999).
Bacteria are a major cause of disease and even human death.
Disinfectant as an effective agent to kill or eliminate bacteria
is widely used in various ways, especially in academic,
industrial and microbial laboratory. Disinfectants can be mainly
divided into five agents: alkylating, sulfhydryl combining,
oxidizing, dehydrating and permeable. Mounting concerns over the
potential for microbial contamination and infection risks in the
classroom, laboratory, atmospheric air and general house hold and
industries have also led to increased use of antiseptics,
antibiotics and disinfectants by the public. Antiseptics and
disinfectants are used extensively in hospitals and other health
care settings for a variety of topical and hard-surface
applications. In particular, they are an essential part of
infection control practices and aid in the prevention of
4
nosocomial infections (Mcdonnell and Russell, 1999). A wide
variety of active chemical agents (or “biocides”) are found in
these products, many of which have been used for hundreds of
years for antisepsis, disinfection, and preservation (Block,
1991). In order to test which disinfectant inhibits both bacteria
the best, bacterial inhibition will be performed. A bacterial
inhibition assay is a test done to measure zones of inhibition of
a certain bacteria (Daughterty, 2007). Filter disks are put on an
agar plate that contains a solution of the bacteria that you are
attempting to inhibit. Before the disks are put on the agar, they
are soaked in a desired chemical. In the experiment, filter disks
are going to be soaked in solutions of the brand name
disinfectants. After placing disks on the agar the plates are
incubated so the bacteria can grow on the plates. The purpose of
bacterial inhibition is to see where bacteria do not grow on the
plate. The area around discs is where the bacteria on the plate
were inhibited.This ring of inhibition shows that the chemical
has successfully inhibited that bacterium, meaning that the
bacteria were not able to grow and thrive because that chemical
killed the bacteria. After several days have passed a metric
5
ruler is used to measure the diameter of the zone of inhibition
around each filter disks. This “zone” will just appear to be a
clear circle around the disk where no bacterial growth is
apparent. Zones can be measures in several ways. Sometimes it is
accepted to measure the radius from the filter disc to the edge
of the zone of inhibition. Other times, like in this experiment,
zones of inhibition will be measured from the edge of the filter
disc to the edge of the ring of inhibition. Products include:
Clorox, Lysol, Green Works, Mr. Clean, and Seventh Generation.
DEFINITIONS
Antibiotics: are defined as naturally occurring or synthetic
organic substances which inhibit or destroy selective bacteria or
other microorganisms, generally at low concentrations.
Antiseptics - Chemicals that kill microorganisms on living skin
or mucous membranes.
Bactericidal - Chemical agents capable of killing bacteria.
Similarly agents that are virucidal, fungicidal or sporicidal are
agents capable of killing these organisms.
6
Bacteriostatic - Chemical agents that inhibit the growth of
bacteria but do not necessarily kill them.
Biocide: is a general term describing a chemical agent, usually
broad spectrum, that inactivates microorganisms. Because biocides
range in antimicrobial activity, other terms may be more
specific, including “-static,” referring to agents which inhibit
growth (e.g., bacteriostatic, fungistatic, and sporistatic) and
“-cidal,” referring to agents which kill the target organism
(e.g., sporicidal, virucidal, and bactericidal).
Cleaning - the physical removal of foreign material, e.g., dust,
soil, organic material such as blood, secretions, excretions and
microorganisms. Cleaning generally removes rather than kills
microorganisms. It is accomplished with water, detergents and
mechanical action. The terms “decontamination” and “sanitation”
may be used for this process in certain settings, e.g., central
service or dietetics. Cleaning reduces or eliminates the
reservoirs of potential pathogenic organisms.
Critical items: instruments and devices that enter sterile
tissues, including the vascular system. Critical items present a
high risk of infection if the item is contaminated with any
7
microorganisms. Reprocessing critical items involves meticulous
cleaning followed by sterilization.
Decontamination: the removal of disease-producing microorganisms
to leave an item safe for further handling.
Disinfection: the inactivation of disease-producing
microorganisms. Disinfection does not destroy bacterial spores.
Disinfectants are used on inanimate objects in contrast to
antiseptics, which are used on living tissue. Disinfection
usually involves chemicals, heat or ultraviolet light. The nature
of chemical disinfection varies with the type of product used.
High level disinfection: High level disinfection processes
destroy vegetative bacteria, mycobacteria, fungi and enveloped
(lipid) and nonenveloped (non lipid) viruses, but not necessarily
bacterial spores. High level disinfectant chemicals (also called
chemical sterilants) must be capable of sterilization when
contact time is extended. Items must be thoroughly cleaned prior
to high level disinfection.
Intermediate level disinfection: Intermediate level disinfectants
kill vegetative bacteria, most viruses and most fungi but not
resistant bacterial spores.
8
Noncritical items: those that either come in contact with only
intact skin but not mucous membranes or do not directly contact
the patient. Reprocessing of noncritical items involves cleaning
and/or low level disinfection.
Sanitation: a process that reduces microorganisms on an inanimate
object to a level below that of infectious hazard (e.g., dishes
and eating utensils are sanitized).
Semicritical items: devices that come in contact with nonintact
skin or mucous membranes but ordinarily do not penetrate them.
Reprocessing semicritical items involves meticulous cleaning
followed preferably by high-level disinfection.
Sterilization: the destruction of all forms of microbial life
including bacteria, viruses, spores and fungi. Items should be
cleaned thoroughly before effective sterilization can take place.
Regardless of how well clean rooms function, potential
contaminants can be continuously introduced into production
facilities through entry of materials and equipment.
Operators are another major source of particulates and
microorganisms, shedding particles and microbes from skin, mucous
membranes, and through respiratory secretions. Manufacturing
10
procedures such as mixing, concentration, centrifugation, or
transfer may also generate spills or aerosols that spread widely
through production areas. Where bacteria and fungi are allowed to
grow in recesses or when cleaning and sanitation procedures are
ineffective, continuous or even resistant environmental strains
can be developed. Disinfectant cleaners contain surfactants and
builders that work to exterminate viruses and bacteria from
various surfaces. Surfactants are compounds that lower the
surface tension of a liquid and by doing so, increase the contact
between the liquid and another substance. Common household
surfactants include: alcohols, aldehydes, bleaches, hydrogen,
peroxide, iodine, and potassium permanganate (Senior, 2011).
11
Antibiotics are commonly used to treat infections caused by
bacterial pathogens. The high prevalence of indigenous antibiotic
resistant bacteria harbouring diverse resistance traits could
represent potential health risks. Antibiotic resistant genes
might be transferred to transnational pathogenic bacteria
infecting humans, particularly under the selection pressure of
antibiotics as well as via the “SOS” response (Beaber et al., 2002;
Ubeda et al., 2005). The administration of a single or a long term
exposure of microorganisms to high concentration of antibiotics
can select for multidrug resistant strains (Li et al., 2002).
Research has also shown that there has been a sigmoidal rise in
resistance over time in the presence of a constant rate of
antibiotic consumption and a threshold level of antibiotic usage
needed to trigger the emergence of resistance to significant
levels‟ (Austin et al., 1999). The mechanisms by which bacteria
become resistant are either by modification of the antibiotic or
the target site or its removal from the cell (DubMendal, 2005).
Environmental bacteria have been shown to be reservoirs and
sources of antibiotic resistance genes in clinical pathogens (Li
et al., 2010). Acquisition of resistance genes through horizontal
12
transfer facilitated by plasmid has been found to be ubiquitous
in clinical pathogens (Mendal et al., 2003, 2004). While most
antimicrobial efforts are geared toward finding new ways to kill
bacteria, it is not lack of antibiotics per se, but rather the
increase of resistant bacteria that has made bacterial infections
difficult to treat (DeNap and Hergenrother, 2005). In view of the
fact that everybody would use a toilet, whether public or
private, the rate of disease development would be dependent on
people’s attitude after the use of a toilet.
MECHANISMS OF ACTION OF DISINFECTANT ON MICROORGANISMS
Considerable progress has been made in understanding the
mechanisms of the antibacterial action of antiseptics and
disinfectants (Li et al., 2010). By contrast, studies on their modes
of action against fungi , viruses , and protozoa have been
rather sparse. Furthermore, little is known about the means
whereby these agents inactivate prions. Whatever the type of
microbial cell (or entity), it is probable that there is a common
sequence of events. This can be envisaged as interaction of the
antiseptic or disinfectant with the cell surface followed by
penetration into the cell and action at the target site(s) (Li et
13
al., 2010). The nature and composition of the surface vary from
one cell type (or entity) to another but can also alter as a
result of changes in the environment . Interaction at the cell
surface can produce a significant effect on viability (e.g. with
glutaraldehyde) , but most antimicrobial agents appear to be
active intracellularly . The outermost layers of microbial cells
can thus have a significant effect on their susceptibility (or
insusceptibility) to antiseptics and disinfectants; it is
disappointing how little is known about the passage of these
antimicrobial agents into different types of microorganisms(DeNap
and Hergenrother, 2005). Potentiation of activity of most biocides
may be achieved by the use of various additives,
MECHANISMS OF RESISTANCE OF MICROORGANISM TO DISINFECTANTS
Different types of microorganisms vary in their response to
antiseptics and disinfectants. This is hardly surprising in view
of their different cellular structure, composition, and
physiology. Traditionally, microbial susceptibility to
antiseptics and disinfectants has been classified based on these
differences; with recent work, this classification can be further
14
. Because different types of organisms react differently, it is
convenient to consider bacteria, fungi, viruses, protozoa, and
prions separately(DeNap and Hergenrother, 2005).
Bacterial Resistance to Antiseptics and Disinfectants
In recent years, considerable progress has been made in
understanding more fully the responses of different types of
bacteria (mycobacteria, nonsporulating bacteria, and bacterial
spores) to antibacterial agents(DeNap and Hergenrother, 2005) .As a
result, resistance can be either a natural property of an
organism (intrinsic) or acquired by mutation or acquisition of
plasmids (self-replicating, extrachromosomal DNA) or transposons
(chromosomal or plasmid integrating, transmissible DNA
cassettes). Intrinsic resistance is demonstrated by gram negative
bacteria, bacterial spores, mycobacteria, and, under certain
conditions, staphylococci. Acquired, plasmid mediated resistance
is most widely associated with mercury compounds and other
metallic salts. In recent years, acquired resistance to certain
15
other types of biocides has been observed, notably in
staphylococci (DeNap and Hergenrother, 2005).
Intrinsic Bacterial Resistance Mechanisms
For an antiseptic or disinfectant molecule to reach its target
site, the outer layers of a cell must be crossed. The nature and
composition of these layers depend on the organism type and may
act as a permeability barrier, in which there may be a reduced
uptake (Li et al., 2010). Alternatively but less commonly,
constitutively synthesized enzymes may bring about degradation of
a compound. Intrinsic (innate) resistance is thus a natural,
chromosomally controlled property of a bacterial cell that
enables it to circumvent the action of an antiseptic or
disinfectant. Gram-negative bacteria tend to be more resistant
than gram-positive organisms, such as staphylococci (Li et al.,
2010).
Intrinsic Resistance of Bacterial Spores.
Bacterial spores of the genera Bacillus and Clostridium have been
widely studied and are invariably the most resistant of all types
of bacteria to antiseptics and disinfectant. Although Bacillus
species are generally not pathogenic, their spores are widely
16
used as indicators of efficient sterilization. Clostridium species
are significant pathogens; for example, C. difficile is the most
common cause of hospital acquired diarrhea(Li et al., 2010). Many
biocides are bactericidal or bacteristatic at low concentrations
for nonsporulating bacteria, including the vegetative cells of
Bacillus and Clostridium species, but high concentrations may be
necessary to achieve a sporicidal effect (e.g., for
glutaraldehyde and CRAs). By contrast, even high concentrations
of alcohol, phenolics, QACs, and chlorhexidine lack a sporicidal
effect, although this may be achieved when these compounds are
used at elevated temperatures.
Intrinsic Resistance of other gram-positive Bacteria.
The cell wall of staphylococci is composed essentially of
peptidoglycan and teichoic acid. Neither of these appears to act
as an effective barrier to the entry of antiseptics and
disinfectants. Since highmolecular-weight substances can readily
traverse the cell wall of staphylococci and vegetative Bacillus
spp., this may explain the sensitivity of these organisms to many
antibacterial agents including QACs and chlorhexidine (Li et al.,
2010) .
17
However, the plasticity of the bacterial cell envelope is a well-
known phenomenon. Growth rate and any growth limiting nutrient
will affect the physiological state of the cells. Under such
circumstances, the thickness and degree of crosslinking of
peptidoglycan are likely to be modified and hence the cellular
sensitivity to antiseptics and disinfectants will be
altered. Therefore, the cell wall in whole cells is responsible
for their modified response.
In nature, S. aureus may exist as mucoid strains, with the cells
surrounded by a slime layer. Nonmucoid strains are killed more
rapidly than mucoid strains by chloroxylenol, cetrimide,
and chlorhexidine, but there is little difference in killing by
phenols or chlorinated phenols removal of slime by washing
rendered the cells sensitive. Therefore, the slime plays a
protective role, either as a physical barrier to disinfectant
penetration or as a loose layer interacting with or absorbing the
biocide molecules.
Intrinsic resistance of gram-negative bacteria.
18
Gram-negative bacteria are generally more resistant to
antiseptics and disinfectants than are nonsporulating,
nonmycobacterial grampositive bacteria. there is a marked
difference in
the sensitivity of S. aureus and E. coli to QACs (benzalkonium,
benzethonium, and cetrimide), hexachlorophene, diamidines, and
triclosan but little difference in chlorhexidine susceptibility.
The outer membrane of gram-negative bacteria acts as a barrier
that limits the entry of many chemically unrelated types of
antibacterial agents. This conclusion is based on the relative
sensitivities of staphylococci and gram-negative bacteria and
also on studies with outer membrane mutants of E. coli, S.
typhimurium, and P. aeruginosa. Smooth, wild-type bacteria have a
hydrophobic cell surface; by contrast, because of the
phospholipid patches on the cell surface, deep rough (heptose-
less) mutants are hydrophobic. These mutants tend to be
hypersensitive to hydrophobic antibiotics and disinfectants. Low-
molecular-weight (Mr ,ca. 600) hydrophilic molecules readily
pass via the porins into gram-negative cells, but hydrophobic
19
molecules diffuse across the outer membrane bilayer. The presence
of a less acidic type of
outer membrane LPS could be a contributing factor to this
intrinsic resistance. The possibility exists that the cytoplasmic
(inner) membrane provides one mechanism of intrinsic resistance.
This membrane is composed of lipoprotein and would be expected to
prevent passive diffusion of hydrophilic molecules. It is also
known that changes in membrane composition affect sensitivity to
ethanol proposed that decreased susceptibility of Serratia
marcescens to chlorhexidine was linked to the inner
membrane( Lannigan and Bryan 2007) .
Acquired Bacterial Resistance Mechanisms
As with antibiotics and other chemotherapeutic drugs, acquired
resistance to antiseptics and disinfectants can arise by either
mutation or the acquisition of genetic material in the
form of plasmids or transposons. It is important to note that
“resistance” as a term can often be used loosely and in many
20
cases must be interpreted with some prudence. This is
particularly
true with MIC analysis. Unlike antibiotics, “resistance,” or an
increase in the MIC of a biocide, does not necessarily correlate
with therapeutic failure. An increase in an antibiotic MIC can
may have significant consequences, often indicating that the
target organism is unaffected by its antimicrobial action.
Increased biocide MICs due to acquired mechanisms have also been
reported and in some case misinterpreted as indicating
resistance. It is important that issues including the pleiotropic
action of most biocides, bactericidal activity, concentrations
used in products, direct product application, formulation
effects, etc., be considered in evaluating the clinical
implications of these reports( Lannigan and Bryan 2007).
Factors affecting disinfection and antibiotic efficiency
The effectiveness of antibiotics and disinfectants is limited and
much dependent on application
21
conditions (Bessems 1998). The factors which control the
efficiency of disinfectants are microbial type and growth
condition; interfering substances; acidity-pH; temperature;
contact time; and concentration (Bessems 1998, Chmielewski and
Frank 2003).
Microbial type and growth conditions
Antimicrobial activity of a disinfectant and antibiotics varies
greatly between different types of
Microorganisms and might also differ between different strains of
the same species
(Maillard 2002). Studies found that vegetative cells are more
susceptible to disinfectant
than spores (Kitis 2004) and adhered cells are less sensitive
than plankton cells (Johnston
and Jones 1995, Bredholt et al. 1999, Lindsay and von Holy 1999).
Among vegetative
bacteria, mycobacteria are probably the most resistant to
disinfectant, followed by Gramnegative
22
bacteria and Gram-positive bacteria, which is the most sensitive
(Maillard 2002).
The significant differences in the composition and structure of
the cell and outer walls of
these organisms can account for these phenomena (Maillard 2002).
Interfering substances
The efficiency of disinfectants is reduced in the presence of
organic and inorganic matter.
The influence of the protein load on the killing spectrum of
different disinfectants was
evidently proved in a series of studies such as (Bessems ,1998)
and( Lambert and Johnston
2001). Some disinfectants may also be affected by inorganic
materials such as hard water salts. Among commonly used
disinfectants in the food industry the disinfectant based on
peracetic acid is relatively stable in use (National Seafood
HACCP Alliance 2000). Although quaternary ammonium compound
disinfectant is affected by water hardness it is less affected by
organic matter (National Seafood HACCP Alliance 2000). In
contrast disinfectant based on chlorine compounds is
23
significantly reduced effectiveness by organic soils but is less
affected by hard water (National Seafood HACCP Alliance 2000).
Chemical reaction and spatial non-reaction are two main reasons
that result in the reduction of disinfection efficiency (Lambert
and Johnston 2001). In the former way, organic and inorganic
material may compete with bacteria to react with disinfectants
and thus the concentration of bactericidal compounds in aliquots
is lowered
(Lambert and Johnston 2001). Whilst, in the latter way, organic
and inorganic material may form a spatial barrier such that
microorganisms are protected from the effects of disinfectants
(Lambert and Johnston 2001).
Acidity - pH
The acidity or pH of the contaminated matrials is one of the
factors significantly affecting the
activity of some disinfectants (Schmidt 2003, Kitis 2004).
Therefore to achieve the
highest killing activity disinfectants should only be used within
the pH range specified by
the manufacturer (Springthorpe 2000).
24
Contact time, temperature and concentration
To be effective, disinfectants must find, bind to and transverse
microbial cell envelopes before they reach their target site and
begin to undertake the reactions, which will subsequently lead to
the destruction of the microorganism (Kleperer 1982). Sufficient
contact time is therefore critical to ensure disinfection and
most general purpose disinfectants are formulated to reduce
bacterial populations by at least 5 log orders within 5 minutes
in suspension or a 3 log unit reduction in population of surface-
adherent cells (Holah 1995). Contact time can be increased by
applying the disinfectant as a foam or gel (Schmidt 2003). The
relationship between time and efficiency is dependant upon the
type of microorganism (Bessems 1998). There is a close
relationship between contact time, temperature and bactericidal
efficiency of some disinfectant (Schmidt 2003). The study by
Taylor et al., (1999) found that at 20°C then 13 of 18
disinfectants tested were effective on P. aeruginosa whilst only 11
of them proved their effect at 10°C. The results
by Tuncan (1993) also demonstrated that the efficiency of
quaternary compound at 50ppm and lower concentration against
25
Listeria sp. decreased considerably as the exposure temperature
decreased. However, its effect was improved via increasing the
contact time at cold temperature. Therefore, increasing
temperature is also an alternative method that could be applied
to improve the effectiveness of disinfectants (Langsrud 2003).
The range of temperature applied is typically from 5°C to 55°C.
However in the majority of operations, disinfectants should offer
a recognized performance level at ambient temperature (Schmidt
2003).Concentration of disinfectant is one of the major factors
in biocidal activity (Russell and McDonnell 2000). The
relationship between microbial death and disinfectant
concentration is not linear, but usually follows a typical
biological sigmoidal death curve (Bessems 1998). The results by
Tuncan (1993) indicated that the effectiveness of quaternary
ammonium compound and chlorine on Listeria sp. was improved when the
concentration was increased from 50ppm to 100-200ppm. Bessems
(1998) found that at a constant test concentration, the rate of
killing was increased with an increase in time and the relation
between time and concentration for membrane-active disinfectant,
i.e.quaternary ammonium compound was regulated by Gram-negative
26
bacteria, whereas Gram-positive bacteria regulated the
application of disinfectants having oxidizing properties, i.e.
halogen containing disinfectant. The recommendation
concentrations of chlorine compounds, QACs and PAA commonly used
in classroom doors, laboratory table are presented in Table 1.
Table 1: The recommended concentrations of common disinfectants
(Huss 2003).
Sanitizers
Food contact surfaces Non-food contact
Chlorine (ppm)
100-200* 400
Quats (ppm)
200* 400-800
Peroxyacetic acid (ppm)
200-315* 200-315
* The higher end of the listed range indicates the maximum concentration
permitted without a required
Methods for testing disinfectant efficiency
Catalogues of test methods
27
There is a range of test methods for evaluating disinfectant
efficiency. Although these
methods differ in experimental detail they are all based on the
same principle, which
involves adding the test organism to a sample of disinfectant.
The test mixture is sampled
at the prescribed contact time and, following neutralisation of
the disinfectant, the
number of survivors in the sample is estimated (Bloomfield et al.
1995). Disinfection
tests are subdivided into suspension tests, carrier tests,
surface disinfection tests and other practice-mimicking
tests.
Background of Study
Pathogenic micro-organisms and fungal spores entering the home
can survive on surfaces for significant periods of time and can
be transferred to the hands when touched. Germs from hands can
then be transferred to other surfaces and other people, leading
to infection. To break this chain of infection, household
surfaces should be cleaned thoroughly with an antibacterial
28
cleanser or disinfectant on a regular basis, reducing the risk of
cross-contamination and lowering the risk of illness. Statistics
are showing that incidences of illness and disease spread by food
contamination are
29
common and increasing dramatically even in academic
environments , laboratory personnel and the environment out
large. Apart from direct infection from contaminated food, it is
known that infection from contaminated surfaces , doors, door
handles, chairs, laboratory tables or utensils is also playing a
major role. Sensitivity of microorganism to antibiotics and
disinfectant becomes highly indispensible.Environmental bacteria
have been shown to be reservoirs and sources of antibiotic
resistance genes in clinical pathogens (Li et al., 2010).
Acquisition of resistance genes through horizontal transfer
facilitated by plasmid has been found to be ubiquitous in
clinical pathogens (Mendal et al., 2003, 2004). While most
antimicrobial efforts are geared toward finding new ways to kill
bacteria, it is not lack of antibiotics per se, but rather the
increase of resistant bacteria that has made bacterial infections
difficult to treat (DeNap and Hergenrother, 2005)
Aim and Objectives of study
The aim of this work is to investigate the sensitivity of
bacteria isolate from classroom doors, laboratory tables and
30
organisms from air to disinfectant and antibiotics. To achieve
this aim, the following objectives were pursued.
1) Identification of the bacteria present in classroom doors,
laboratory tables and air.
2) The effects of several parameters such as time, concentration,
and interfering substances on
disinfectant and antibiotics.
3) Determination of the sensitivity of antibiotics on the
bacteria isolates
4) Disinfectants and antibiotic susceptibility test
Justification of Study
We sought to characterize and quantify bacteria of medical
interest on commonly touched classroom, laboratory surfaces and
air to evaluate the sensitivity of disinfectant and antibiotics
to the bacteria isolates from class room doors, laboratory and
air. Naturally, this organism is endowed with weak pathogenic
31
potentials. However, its profound ability to survive on inert
materials, minimal nutritional requirement, tolerance to a wide
variety of physical conditions and its relative resistance to
several unrelated antimicrobial agents and antiseptics,
contributes enormously to its ecological success and its role as
an effective opportunistic pathogen
Review of Related Literature
Evaluation of hospital environment disinfection as a
means of controlling endemic nosocomial pathogens in a University
Teaching Hospital in Nigeria was evaluated. Disinfectant used in
the Hospital was collected from the Infection Control unit and
prepared in different concentrations. The isolated bacterial
species from the hospital environment were exposed to graded
concentrations of the disinfectants and the most effective
concentration on each isolate was noted. This procedure was
carried out in two successive years (2006 and 2007). Killing rate
of the isolates that were resistant to the disinfectants was also
carried out and likely effective exposure time was determined.
The following bacterial species were isolated: Staphylococcus
32
epidermidis, Klebsiella Pneumoniae, Klebsiella spp., Bacillus subtilis, Enterobacter spp.,
Serratia marcescens, Pseudomonas aeruginosa, Pseudomonas spp., Escherichia coli,
Serratia spp., Bacillus cereus, Citrobacter freundii, Proteus mirabilis, Staphylococcus
aureus, Bacillus megaterium, Streptococcus pyogenes, and Streptococcus spp.
Minimum Effective Dilution (MED) of the disinfectant on all
isolates ranged from 1:300 to1:1000. Staphylococcus aureus and
Pseudomonas aeruginosa were the most resistant isolate with MED of
1:400 and 1:300 respectively. Result of killing rate on the two
most resistant isolates showed that Staphylococcus aureus and
Pseudomonas aeruginosa required 80 and 120 minutes of exposure
respectively to the disinfectant to bring about almost total
killing of these resistant isolates. The results show that
improper disinfections, degradation of disinfectant and lack of
routine standardization of disinfectants are responsible for
failure of chemical disinfection as a means of controlling
nosocomial infections in the hospital (ihejirika et al,. 2011).
(DubMendal, 2005) Studies on efficacy of some locally sold
disinfectants on bacterial pathogens isolated from cold rooms was
determined. Five milliliters of different dilutions of
chloroxylenol (Dettol), 5-chloro-2-hydroxy-diphenyl methane
33
(Septol), and chlorohexidine gluconate (Purit) respectively were
used. Organisms were isolated from swab and effluent samples
collected in triplicates from 10 major cold rooms under standard
microbiological techniques. Staphylococcus aureus, Streptococcus pyogenes
and Citrobacter freundii were isolated. A 0.1ml of each dilution was
delivered into a culture plate of individual isolate
respectively, and zones of inhibition measured. Zones diameter of
inhibition produced by disinfectants on isolates revealed
increasing inhibition as concentrations increased with
significant difference in inhibition of individual isolates by
the different disinfectants used at P<0.05. The trend of the
zones of inhibition showed that Dettol>Septol>Purit. Bacterial
pathogens present in cold rooms can cause food contamination and
poisoning, and infection of exposed individuals. Proper sanitary
application of Dettol, Septol and Purit will help prevent public
health consequences of food contamination.
Aboh et al.,(2000) worked on antibacterial activities of
five brands of household disinfectants were obtained from
different locations within the Federal Capital Territory, Nigeria
and comparatively studied using the Rideal Walker Phenol
34
coefficient test and quantitative suspension test. The
quantitative suspension test was carried out at the recommended
concentrations of the manufacturers for household and utensil
disinfection. The active compounds of the products according to
their respective labels were: D1-(Chloroxylenol 4.8%), D2-
(Dichloroxylenol 2%), D3- (Chlorhexidine gluconate 0.3% and
cetrimide 3%), D4 - (Dichlorometaxylenol 2.5%), D5-(Chlorhexidine
gluconate 0.3% and cetrimide 3%). The test organisms were
clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa,
Klebsiella aerogenes and Escherica coli. The phenol coefficient of the
disinfectants ranged between 5.0 – 9.0. All the disinfectants
showed strong bactericidal effect against the organisms used with
exception to Pseudomonas aeruginosa to which only D5 and D3 were
effective.
Manan and Sharma, 2003 studied household antibiotics and
disinfecting treatments to reduce bacteria, yeasts and molds on
kitchen sponges were evaluated. Sponges were soaked in 10% bleach
solution for 3 min, lemon juice (pH 2.9) for 1 min, or deionized
water for 1 min, placed in a microwave oven for 1 min at full
power, or placed in a dishwasher for full wash and drying cycles,
35
or left untreated (control). Microwaving and dishwashing
treatments significantly lowered (P < 0.05) aerobic bacterial
counts (<0.4 log and 1.6 log CFU/sponge, respectively) more than
any chemical treatment or control (7.5 CFU/sponge). Counts of
yeasts and molds recovered from sponges receiving microwave (<0.4
log CFU/sponge) or dishwashing (0.4 log CFU/sponge) treatments
were significantly lower than those recovered from sponges
immersed in chemical treatments. Studies shows that microwaving
and dishwashing treatments may kill food borne pathogens in a
household kitchen environment. The effects of several parameters
such as time, concentration, and interfering substances on
disinfection were discussed
Rasha et al,.(2012) studied the efficiency of traditional
disinfectants and antiseptics used in the laboratory against
bacterial isolates was detected with help of Broth dilution
method (for determination of minimum inhibitory concentration-
MIC), Disc and Well diffusion method.The results of MIC method
show that chlorohexidine gluconate Hibitene) was most effective
disinfectants on tested bacteria and it followed by chloroxylenol
(S1)type (while chloroxylenol(S2)and(Sp)type showed lower
36
activity), hydrogen peroxide, sodium hypochlorite, formaldehyde,
sodium dichloroisocyanurate and PVP-I, while chlorohexidine
cetramid (Savlon) showed no efficiency against all tested
bacteria . Generally, B.subtilis and methicillin resistant S.aureus
were found to be the most sensitive bacteria being tested in this
study against disinfectants and antiseptics while P.aeruginosa was
the most resistant bacteria to these agents.
Disc and well diffusion methods showed correlation between the
concentrations of disinfectants and the inhibition zones of
bacterial growth increase significantly P <0.05.
37
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