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

9

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).

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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) .

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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.

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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

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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

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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

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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

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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

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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).

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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