Download 13-7-ET-V1-S1__labor..

Laboratory evaluation of antimicrobial agents
1. Introduction:
Search of potential antimicrobial and biocides and their evaluation is continues process in research
laboratories, as the microorganism acquires resistance to the presently available antimicrobial and
biocide agents. Resistance acquired to biocide and antimicrobial agents among bacteria is a natural
evolution phenomenon. Apart from blood born viruses and bacteria and their resistance, their
contamination on medical devices and instruments and devices in hospitals has to be cleaned by
suitable liquid disinfectant or sterilizing agent. Evaluation of potency of new antimicrobial or biocidal
agents to be used in human, animal and in routine medical and environmental cleaning has gained
significant laboratory considerations.
2. Factors affecting the antimicrobial activity of disinfectants:
It is important to prove the safety and potency of antimicrobial activity of chemicals in laboratory. As
studied by Krönig and Paul in the late 1890s, bacterial do not died simultaneously on exposure to
bactericidal agent but follow an orderly sequence, which includes kinetics of chemical reaction
between bacterium and an agent. Variety of factors plays an important role in the interaction of
antimicrobial agents and microbes. Chicke’s pioneer work has open up a new door to disinfecting
agents and their evaluation of potency in early 20th century.
2.1. Natural (innate) resistance of Microorganism:
Some of vegetative microorganism and viruses are highly susceptible to chemical disinfectants while
others like prions, bacterial endospores and mycobacterial possess most innate resistance.
Microorganism adhering to biofilm or presents within other cell or capsule shows increased
resistance to same agents too. Range of different microorganism and environmental conditions are
selected to evaluate the potency of new agent.
2.2. Microbial mass:
Microbial killing process initiated by interaction of killing agent with bacterial cell surface, huge cell
biomass utilizes all disinfectant and reduce the killing efficacy. Denser the bacterial density (Biomass)
more time to kill all bacteria. If we use identical conditions, it has been demonstrated that 10 spores
of the anthrax bacillus were destroyed in 30 minutes but it takes 3 hours to kill 100,00 spores.
Cleaning and washing removes most of the bacteria before disinfection in routines practice. Unlike
sterilization, evaluation of disinfectant did not show linear killing curve, rate of killing reduced at
lower cell number. Hence, 3-log killing may be quickly achieved with 108 cell rather than 104 cells.
Standardized cell number will be preferred to perform disinfectant efficacy test and degree of killing
for required time period.
2.3. Disinfectant concentration and exposure time:
Dilution or concentration of active ingredients has significant effect on potency. With exception of
iodophore, the more concentrated the product, more efficacy and less time of exposure to kill the
microorganism. Linear graph observed when plot Log10 of a death time (time required to destroy a
standard inoculum) against Log10 of concentration. Slop of graph indicates concentration exponent
(η) and expressed as an equation
𝜂=
(𝐿𝑜𝑔 𝑑𝑒𝑎𝑡ℎ 𝑡𝑖𝑚𝑒 𝑎𝑡 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑖𝑜𝑛 𝐶2 ) − (𝐿𝑜𝑔 𝑑𝑒𝑎𝑡ℎ 𝑡𝑖𝑚𝑒 𝑎𝑡 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑖𝑜𝑛 𝐶1 )
𝐿𝑜𝑔 𝐶1 − 𝐶2
Concentration exponent (η) can be calculated by above equation or derived graphically.
All disinfectant shows different cidal attributes on same serial dilution. e.g. mercury chloride has
concentration exponent 1 so 31 time less active at 3 fold dilution; whereas phenol has η = 6 so 3 fold
dilution shows 36 or 729 less activity compared to original.
2.4. Physical and Chemical Factor:
Temperature, pH, and water hardness has proven influence on potency of disinfectant.
2.4.1. Temperature
Within certain limits; as the temperature raises the cidal activity of disinfectant raises. Quantitatively
it can be expressed as temperature coefficient, either temperature coefficient per degree raise in
temperature (θ) or per 10° raise (Q10). Koch results shows that the increase of temperature from 20
to 30° increase 4 fold efficacy of phenol to kill anthrax spores (Q10). Temperature coefficient can be
calculated from the following equation. It is also possible to plot the rate of kill against the
temperature.
𝜃 (𝑇1 −𝑇2 ) =
𝑡1
⁄𝑡
2
Where t1 is the extinction time at T1°C, and t2 the extinction at T2°C (i.e. T1 + 1°C).
𝑄10 =
𝑇𝑖𝑚𝑒 𝑡𝑜 𝑘𝑖𝑙𝑙 𝑎𝑡 𝑇°
𝑇𝑖𝑚𝑒 𝑡𝑜 𝑘𝑖𝑙𝑙 𝑎𝑡 (𝑇 + 10°)
2.4.2.pH
pH change the ionization state of disinfectant and may alter the activity. Many times alteration of pH
may shows better results as ionized molecules may react with cell surface more efficiently. With
some antimicrobials such as phenols, acetic acid and benzoic acid it reverse, it means ionized state
reduced the cidal capability of these compounds. In contrast glutaraldehyde, quaternary ammonium
compounds (QACs) shows better activity at increased pH, as the pH increases activity increases.
Increased pH renders the cell surface and makes it more negative charged and hence better
adherence of cationic compounds like chlorhexidine and QACs.
2.4.3.Divalent cations
Divalent cations such as Mg2+, Ca2+, etc. present in hard water may interact with microbial cell
surface and block the binding site and reduce the cidal power of disinfectants.
2.5. Presence of extraneous organic material
Presence of extraneous organic material may react with disinfectant like halides (sodium
hypochloride) and reduce its efficacy as cidal. On the other way organic material interact with cell
surface and block the active site too. To clear the actual picture, cidal efficacy of agent should be
studied in both clean and dirty conditions. Albumin or sheep blood or mucin are used to create dirty
conditions in experiments.
3. Evaluation of liquid disinfectants
3.1. General
To evaluate the disinfectant capability of phenol, phenol coefficient test was proposed in early 20th
century. However non-phenolic disinfectants are more widely available then phenolics. The method
so called “capacity use dilution test” proposed by kalsey and co-worker in 1970, gives appropriate
results when incubated with bacterial culture. The results are simply pass or fail and not numbers or
coefficient. This test is used to evaluate the disinfection capability of wide range of compounds.
Soon it is easily understood that the viable count calculation will be employed after successful
exposure of disinfection at unit time interval and data can be expressed by many ways like
percentage kill, log10 reduction in number or Log10 survival expressed in percentage. It is difficult
for the researcher to set the standardization of the method as there is no standard limit for the
acceptable results. Unlike present antibiotics (chemotherapeutic agents) where minimum inhibitory
concentration (MIC) were found. Appropriate cidal levels were measured in case of disinfectants.
Different regulatory agencies required different level of killing efficiency at given time interval.
Disinfection efficiency varies between regulatory agencies and in general 5log killing of bacteria (i.e.
starting with 106 CFU/mL) being suggested for suspension test but some agencies asks for 6log
killing. 4log killing efficiency is acceptable for viruses. With proper cleaning, 10 4 prion losses are
acceptable as a disinfectant and considered as appropriate disinfectant.
3.2. Antibacterial disinfectant efficacy test
To establish standardized procedure to evaluate antibacterial, disinfectant efficacy test, various
regulatory agencies like US food and drug administration (USFDA), environmental protection
authority (EPA), association of official analytical chemists (AOAC) and some of European agencies
like European standards or norms, EN; British Standards, BS; Germany, DGHM; France, AFNOR; etc.
work published by Kampf and co-workers (2002) provides detailed guideline and methodology
regarding the evaluation of bactericidal, fungicidal, tuberculocidal, and virucidal efficacy of
disinfectant with reference to EN procedures, however the significant results were easily justified by
statistical analysis.
3.2.1.Suspension tests
Water and albumin solution suspending microorganism and appropriate dilution of disinfectant are
employed in all the proposed methodology with some minor changes at controlled temperature
(generally at 20°C). At unit time interval sample are lake out and no. of microorganisms are count.
Inactivation of disinfectant in withdrawal sample can be done by further dilution or adding specific
inhibiting agent. 99.99% of microbial inhibition activity of disinfection can be calculated by viable
count. 10 colonies represent the 99.99% inhibition capability of disinfectant in 106 cells/mL or 5log
killing efficiency of culture medium. Inactivation of disinfectant with addition of inactivating agent or
dilution may cause decline in number and thus simultaneous test is performed without disinfectant.
Bactericidal effect can be calculated by the following equation.
BE=LogNC – LogND
NC = CFU/mL in control; ND = CFU/mL in disinfectant series.
Here we should take a note that the each colony represents 1 cell and thus disinfectant (e.g.
cetrimide) having tendency to make clumps of bacterial cells may give wrong interpretation. This
problem can be avoided by adding non-ionic surface active agents to diluting fluids.
3.2.2.In use and simulated use tests
In contrast to suspension test, in use test and simulated tests are involved in testing used medical
devices, instruments, and surfaces intentionally contaminated with biological load and suitable
microorganism have been used to check the efficacy of disinfectant.
In 1972, maurer have first expressed an idea of in use test. He tested the jar, bucket, and other
places containing living organism contaminated material and in what number. Sample is collected
from in use container, instruments and medical devices and diluted with suitable diluent, viable
count of resulting suspension is then performed. Duplicate plates are incubated, one at 32°C for 3
days (recovery of damaged bacterial at lower temperature is significantly rapid then 37°C) and
second plate is incubated at 37°C for 7 days. One or two colonies are avoided during interpretation
and 10 or more colonies suggest weak action of disinfectant.
Simulated use test performed on instruments inanimate surface or skin previously contaminated
with known microorganisms. This test can be performed on both clean and dirty conditions.
Deliberately contaminated objects are left to dry, contaminated surfaces are being exposed to
disinfectant for unite time interval. Microbes are then recovered from the surface and re-suspending
appropriate neutralizing medium and assessed viability as like suspension test. Effectiveness of new
products is assessed by comparing them with old products or compounds.
3.2.3.Problematic bacteria:
Preparation of homogeneous suspension is problematic for Mycobacteria as of its hydrophobic
nature. Slow growth of mycobacterium tuberculosis can be a problem for evaluation;
Mycobacterium terrae can be substituted as M. tuberculosis to get rapid growth.
Spores are the highly resistive form of bacterium and are used as inoculum in test. Prolonged
inoculum time is allowed for germination and growth in such conditions.
Bacterial biofilms are more prone to resistant then planktonic cells against antimicrobial agents.
Assessment can be done by the modified suspension test, briefly small object like glass or metal
substrate or bottom of microtitre plate are used as a support to produce biofilm, exposure of this
biofilm is done with disinfectant for unit time interval. Exposed cells are treated with appropriate
diluent and re suspended as separate cell by sonication, planktonic cells are then counted by viable
count method.
Biocidal activity of compound is significantly reduced in case of parasitic bacteria which are grown in
some host. Biocidal activity of bacteria in such organism can be performed in both suspension and
suspension involving amoeba containing bacteria after lysis of host bacteria viable count is
performed to check the cidal activity.
3.3. Other microbe disinfectant tests
Like antimicrobial activity, antifungal and antiviral activity of compounds can also be tested by
suspension test.
3.3.1.Antifungal test
Unicellular yeast can be treated like bacteria; but to use spore or pieces of hyphae with the
filamentous, moulds has not been clear to assess antifungal activity. Currently spore suspension
grown in normal saline solution for 7 days is used to check antifungal activity. Spore suspension of
106 CFU/Ml is recommended for test.
EN 1275 guideline recommends the Candida albicans and Aspergillums niger, activity proves the
significant reduction by the factor of 104 within 60 minutes.
3.3.2.Antiviral activity
Evaluation of antiviral activity of disinfectant is not an easy task as viruses are an intracellular
parasites and unable to grow in artificial culture media.
Virucidal activiry can be done on retrovirus, adenovirus, poliovirus, herpes simplex viruses, human
immunodeficiency virus, pox viruses and papova virus. Virucidal activity can be assessed by growing
virus on appropriate cell line. After appropriate growth it is treated with water containing organic
load and disinfectant under test. At unit time interval residual viral infectivity is determined by
appropriate technique. Acceptable virucidal activity can be considered at 104 fold reduced infectivity
of viruses.
3.3.3.Prion disinfection test
Prions are misfolded protein pathogens and completely lacking nucleic acid (either DNA or RNA or
both) Stanley (1982) gets the prestige to give the term prions for this unique class of pathogens.
Prions are non-living misfolded proteins and have tendency to transmit and propagate in hosts. They
have high resistance to conventional chemical and physical disinfectants mostly they are found in
tissue homogenates and have protection.
Slow, laborious and cost are the limitations of currently available prion disinfection assays, except
sodium hydroxide and sodium hypochloride, most of the disinfectant are inadequate to eliminate
prions.
4. Evaluation of solid disinfectants
Solid disinfectants are an appropriate combination of disinfectant and diluents. Phenolic adsorbent
onto kieselguhr, sodium dichloroisocyanurate, zinc undecenoate or salicylic acid are the known solid
disinfectants.
Evaluation can be done by sprinkling these agents on solid agar medium inoculated with organism.
Disc of agar is cut and organism is sub cultured for evaluation of survivors.
5. Evaluation of air disinfectants
Numbers of microorganism are spread via air and cause infection. They may be starting their travel
from hospital, cinemas, aeroplanes, etc. many microorganism present in air can be assessed by
simply exposing the solid nutrient medium plate in air for unite time interval as studied by Robert
Koch (1881).
Microorganism can be easily assessed by these methods but not viruses, by simple exposure and
gravitational force. One step forward industries are used mechanical impactor rather depend on
gravitational force to collect air born particles. It may be done by blowing air on plate or bubbling
through liquid medium or passing air through membrane filter and then filter paper is incubated on
nutrient medium.
To evaluate air disinfectant, air suspension of appropriate organism is created in close chamber, and
then exposed to air disinfectant. Survival of organism is then checked by regular sampling at unit
time interval by appropriate sampler. Two major problems with this technique are associated one is
preparation of microbial air suspension and second is neutralization of disinfectant remain in air.
Solar heating for a period of 6 hours reduced the biological load at 7 log and may be best and
economic option for developing countries.
6. Evaluation of preservatives
Preservatives are inseparable part of pharmaceutical, food, cosmetics and other variety of
industries. Probable cidal or inhibitory activity of preservatives alone and in product can be
evaluated in vitro. It is difficult with the product having two or more phases where preservative
present in one phase, such as emulsion where preservatives may present in one phase and other
facilitates the growth of organism. There products required simulated use challenge test to prove
potency of preservatives used in these product.
7. Rapid evaluation procedures
All tests discussed earlier are time consuming and required 18 hours or more. Do we have test which
gives results earlier than 18 hours? Answer is yes, as per the study of Hugo and Russell in 1998,
epifluorescent and bioluminescence are help full to get the quick results. Bioluminescence work on
the principle that organism treated with DNA specific fluorescent dye (e.g. acridine organge) will give
different color fluorescence under UV exposure. A dead cell shows orange coloration while live cell
shows green fluorescence.
8. Evaluation of potencial chemotherapeutic antimicrobials
Minimum inhibitory concentration (MIC) is prime focus to evaluate the chemotherapeutic agents
whereas cidal activity is the paramount importance for disinfectants.
8.1. Bacteriostatic activity
The new and improved quantitative techniques such as disc diffusion, broth and agar dilution and Etest are extensively used by modern labs compared to gradient dilution test, ditch-plate and
cup/well plate technique. These are performed by using a chemically defined media such as Muller
Hilton or iso-sensitest at pH range of 7.2 to 7.4.
Many countries have followed the guideline provided by national committee for clinical laboratory
standards (NCCLS), who time to time update the guideline for batter evaluation.
8.1.1.Disc tests
Disc test are the upgraded version of the filled cups and well method, where filter paper disc loaded
with test material are used. Standard suspension of microorganism are prepared and inoculated on
appropriate agar plate to form lawn. Filter paper containing known concentration of antimicrobial
agents are placed on dried lawn and incubated at 35 °C for 18 hours. Confluent growths of bacteria
are seen after incubation. Zone of inhibition if occurred surrounding disc is then measured and
compared with standard. Particular microbial susceptibility can be checked by placing different
standard disc in single plate.
8.1.2.Dilution test
Different ranges of concentration of antimicrobial under test are prepared in a suitable broth
medium. Log phase culture is added to each tube in such a way that the final density becomes 5 X
105 CFU/mL and incubated in 35 °C for 18 hours. Lowest concentration at which clear tube is
observed is considered as MIC of that compound.
To prove the correctness of experiment various controls are put with the test series for eg. Sterility
control, growth control and compound having known MIC.
Dilution test can be carried out on a series of agar plate previously mixed with an antimicrobial agent
in a serially dilution, known bacterial suspension is then inoculated and growth is observed after
appropriate incubation.
8.1.3.e-test
This test is the most convenient, more advanced and presently acceptable method of determining
MIC. This is performed similarly like disc test but in this test nylon strip gradually loaded with
antimicrobial agent is used instead of filter paper impregnated with antimicrobials.
Nylon strip marked with series of lines and figures which indicates MIC value at different
concentration. Nylon strip was placed over the agar plate freshly prepared with microbial lawn and
incubated. After incubation MIC is determined by where the pear shaped inhibition zone crosses the
strip.
8.2. Test for bactericidal activity
Apart from MIC, novel antimicrobial agents are also required to test minimum bactericidal
concentration (MBC). It is the lowest concentration of agent to kill ≥99.9% of bacterial under test
conditions.
0.1 mL (out of 100 mL of total) volume of clear tube from MIC is spread onto agar plate to determine
the MBC. Plate is incubated at 35 °C for 18 hours; after incubation, colonies are growing on each
plate are counted. More than 50 colonies at first lowest concentration is considered as MBC of that
compound. As we have discussed in MIC that the bacterium inoculum is about 5 X 105 CFU/mL, at
99.9% (3 log) killing may allow to survive more than 50 colonies on subsequent plates.
8.3. Fungistatic and fungicidal activity
Fungi can be considered as more prominent human pathogen. Determination of fungistatic and
fungicidal activity, same methods denoted previous are used but media, inoculation condition and
inoculum density are different. To evaluate fungistatic or fungicidal activity RPMI 1640
supplemented with 2% dextrose is used as a medium and 104 CFU/mL cell density is suitable.
8.4. Synergistic antimicrobial combinations.
To evaluate the potential of synergistic activity of two or more antimicrobial agents, organism are
exposed to various concentration of test compound separately and in combination. Disc diffusion
method or kinetic kill curve assay will be performed.
8.5. Kinetic kill curves
Four different test tubes are prepared namely single concentration of each drug, same
concentration in combination, and control tubes have no antimicrobial agent. All tubes are
incubated and time to time viable counts are performed to evaluate killing efficacy. The results are
plotted on semialgorithmic paper. In this test >100 fold increase in killing efficiency defined as
synergistic activity.