2. Topics to cover…..
• Definition of terms:
• Antibiotics and chemical biocides
• Disinfectants and disinfection
• Antiseptics and antiseptants
• Preservatives and preservation
• Chemical biocides and mechanism of action
• Factors affecting their activation and mechanism of action
• Evaluation of disinfectant efficacy
• Evaluation of preservatives
• Evaluation of chemotherapeutic activity
• Bactericidal, bacteriostatic
3. Terminologies
• Antimicrobial agents
• Antibiotics and non-antibiotic antimicrobials (Chemical biocides)
• Disinfectants: Broad antimicrobial activity but will have toxicity issues
limiting their use to inanimate surfaces
• Disinfection
• High-level disinfection (chemosterilants)
• Intermediate-level disinfection
• Low-level disinfection
• Antiseptics: Broad spectrum of antimicrobial activity but are
sufficiently non-toxic to allow them to be used on broken skin
• Preservatives: Broad spectrum antimicrobial agents incorporated into
pharmaceutical and other products
• Bacteriostatic: Those that arrest the growth of bacteria
• Bactericidal: Those that kill bacteria
• Fungistatic and fungicidal
• Virustatic and viricidal
4. Chemical biocides (Types)
• Acids and esters
• Antimicrobial activity is generally found only in the organic acids (weaker acids).
• The ionization constant, Ka and the pKa of the acid must be considered, especially in
formulation of the agent.
• Benzoic acids
• Organic acid, C₆H₅COOH, can be included, alone or in combination with other
preservatives.
• pKa of benzoic acid is 4.2 at which pH 50% of the acid is ionized.
• A disadvantage of the compound is the development of resistance by some
organisms.
• Sorbic acid
• The pKa is 4.8.
• It is most effective at pH 4 or below.
• Pharmaceutical products such as gums, mucilages and syrups are usefully preserved
with this agent
5. Acids and esters
• Sulphur dioxide, sulphites and metabisulphites
• Sulphur dioxide has extensive use as a preservative in the food and
beverage industries.
• Sodium sulphite and metabisulphite or bisulphite have a dual role,
acting as preservatives and antioxidants.
• Esters of p-hydroxybenzoic acid (parabens)
• Have pKa values in the range 8 – 8.5
• Exhibit good preservative activity even at pH levels of 7 – 8
• They are active against a wide range of fungi but are less so against
pseudomonas
• They are frequently used as preservatives of emulsions, creams and
lotions where two phases exist.
6. Alcohols
• As disinfectants and antiseptics
• Ethanol and isopropanol
• They are bactericidal against vegetative forms, including
Mycobacterium species, but are not sporicidal.
• Alcohols have poor penetration of organic matter and their use is,
therefore, restricted to clean conditions.
• Have been widely used for skin preparation before injection or other
surgical procedures.
• Ethanol (70%) solution is usually employed for the disinfection of skin,
clean instruments or surfaces. At higher concentrations, e.g. 90%,
ethanol is also active against fungi and most lipid - containing viruses,
including HIV.
• Mixtures with other disinfectants such as formaldehyde (100 g/L), are
more effective than alcohol alone.
• Isopropyl alcohol (isopropanol, CH₃CHOHCH₃) has slightly greater
bactericidal activity than ethanol but is also about twice as toxic.
7. Alcohols
• As preservatives
• Benzyl alcohol (C6H5CH2OH). This has antibacterial and weak local
anaesthetic properties
• Chlorbutol (chlorobutanol; trichlorobutanol; trichloro -t - butanol) is
typically used as a preservative in injections and eye drops.
• Phenylethanol (phenylethyl alcohol; 2 – phenylethanol) is reported to
have greater activity against Gram – negative organisms
• Phenoxyethanol (2 - phenoxyethanol) which is more active against P.
aeruginosa
8. Aldehydes
• Most aldehydes possess broad - spectrum antimicrobial
properties, including sporicidal activity (chemosterilants).
• Glutaraldehyde(CHO(CH₂)₃CHO)
• It has a broad spectrum of antimicrobial activity and rapid rate of kill.
• It has the further advantage of not being affected significantly by
organic matter.
• At a pH of 8, biocidal activity is greatest but unstable due to
polymerizaton. In contrast, acid solutions are stable but less active.
• Employed mainly for the cold liquid chemical sterilization of medical
and surgical materials such as the endoscopes.
• The contact time for sterilization can be as long as 10 hours.
• Times for general disinfection generally range from 20 – 90 minutes at
20 ° C depending on formulation and concentration.
9. Aldehydes
• Ortho- phthalaldehyde (OPA)
• This agent has demonstrated excellent mycobactericidal activity with
complete kill of M. tuberculosis within 12 minutes at room temperature.
• It requires no activation and has excellent stability over the pH range 3
– 9.
• Formaldehyde (HCHO)
• Used in either the liquid or the gaseous state for disinfection purposes.
• In the vapour phase it has been used for decontamination of isolators,
safety cabinets and rooms.
10. Biguanides
• Chlorhexidine
• Exhibits the greatest antibacterial activity at pH 7 – 8 where it exists
exclusively as a dication.
• Polyhexamethylene biguanides
• The antimicrobial activity of the bisbiguanide chlorhexidine exceeds
that of monomeric biguanides.
11. Halogens
• Chlorine (liquid chlorine)
• Hypochlorites (bleach)
• Readily available, inexpensive and compatible with most anionic and
cationic surface - active agents.
• They exhibit a rapid kill against a wide spectrum of microorganisms,
including fungi and viruses and mycobacteria and bacterial spores
when in high levels of available chlorine.
• The disadvantages are that they are corrosive, suffer inactivation by
organic matter and can become unstable.
• Chloroform
• Chloroform (trichloromethane, CHCl₃) has a narrow spectrum of
activity.
• It has been used extensively as a preservative.
12. Halogens
• Iodine (I₂)
• Iodine has a wide spectrum of antimicrobial activity against all
microbes.
• Iodine is also less susceptible to inactivation by organic matter.
• Disadvantages in the use of iodine in skin antisepsis are staining of skin
and fabrics
13. Hydrogen peroxide and peracetic acids
• Hydrogen peroxide and peracetic acid are high - level disinfectants
because of their production of the highly reactive hydroxyl radical.
• Hydrogen peroxide (H₂O₂)
• Used for disinfection of soft contact lenses.
• Concentrations of 3 – 6% are effective for general disinfection purposes.
• At high concentrations (35%) and increased temperature, hydrogen
peroxide is sporicidal.
• Peracetic acid (CH₃COOOH)
• It is a peroxide of acetic acid and is a more potent biocide than hydrogen
peroxide, with excellent rapid biocidal activity against bacteria, including
mycobacteria, fungi, viruses and spores.
• Its disadvantages are that it is corrosive to some metals and highly irritant.
14. Phenols
• Widely used as disinfectants and preservatives.
• They have good antimicrobial activity and are rapidly
bactericidal but generally are not sporicidal.
• Their activity is markedly diminished by dilution and is also
reduced by organic matter.
• They are more active at acid pH.
• Major disadvantages include their caustic effect on skin and
tissues and their systemic toxicity.
• Carbolic acid
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20. Mechanism of action of biocides
• Antibiotics have specific targets whereas biocides have a
general mechanism of action;
• Disruption of cell wall and membrane structure and function
• Intracellular coagulation
• Cross-linking reactions
• Oxidation reaction
• Enhancing activity
21. Oxidation reaction
• Biocides with oxidizing (electron - withdrawing) ability are used
as disinfectants and chemical sterilants (halogens - chlorine,
hypochlorites, bromine, iodophors) and peroxygens (hydrogen
peroxide, peracetic acid).
• This causes strand breakage and adduct formation on DNA and
RNA; degradation of unsaturated fatty acids leading to loss of
membrane fluidity; and specific modifications to amino acid
residues.
22. Cross-linking reactions
• The aldehydes and the sterilant alkylating agents ethylene oxide
and propylene oxide exhibit particularly strong reactions with
guanine residues causing cross - linking between DNA strands,
inhibiting DNA unwinding and RNA translation.
• The amino, carboxyl, sulphydryl and hydroxyl groups of
structural or enzymic proteins are also susceptible to alkylation,
causing cross -links between adjacent amino acid chains and
also with other amino acid - containing structures such as
peptidoglycan.
23. Intracellular coagulation
• The cross - linking reactions give rise to macromolecule
denaturation which can be recognized as intracellular
coagulation.
• High concentrations of disinfectants such as chlorhexidine,
phenol, ethanol and mercuric salts will coagulate the cytoplasm.
• This most likely arises from the precipitation of protein caused
by a variety of interactions including ionic and hydrophobic
bonding and the disruption of hydrogen bonds.
24. Disruption of functional structures
• Disruption of cell wall
• Low concentrations of disinfectant substances cause enzymes whose
normal role is to synthesize the cell wall to reverse their role in some
way and effect its disruption or lysis.
• These low concentrations of disinfectants (formalin, 0.12%; phenol,
0.32%; mercuric chloride, 0.0008%; sodium hypochlorite, 0.005% and
merthiolate, 0.0004%) caused lysis of Escherichia coli, streptococci, and
staphylococci.
• Disruption of cell membrane
• Uncoupling agents are believed to act by partitioning into the membrane
and rendering it permeable to protons, hence short - circuiting the
potential gradient or proton motive force.
25. Enhancing activity
• Much effort has also been expended in the search for synergistic
combinations of biocides which, when added together, will greatly
amplify the bactericidal effect.
• Combinations of phenylmercuric acetate with benzalkonium
chloride, lipophilic weak acids with fatty alcohols, and
chlorocresol with phenylethanol have been reported.
• The loss of outer membrane integrity and subsequent
permeabilization has been exploited in the potentiation of
biocides, including combinations of EDTA with chloroxylenol,
cetrimide, phenylethanol and the parahydroxy benzoic acid esters
26. Viricidal effect of biocides
• Viruses can be divided into two groups according to their
susceptibility to biocides.
• Lipophilic viruses that possess a viral envelope derived from their host
(e.g. HIV, herpes simplex virus, influenza virus) are the most susceptible
to biocides.
• The hydrophilic viruses comprise all the non - enveloped viruses and
differ tremendously in size and structure (poliovirus, hepatitis A virus,
foot - and - mouth disease virus) are often considered to be the least
susceptible to biocide.
• The biggest challenges for biocide activity against viruses is that
viruses on surfaces are often associated with soiling and fomites.
• In terms of mechanisms of action, the goal of a viricide should be
the destruction of the viral nucleic acid. Only oxidizing agents
have been observed to damage the viral nucleic acid within the
capsid.
27. Biocides and fungi
• The activity of biocides against fungi has not been widely
documented.
• Available information links cell wall glucan, wall thickness and
consequent relative porosity to the sensitivity of Saccharomyces
cerevisiae to chlorhexidine.
• Moulds tend to be less susceptible to biocides than yeasts.
28. Factors affecting biocide activity
• The activity of antimicrobial agents on a given organism or
population of organisms will depend on a number of factors
which must be reflected in the tests used to define their efficacy
• Innate (natural) resistance of microorganism
• Microbial density
• Disinfectant concentration and exposure time
• Physical and chemical factors
• Temperature
• pH
• Divalent cations
• Presence of extraneous organic matter
29. Innate (natural) resistance of microorganism
• The susceptibility of microorganisms to chemical disinfectants
and biocides exhibits tremendous variation across various
classes and species.
• Bacterial endospores and the mycobacteria possess the most
innate resistance, while many vegetative bacteria and some
viruses appear highly susceptible.
• Microorganisms adhering to surfaces as biofilms or present
within other cells may reveal a marked increase in resistance to
disinfectants and biocides
30. Microbial density
• Many disinfectants require adsorption to the microbial cell
surface prior to killing, therefore dense cell populations may
sequester all the available disinfectant before all cells are
affected.
• The larger the number of microorganisms present, the longer it
takes a disinfectant to complete killing of all cells.
31. Disinfectant concentration and exposure time
• With the exception of iodophors, the more concentrated a
disinfectant, the greater its efficacy, and the shorter the time of
exposure required to destroy the population of microorganisms.
• A graph plotting the log10 of the death time against the log10 of
the concentration is typically a straight line, the slope of which is
the concentration exponent (η).
• It is important to note that dilution does not affect the cidal
attributes of all disinfectants in a similar manner.
32.
33. Physical and chemical factors
• Temperature
• As with most chemical/biochemical reactions, the cidal activity of most
disinfectants increases with increase in temperature.
• Increasing the kinetic energy of a reaction system increases the rate of
reaction by increasing the number of collisions between reactants per
unit time.
• Raising the temperature of phenol from 20 ° C to 30 ° C increased the
killing activity by a factor of 4.
• pH
• Where the biocidal agent is an acid or a base), the ionization state (or
degree of ionization) will depend on the pH.
• As is the case with some antimicrobials (e.g. phenols, acetic acid,
benzoic acid), the non - ionized molecule is the active state (capable of
crossing the cell membrane/partitioning) and alkaline pHs which favour
the formation of ions of such compounds will decrease the activity.
34. Physical and chemical factors
• Divalent ions
• The presence of divalent cations (e.g. Mg2 +, Ca2 +), for example in
hard water, has been shown to exert an antagonistic effect on certain
biocides while having an additive effect on the cidal activity of others.
• Metal ions such as Mg2 + and Ca2 + may interact with the disinfectant
itself to form insoluble precipitates and also interact with the microbial
cell surface and block disinfectant adsorption sites necessary for
activity.
• Biguanides, such as chlorhexidine, are inactivated by hard water.
35. Presence of extraneous organic matter
• The presence of extraneous organic material such as blood,
serum, pus, faeces or soil is known to affect the cidal activity of
many antimicrobial agents.
• It is necessary to determine the likely interaction between
organic matter and the disinfectant by including this parameter
in laboratory evaluations of their activity.
• In order to simulate ‘ clean ’ conditions disinfectants are tested
in hard water containing 0.3 g/L bovine albumin, with the
albumin being used to mimic ‘ dirty ’ conditions.
36. Evaluation of disinfectants
• Evaluation of a disinfectant ’ s efficacy was based on its ability
to kill microbes, i.e. its cidal activity, under environmental
conditions mimicking as closely as possible real life situations
• Capacity - use dilution test or in-use test (Kelsey-Sykes test)
• This measures the ability of a disinfectant at appropriate concentrations
to kill successive additions of a bacterial culture.
• Tests employed disinfectants diluted in hard water (clean conditions)
and in hard water containing organic material (yeast suspension to
simulate dirty conditions), with the final recovery broth containing 3%
Tween 80 as a neutralizer.
• Capacity tests mimic the practical situations of housekeeping and
instrument disinfection, where surfaces are contaminated, exposed to
disinfectant, recontaminated and so forth.
• Results are reported simply as pass or fail and not a numerical
coefficient.
37. Evaluation of disinfectants
• Suspension test
• Most of the proposed procedures tend to employ a standard suspension
of the microorganism in hard water containing albumin (dirty conditions)
and appropriate dilutions of the disinfectant.
• Tests are carried out at a set temperature (usually around room
temperature or 20 ° C), and at a selected time interval samples are
removed and viable counts are performed following neutralization of any
disinfectant remaining in the sample.
• Using viable counts, it is possible to calculate the concentration of
disinfectant required to kill 99.999% (5 - log kill) of the original
suspension. Thus 10 survivors from an original population of 10⁶ cells
represents a 99.999% or 5 - log kill.
38. Evaluation of disinfectants
• Simulated use tests
• This involve deliberate contamination of instruments, inanimate
surfaces, or even skin surfaces, with a microbial suspension. This
may either be under clean conditions or may utilize a diluent
containing organic material (e.g. albumin) to simulate dirty
conditions.
• After being left to dry, the contaminated surface is exposed to the
test disinfectant for an appropriate time interval. The microbes are
then removed (e.g. by rubbing with a sterile swab), resuspended in
suitable neutralizing medium, and assessed for viability as for
suspension tests.
• New products are often compared with a known comparator
compound (e.g. 1 minute application of 60% v/v 2 - propanol for
hand disinfection products to show increased efficacy of the novel
product.
39. Fungicidal tests
• In order for disinfectants to claim fungicidal activity, a range of
standard tests have been devised.
• The main problem with fungi concerns the question of which
morphological form of fungi to use as the inoculum.
• Spore suspensions (in saline containing the wetting agent Tween 80)
obtained from 7 - day - old cultures are presently recommended.
• Aspergillus niger, Trichophyton mentagrophyes, Penicillium variabile
are also employed.
• Spore suspensions of at least 10⁶ CFU/ml have been recommended.
• Viable counts are typically performed on a suitable media (e.g. malt
extract agar, sabouraud dextrose agar) with incubation at 20 ° C for
48 hours or longer.
40. Viricidal tests
• The evaluation of disinfectants for viricidal activity is a
complicated process requiring specialized training and facilities
• They require some other system employing living host cells.
• Suggested test viruses include rotavirus, adenovirus, poliovirus,
herpes simplex viruses, HIV, pox viruses and papovavirus
• The virus is grown in an appropriate cell line that is then mixed
with water containing an organic load and the disinfectant
under test. After the appropriate time, residual viral infectivity is
determined using a tissue culture/plaque assay or other system
(e.g. animal host, molecular assay for some specific viral
component).
• A reduction of infectivity by a factor of 10⁴ has been regarded
as evidence of acceptable viricidal activity.
41. Evaluation of preservatives
• Adequate preservation (and validation of effectiveness) is a
legal requirement for certain formulations.
• Effective preservation prevents microbial growth and, as a
consequence, related chemical, physical and aesthetic spoilage
that could otherwise render the formulation unacceptable for
patient use, therapeutically ineffective or harmful to the patient.
• While the inhibitory or cidal activity of the chemical to be used
as the preservative can be evaluated using an appropriate in
vitro test system its continued activity when combined with the
other ingredients in the final manufactured product must be
established.
• A suitably designed simulated use challenge tests involving the
final product are, therefore, required in addition to direct
potency testing of the pure preservative.
42. Evaluation of preservatives
• In the challenge test, the final preserved product is deliberately
inoculated with a suitable environmental microorganism which
may be fungal or bacterial
• For oral preparations with a high sucrose content, the
osmophilic yeast Zygosaccharomyces rouxii is a recommended
challenge organism. The subsequent survival (inhibition), death
or growth of the inoculum is then assessed using viable count
techniques.
43. Evaluation of potential chemotherapeutic
antimicrobials
• Unlike tests for the evaluation of disinfectants, where
determination of cidal activity is of paramount importance, tests
involving potential chemotherapeutic agents (antibiotics)
invariably have determination of MIC as their main focus.
• Tests for bacteriostatic activity
• Disc test: For disc tests, standard suspensions
(e.g.0.5 McFarland standard) of log – phase
growth cells are prepared and inoculated
on to the surface of appropriate agar plates
to form a lawn.
• Commercially available filter -paper discs
containing known concentrations of
antimicrobial agent are then placed on the
dried lawn and the plates are incubated
aerobically at 35 ° C for 18 hours.
44. Evaluation of potential chemotherapeutic
antimicrobials
• Any zone of inhibition occurring around the disc is then
measured, and after comparison with known standards, the
bacterium under test is identified as susceptible or resistant to
that particular antibiotic.
• Use of such controls endorses the suitability of the methods
(e.g. medium, inoculum density, incubation conditions)
employed.
• Also known as the Kirby-Bauer method
45. Evaluation of potential chemotherapeutic
antimicrobials
• Dilution tests
• Doubling dilutions, usually in the range 0.008 – 256 mg/L of the
antimicrobial under test, are prepared in a suitable broth medium,
and a volume of log - phase cells is added to each dilution to
result in a final cell density of around 5 × 10⁵ CFU/ml.
• After incubation at 35 ° C for 18 hours, the concentration of
antimicrobial contained in the first clear tube is read as the MIC
• Dilution tests require a number of controls, e.g. sterility control,
growth control, and the simultaneous testing of a bacterial strain
with known MIC to show that the dilution series is correct.
46.
47. Evaluation of potential chemotherapeutic
antimicrobials
• E-test (Epsilometer) - test
• The most convenient and presently accepted
method of determining bacterial MICs.
• The concept and execution of the E - test is
similar to the disc diffusion test except that a
linear gradient of lyophilized antimicrobial in
twofold dilutions on nylon carrier strips on
one side are used instead of the filter - paper
impregnated antimicrobial discs.
• MIC is determined by noting where the
ellipsoid (pear -shaped) inhibition zone
crosses the strip
48. Evaluation of potential chemotherapeutic
antimicrobials
• Tests for bactericidal activity
• MBC testing is required for the evaluation of novel antimicrobials. The
MBC is the lowest concentration (in mg/L) of antimicrobial that results
in 99.9% or more killing of the bacterium under test.
• MBCs are determined by spreading 0.1 ml (100 μl) volumes of all
clear (no growth) tubes from a dilution MIC test onto separate agar
plates (residual antimicrobial in the 0.1 ml sample is ‘ diluted ’ out
over the plate).
• After incubation at 35 ° C overnight (or longer for slow - growing
bacteria), the numbers of colonies growing on each plate are
recorded.
• The first concentration of drug that produces <50 colonies after
subculture is considered the MBC.
