COMMON INFECTIOUS MICRO-ORGANISMS

CLASSIFICATION OF ANTI-MICROBIALS
 SIGNIFICANCE OF SCREENING OF
ANTI-BACTERIALS & ANTI-FUNGALS
Need for effective anti-bacterial & anti-fungal
drugs due to emergence of AIDS & ARC that
are associated with opportunistic infections
Search for novel, safer, more potent &
effective antimicrobials from various sources-
terrestrial, plants, marine flora & fauna, &
synthetic
SCREENING OF ANTI-BACTERIALS
& ANTI-FUNGALS
2 PHASES:
In vitro:-
Organism under laboratory conditions
In vivo:-
Animal models of infections
e.g.. Rodents, lagomorphs, non-human primates, etc.
 IN VITRO ANTI-MICROBIAL
SCREENING
AIM:
To find out the minimum
inhibitory concentration (MIC)
To determine susceptibilities of non-target bacterial
flora
MIC Min. quantity of a compound required to
inhibit (static effect) or kill (cidal effect) the growth
of an organism
 FACTORS CONTRIBUTING TO
SENSITVITY OF IN VITRO METHODS
 FACTORS CONTRIBUTING TO SENSITVITY

SOLUBILITY:
Solubility in an aqueous solution or in a
particular solvent (DMF, DMSO, PEG, etc.)

Important factor for getting a correct picture of

activities & also to achieve sensitivity of test
product to temperature, pH, light, etc.
 FACTORS CONTRIBUTING TO SENSITVITY

MEDIA USED & pH:

Choice depends on type of organism & nature of the
test compound
ORGANISM TYPE MEDIA USED pH

Nutrient agar/ broth 7.4

Bacteria
Muller Hinton agar 7.4

Sabourauds dextrose 5.5 - 6

agar/ broth

Fungi Yeast nitrogen base 5.6

Synthetic casitone 6.6

liquid media
 FACTORS CONTRIBUTING TO SENSITVITY

PREPARATION OF INOCULA:
Fresh inocula
CFU of test inocula for testing is 105 cells/ ml
 FACTORS CONTRIBUTING TO SENSITVITY

TEMPERATURE & PERIOD OF

INCUBATION:

ORGANISM INCUBATION TEMPERATURE

TYPE PERIOD OF INCUBATION

Bacteria 18 24 hrs 37C

Yeast 24 48 hrs 28C

Filamentous 72 95 hrs 28C

fungi
 IN VITRO SCREENING
IN VITRO SCREENING METHODS

Poison food technique (PFT)

Disc diffusion method

Tube dilution (broth dilution) method

Microtitre technique
 IN VITRO SCREENING

POISON FOOD TECHNIQUE

PURPOSE & RATIONALE:
A qualitative method
Can be made quantitative with the help of dose-
response curves (DRC)
Applicable to testing of bacteria as well as
filamentous fungi
Useful for filamentous fungi because of their
rapid radial growth
Sensitivity of several bacterial cultures can be
tested in a single plate
 IN VITRO SCREENING
PROCEDURE (FOR BACTERIA):
 IN VITRO SCREENING
PROCEDURE (FOR FUNGI):
 IN VITRO SCREENING

EVALUATION:

Bacteria: Inhibition of growth

Fungi: Inhibition of linear growth

 IN VITRO
SCREENING

DISC DIFFUSION METHOD

PROCEDURE:
IN VITRO SCREENING
 IN VITRO SCREENING

EVALUATION:
Diameter of zone of
inhibition (in mm)
 IN VITRO SCREENING
TUBE DILUTION (BROTH DILUTION) METHOD
(two-fold serial dilution technique)

Two-fold serial dilution of drug in a series

PROCEDURE:
1.Stock solutions: 10 mg/ml (natural products)
1 mg/ ml (synthetic products)
Solvents: DMSO
2.Media:
Bacteria- Nutrient broth
Fungi- Sabourauds broth
 IN VITRO SCREENING

3. Dilutions:
First dilution 0.2 ml test solution + 1.8 ml seeded broth
Second dilution 1 ml above mixture + 1 ml seeded broth
10 12 such dilutions
4. Control:
Set of tubes containing only seeded broth
+ suitable solvent
5. Incubation:
Bacteria 37C for 24 hrs
Yeasts 28C for 24 48 hrs
Mycelial Fungi 28C for 72 96 hrs
 IN VITRO SCREENING

EVALUATION:
MICs (in g/ml ) The last tube with no apparent
growth of micro-organisms represents the MIC of test
compound
 IN VITRO SCREENING

MICROTITRE TECHNIQUE
PURPOSE & RATIONALE:
Modern, sensitive, rapid, automated, economical, quantitative
compared to tube dilution method
PROCEDURE:
Micro broth (270 l per well) with drug dilutions (30l) are
made serially with a multichannel Appendorf pipette in
microtitre plate with 96 wells
Test inoculum is added (20 ml) in each well separately
Appropriate controls
 IN VITRO SCREENING
EVALUATION:
Automated ELISA Reader based on optical
density (OD at 492 nm, matrix 0.2-2)
 SCREENING MODELS FOR LEPROSY

SCREENING MODELS FOR

LEPROSY
Delay in widespread use of anti-leprotic drugs
Inability to cultivate M. leprae on laboratory medium
Inability to produce experimental model by injecting
the organism
First truly useful and reproducible animal model of
M. leprae infection
MOUSE FOOT PAD MODEL
 SCREENING MODELS FOR LEPROSY

MOUSE FOOT PAD MODEL

PURPOSE & RATIONALE:
Demonstrates whether the drug has partial or complete
activity against M. leprae
Determination of the conc. Of drugs required to achieve
the desired levels
Demonstration of identity of organism, viability or non-
viability of organisms obtained from patients, drug
resistance and the degree of resistance
Other possible models:
mouse ear model
Ear/ foot pad of golden hamster
Rat foot pad
 SCREENING MODELS FOR LEPROSY

PROCEDURE:
 SCREENING MODELS FOR LEPROSY

EVALUATION:
CONTINUOUS METHOD: active drugs are those that inhibit
multiplication of the organisms

KINETIC METHOD:
The activity of the drug is assessed in terms of the
growth delay number of days required for
multiplication to 106 Acid-fast bacilli per foot among the
treated mice compared to that among the untreated controls.
Purely bacteriostatic drug Inhibits multiplication of M.
leprae only as long as the drug is administered
Purely bactericidal drug A growth delay signicantly
longer than the period of drug administration, i.e. failure of
bacterial multiplication to resume immediately following
cessation of drug-administration
Clinically important fungi may be classified into four
main types on the basis of their morphological and
other characteristics:
yeasts (e.g. Cryptococcus neoformans)
yeast-like fungi that produce a structure
resembling a mycelium (e.g. Candida albicans)
filamentous fungi with a true mycelium (e.g.
Aspergillus fumigatus)
'dimorphic' fungi that, depending on nutritional
constraints, may grow as either yeasts or
filamentous fungi (e.g. Histoplasma capsulatum).
Superficial fungal infections can be classified as:
dermatomycoses ( include infections of the
skin, hair and nails )
Candidiasis(the commonest systemic fungal
disease )
ANTIBIOTICS
Polyenes: Amphotericin B, Nystatin, Natamycin
Heterocyclic Benzofurans: Griseofulvin
ANTIMETABOLITE
Flucytosine
AZOLES
Ketoconazole
Fluconazole
Itraconazole
Miconazole
ALLYLAMINE
terbinafine
 EXPERIMENTAL RING WORM
INFECTION/ TRICHOPHYTOSIS MODEL
Animals Used : guinea pigs of either
sex
Weight: 350 g
PURPOSE AND RATIONALE
The guinea pig trichophytosis model
has been used to evaluate
antimycotic compounds
 PROCEDURE

Note: Ringworm infection is noticed between 4 &6 days after

inoculation
EVALUATION:
Infection grade and culture recovery from skin scrapings & hair
at periodic intervals are the 2 major parameters for evaluation
 CORNEAL INFECTION
Animals Used : rabbits
PROCEDURE:

EVALUATION:
Evaluation is done on the basis of :
Infection grading
culture recovery from the site of infection at periodic time
intervals
 MODEL OF VULVO-VAGINAL
CANDIDIASIS IN MOUSE
Animals used : Swiss albino female mice
PROCEDURE

EVALUATION:
culture recovery from vaginal swabs
CFUs in vaginal tissue
 MODEL OF SYSTEMIC CANDIDIASIS/
ASPERGILLOSIS IN MOUSE

PROCEDURE

EVALUATION:
C.F.U.s are determined using kidney tissue homogenatesof the
surviving mice
 MODEL OF PULMONARY
CRYPTOCOCCOSIS IN MOUSE
Fungal sp. Used: C.Neoformans
PROCEDURE
Untreated mice are inoculated with 25l of C.Neoformans
suspensions intranasally
Mice are observed for 7 days and cfu determained from lund
tissue as in the previous model
 MODEL OF BRONCHOPULMONARY
ASPERGILLOSIS
Animals Used : Rhesus monkey
PROCEDURE

EVALUATION
Periodic culture recovery from lung aspirates
X- ray
Hematology & Histopathology
 REFERENCES
Zafar K. Khan, Shukla, P.K., Division of Mycology, Central Drug
Research Institute, Lucknow
Advances in Pharmacology & Chemotherapy, 1969, Vol 7: 212-216
Drake, T. A., & M. A. Sande. 1983. Studies of the chemotherapy of
endocarditis: correlation of in vitro, animal model, & clinical studies. Rev.
Infect. Dis. 5:S345-S354.
Zak, O., W. Tosch, & M. A. Sande. 1985. Correlation of Anti-bacterial
activities of antibiotics in vitro & in animal models of infection. J.
Antimicrob. Chemother. 15 (Suppl. A): 273-282.