CHAPTER 18BIOCIDE – chemical or physical agent which kills viable organisms, both pathogenic and non-pathogenicMICROBICIDE – the same as biocide but generally refers to pathogenic organisms-CIDAL – an agent with killing activity against an org.-STATIC – inhibit the growth of bacteria or fungiMIC (Minimum inhibitory conc.) – lowest conc. Of antimicrobial which will inhibit the visible growth of a microorganism after overnight cultivation MBC (Minimum bactericidal inhibition) – lowest conc of antimicrobial that will prevent the growth of a microorganism after subculture onto antibiotic-free media.Minimum biofilm eradication concentration (MBEC) – minimum concentration of an antimicrobial agent required to kill a microbial filmTOLERANCE – survival of microbes, but not grow, at levels of antimicrobial agentIf MIC/MBC ratio > 32, the term TOLERANCE is used.EAGLE PHENOMENON (paradoxical effect)–inc. conc of antimicrobial result in reduced killing rather than increase in cidal activity.RESISTANCE – ability to withstand the effects of a harmful chemical agent, with the org neither killed nor inhibited*MECHANISM- modification of the normal target of the antimicrobial agent either by mutation, enzymatic changes, target substitution, antibiotic destruction or alteration and restricted permeability to antibioticsFACTORS AFFECTING THE ANTIMICROBIAL ACTIVITY OF DISINFECTANTS

- Nature of the agent

- Nature of the challenge organism

- Mode of growth of the challenge organism

- Concentration of agent

- Size of the challenge population

- Duration of exposure of population to the active agent

- Environmental/physical conditionsINNATE RESISTANCE OF MICROORGANISMBacterial endospores /mycobacteria – possess most innate resistanceVegetative bacteria and some viruses – highly susceptible

Microbes adhering to surfaces as biofilms or present within other cells – increase in resistance to disinfectantsMICROBIAL DENSITY- many disinfectants require adsorption to the

microbial cell surface prior to killing- the larger number of microbes present, the

longer it takes a disinfectant to complete killing of all cells

DISINFECTANT CONC. AND EXPOSURE TIME- the more concentrated a disinfectant, the greater

efficacy, and the shorter the time of exposure required to destroy the population of microbes, except for iodophors.

- Dilution does not affect the cidal attributes of all disinfectants in a similar manner

PHYSICAL AND CHEMICAL FACTORSTemperature – cidal activity inc, with increase in temp.PH – changes in ph may affect the potency of the agent and its ability to interact with cell surface sites-increased cidal activity as the ph rises in alkaline conditions due to enhanced interaction with amino groups on microbial biomolecules- increasing the external pH of cell surface renders more (-) charged and enhances the binding of cationic cmpdsDIVALENT CATIONS – exert an antagonistic effect on certain biocides while having an additive effect on the cidal activity of others.*cationic cmpds – disrupt the outer membrane of gram-negative bacteria and facilitate their own entryEvaluation of Liquid Disinfectants

General • Phenol coefficient tests

– Phenolics: disinfectant – Neutralization of residual disinfectants:

prevent ‘carryover’ toxicity • Disinfectant tests

– Determination of appropriate cidal levels

• Tests involving Chemotherapeutic agents– Antimicrobial concentrations that inhibit

growth• Capacity-use dilution test

– Kelsey, Sykes and Maurer– Kill successive additions of a bacterial

culture– Disinfectants diluted in hard water

(clean), hard water containing organic

material (yeast suspension – simulate dirty conditions)

– 3% Tween 80: neutralizer (final recovery broth)

– BS 6907:1987: estimation of disinfectants used in dirty conditions in hospitals; most widely employed capacity test

Antibacterial Disinfectant Efficacy tests• Suspension Test

– Viable count (one colony develops from one viable cell/CFU; not ideal for disinfectants that promote clumping in bacterial suspensions – overcome by adding non-ionic surface active agents to the diluting liquid) after exposure of a standard suspension of cells to the disinfectant at known concentration for a given time interval

– 5-log killing of bacteria– Bactericidal effect: logNC – logND

o NC and ND – final number of

CFU/mL• In-use and Simulated use Test

– In-use testing• Medical devices• Maurer (1972)

– Simulated use testing• Instruments/surfaces

contaminated with an organic load

• 6-log killing bacteria• Problematic bacteria

– Mycobacteria• Hydrophobic; clump/aggregate

in aqueous media (resistance to chemical disinfection)

• M. tuberculosis – very slow growing; M. terrae, M. bovis, M. smegatis – rapid growing

– Standard crystal violet staining technique – reduction in biomass

– Biocide activity – reduced against intracellular legionellae

Other Microbe disinfectants Antifungal (fungicidal) tests

Spores – more resistant than vegetative mycelium

Spore suspensions – saline containing Tween 80 (wetting agent) from 7-day old cultures

Species:o Contaminant: Aspergillus niger

o Pathogen: Trichophyton

mentagrophyeso Strain: Penicillum variabile

EN 1275:1997 regulations – minimum reduction in viability by a factor of 104 w/in 60 minutes (Test fungi: Candida albicans and A. niger)

EN 1650:1998 – quantitative suspension test for evaluation of fungicidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas

Antiviral (viricidal) tests Viruses – obligate intracellular parasites,

incapable of independent growth and replication

Test viruses: rotavirus, adenovirus, poliovirus, herpes simplex virus, HIV, pox viruses, papovirus, hepatitis B and C

BS EN 13610: quantitative suspension test for the evaluation of viricidal activity against bacteriophages of chemical disinfectants used in food and industrial areas

Prion Disinfection tests Prions: acellular, proteinaceous infectious

agent, devoid of an agent-specific nucleic acid; high resistance to conventional chemical and physical decontamination

Prion Protein: abnormal isoform of a host cellular protein

Disinfectants used for elimination of prion infectivity: NaOH (phenolic formulation), guanidine thiocyanate and chlorine

Evaluation of Solid Disinfectants Disinfectant powders: phenolic substances

adsorbed onto kieselguhr (diatomite) Sodium dichloroisocyanurate: solid disinfectant Antiseptic powders: Acriflavine; Zinc

undecenoate/ salicylic acid mixed with talc (antifungal activity)

Inhibitory activity: dusting the powders onto the surface of seeded agar plates (inert diluents for control)

Evaluation of air disinfectants The decontamination and disinfection of

air is an important consideration for both infection and contamination control

A large number of important infectious diseases are spread via microbial contamination of the air

This cross-infection can occur in:o Hospitals and health care

facilitieso Airplanes

o Public and institutional

buildings Disinfection of air can be carried out by:

o Increased ventilation

o Filtration of air thru HEPA

filterso Chemical aerosol

o UVGI

Efficacy against a range of airborne pathogens and contaminating organisms

The use for formaldehyde vapor is the most commonly employed agent for fumigation procedures

o Should not be used with

hypochlorites The numbers of viable bacteria present

in the air can be assessed by simply exposing plates of solid nutrient media to the air

Any bacteria that fall on to the plates after a suitable exposure time can then be detected following an appropriate period of incubation

o *unsuitable for viruses

More meaningful data can be obtained if force rather than gravity is used to collect airborne particles

o Stream of air directed on to the

surface of a nutrient agar plateo Bubbled thru an appropriate

buffer or culture medium Various commercial impactor samplers

are availableo Filtration sampling

The air is passed thru a porous membrane

o Experimental evaluation of

potential air disinfectants Created in a closed

chamber Airborne microbial population is then

sampled using a slit sampler With viruses, the air can be bubbled thru

a suitable liquid medium then subjected to virological assay system

Evaluation of preservatives Preservatives are widely employed in

the cosmetic and pharmaceutical industries as well as in a variety of other manufacturing industries.

o Prevent microbial growth and

subsequent spoilageo Retard product deterioration

o Restrain growth of

contaminating microorganisms Effective preservation prevents

o Microbial spoilage

o Related chemical spoilage

o Physical spoilage

o Aesthetic spoilage

Suitably designed simulated use challenge tests involving the final product are required in addition to direct potency testing of pure preservative

The final preserved product is deliberately inoculated with a suitable environmental microorganism which may be fungal or bacterial

Zygosaccharomyces rouxii is a recommended challenge organism for oral preparations with high sucrose content

Subsequent survival, death or growth of the inoculums is then assessed using viable count techniques.

Different performance criteria are laid down for

o Injectable and ophthalmic

preparationso Topical preparations

o Oral liquid preparations

*in the British Pharmacopoeia and the European Pharmacopoeia

The range or spectrum of preservation can be extended by using more than one preservative at a time

Combination of parabens with varying water solubilities may protect both the aqueous and oil phases of an emulsion

A combination of German 115 and parabens results in a preservative system with both antibacterial and antifungal activity

Rapid evaluation procedures• With the tests mentioned before, results are not

available until a visible result is obtained which usually takes around 24 hours or more. Obviously, rapid antimicrobial susceptibility screening procedures will have the potential benefits, particularly in aggressive infection or rapidly progressing nosocomial infections of immunocompromised patients. These include epiflourescent and bioluminescence techniques.

Evaluation of potential chemotherapeutic antimicrobials

• Tests for the bacteriostatic activity of antimicrobial agents are valuable tools in predicting antimicrobial sensitivity/tolerance in individual patient samples and for detection and monitoring of resistant bacteria. The determination of MIC values must be conducted under standardized conditions, since deviation from the standard test conditions can result in considerable variation in data.

Tests for bacteriostatic activity• Quantitative techniques such as disc diffusion,

broth and agar dilution, and E-tests have replaced historical gradient plates, ditch-plate, and cup-plate techniques.

• Uses Mueller-Hinton or Iso-Sensi test at a pH of 7.2 or 7.4 or a defined thickness when solid agar plates are employed.

• National Committee for Clinical Laboratory Standards(NCCLS)- updating guidelines for the test.

Disk Test • Standard suspension of log phase growth cell are

prepared and inoculated on the surface of the agar.

• Different antibiotic disks were put on the surface of the agar

• The zone of inhibition was then measured and after this the microorganism resistance to a certain antibiotic would be specify.

Dilution Test • Usually liquid media were used but one can also

use a solid media.• 0.008-256 ml/L is used as doubling dilution of

the antimicrobial.• Log phase cell were added to the dilution

mixture• 5x103 is the final cell density to be recorded.

Epsilometer Test (E-Test) • Most convenient and presently accepted method

of determining MIC’s• The concept and execution is similar to disk

infusion test except 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 disks.

Problematic Bacteria • Due to heterogeneous expression of resistance,

poor agar diffusion of the antimicrobial, slow growth of resistance cell.

• Heterogeneous Expression should be enhanced by lowering temperature followed by an increase in concentration of salt.

Test for Bactericidal Activity • Required for the evaluation of a microbial.• MBC is the lowest concentration of

antimicrobial that results in 99.9% killing of bacterium under test.

• The first concentration the drug produces is considered the MBC.

Test for Fungistatic and Fungicidal Activity • Fungistatic- fungis are not killed but

suppressed.• Fungicidal- fungis are totally killed.• Yeast disk and E-Test were used to identify the

matter then distinct colonies is preferable to confluent growth

• Methylene Blue- enhances the clarity of the edges of the zones of inhibitions.

• Tailing and indistinct zone of inhibitions are usually the common problem for test involving azoles and yeasts.

Evaluation of Possible Synergistic Antimicrobial Combination

• Can be demonstrated by different laboratory procedures.

• Ex: “Chuquerboard” MIC Assay, where the microorganism is exposed to varying dilutions of each drug alone and in combination, disk infusion test and kinetic kill curve assay.

• Isobologram-plotted results of “Chuquerboard” MIC Assay.

Kinetic Kill Curves • Microorganism is inoculated into tubes

containing a single concentration of each antimicrobial alone, same concentration of each antimicrobial in combination and no antimicrobial.

• Tubes must incubated and viable count is to be executed at regular intervals on each system.

• Results is plotted to semilogarithmic paper.• Synergy- greater than 100 fold increase in

killing of the combination when compared to either drug alone.

• Antagonism- at least 100 fold decrease in killing of the combination when compared to the most active ingredient alone.

• Only chemotherapeutic agents and disinfectants are amenable to kill curve assays.

Test for Biofilm Susceptability • It is based on dye absorbance assay that could be

measuring a change in extracellular matrix rather than a change in viable bacterial cell number.

• 96 well plate- instrument used in the test.• Mycobacteria and Fungi-organisms subjected

to the test.Synergy Biofilm Assays

• Reduced susceptibility to antimicrobials means effective in vitro drug concentration to far exceed to a safe or achievable dose.

• Formula: