MICROBIOLOGY II

TO: Assistant Professor HADAYAT RASOOL

COLLEGE OF PHARMACY

Pharm. D 4th semester (evening) SESSION 2007-2012

ANTIBIOTICS---introduction CEPHALOSPORIN
Introduction Fermentation

EXTRACTION

LYOPHILIZATION
PACKAGING AND LABELLING

INTRODUCTION

DEFINITION:

Antibiotics are substances that kill or inhibit growth of microorganisms but causes little or no damage to the host. OR Antibiotics are antimicrobial agents produced naturally by other microbes (usually fungi or bacteria).

Bacteria cause infections in 1. Humans 2. Other higher organisms

Point to be noted
How other plants, animals and fungi protect themselves?

The first antibiotic was discovered in 1896 by Ernest Duchesne and "rediscovered" by Alexander Flemming in 1928 from the filamentous fungus Penicilium notatum.

Ernest Duchesne

A French physician
Discovery: thirty-two years before Alexander Fleming discovery Entered The Military Health Service School of Lyon in 1894

Ernest Duchesne

Thesis:
Contribution to the study of vital competition in micro-organisms
antagonism between molds and microbes

Submitted in: In 1897 get his doctorate degree Important: First study of therapeutic capabilities of molds due to their anti-microbial activity.

Ernest Duchesne Observe: Arab stable boys at the army hospital kept their saddles in a dark and damp room

to encourage mold to grow on them

Ernest Duchesne Asked , why ?

To heal the saddle sores on the horses

Ernest Duchesne Achievement no. 1: Prepared a solution of the mold Injected it into a series of diseased guinea pigs. All recovered

Ernest Duchesne Achievement no. 2: Made interaction between bacteria (Escherichia coli) and fungus (Penicillium glaucum) Fungus eliminated bacteria from medium

Bad Luck:
1.

Ernest Duchesne

He was 23 and unknown

2.

His army service after getting his degree prevented him from doing any further work
Failed to report a connection between the fungus and a substance that had antibacterial properties died at the age of 37

3.

4.

Alexander Flemming
in 1928 Accidentally Discovered penicillin from the filamentous fungus Penicilium notatum.

Penicillin was not purified until the 1940s by Florey and Chain

just in time to be used at the end of the second world war.

Alexander Flemming and Florey and Chain

Share Nobel prize In 1945

INTRODUCTION, CLASSIFICATION, PROPERTIES AND PRODUCTION

DEFINITION: Any of various broad-spectrum B-Lactam antibiotics, closely related to the penicillins, that were originally derived from the fungus Cephalosporium acremonium.

HISTORY Cephalosporin compounds were first isolated from cultures of

Cephalosporium acremonium
in 1948 by Italian scientist Giuseppe Brotzu

HISTORY He noticed that these cultures produced substances that were effective against Salmonella typhi, the cause of typhoid fever,

which had beta-lactamase.

ACTION:

Inhibition of cell wall synthesis

Inactive Against:
1. Enterococci spp. Intrinsic factor 2. MRSA additional substances 3. Legionella spp. Intrinsic factor 4. Mycoplasma spp. No cell wall 5. Chlamydia spp. Intrinsic factor

MRSA= Methicillin-resistant Staphylococcus aureus

Common Use:
In surgical procedures

to reduce the risk of post operative infections

4 GENERATIONS

Based Upon: The spectrum of antimicrobial activity Grouped w.r.t increased gram ive and decreased gram +ive activity, as

Examples: Spectrum:
Cefazolin Cephalexin

First Generation

Use:

Most gram positive cocci (Strep, S. aureus) E. coli Proteus Klebsiella.

S. aureus infection, surgical prophylaxis.

Second Generation

Examples:

Cefoxitin Cefuroxime Cefaclor Cefprozil

Spectrum:
Mainly effective against G- bacteria Modest activity against G+ bacteria

Use: primarily for

Upper Respiratory Tract Infections Lower Respiratory Tract Infections

Third Generation

Examples:
Ceftriaxone Cefotaxime

Spectrum:
enhanced G- activity

Use:
Meningitis highly resistant and multi drug resistant strep

pneumo along with vancomycin.

Fourth Generation

Examples:
Cefepime

Spectrum:
Active against Strep, Staph aerobic gram negatives

INTRODUCTION

Important To Learn:

Isolation Characteristics
Colonial characters
Microscopic characters Without staining With staining

SAMPLE

Cephalosporium acremonium Most common in

1. Soil 2. Plant debris 3. Rotting mushrooms

Found in

1. 2. 3. 4.

Europe Asia Egypt North and Central America

Colonial:
Colour: pink Moist Gradually become more hyphal
Narrow hyphae Phialides Conidia
Narrow Cylindrical

Microscopic:

Arthrospores

INDUSTRIAL SCALE

STEPS:
1. 2. 3. 4.

Fermentation Extraction Lyophilization Packaging and Labelling

METHOD OF FERMENTATION

DEFINITION
Drug producing microbe is grown and cultivated in appropriate culture vessels or in large fermentors on a suitable medium in which respective drug is accumulated.

STEPS 1. Isolation of microbe 2. Characterization of microbe 3. Purification of culture 4. Preparation of inoculum 5. Quality control 6. Lab scale fermentation 7. Quality control 8. Seed cultivation 9. Quality control

SAMPLE Cephalosporium acremonium Most common in 1. Soil 2. Plant debris 3. Rotting mushrooms Found in 1. Europe 2. Asia 3. Egypt 4. North and Central America

SABOURAUD'S AGAR 1. Glucose-------------40g

2. 3. 4.

Filter sterilization

Peptone-------------10g Agar-----------------15g H2O (dist)-q.s1000ml

Autoclave

sterilization

SAMPLE STERILIZED MEDIUM CONDITIONS 1. TEMP= 25-28oC 2. 7 Days 3. Humidity

STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum Quality control Lab scale fermentation Quality control Seed cultivation Quality control

Colonial:
Colour: pink Moist Gradually become more hyphal
Narrow hyphae Phialides Conidia
Narrow Cylindrical

Microscopic:

Arthrospores

STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum Quality control Lab scale fermentation Quality control Seed cultivation Quality control

Malt extract agar (Blakeslee's formula)

1. 2. 3. 4. 5.

Malt extract...............20.0 g Glucose.....................20.0 g Peptone......................1.0 g Agar........................20.0 g Distilled water..............1.0 L

Autoclave at 121C for 15 minutes Add glucose separately sterilized

MacCorteny bottle

Other media used for cephalosporins are

1. Potato dextrose agar 2. Potato carrot agar

Conditions:
1. TEMP= 25-28oC 2. 7 Days 3. Humidity

Repeated growth on these media leads to purified culture.

STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum Quality control Lab scale fermentation Quality control Seed cultivation Quality control

Colonial:
Colour: pink Moist Gradually become more hyphal
Narrow hyphae Phialides Conidia
Narrow Cylindrical

Microscopic:

Arthrospores

PREPARATION OF INOCULUM
5ml water + glass beeds Pour in MacCorteny Bottle Shake Pour out Inoculum

STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum Quality control Lab scale fermentation Quality control Seed cultivation Quality control

Check No. of spores in inoculum

STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum Quality control Lab scale fermentation Quality control Seed cultivation Quality control

"SEED STAGE" MEDIUM (per flask)

1.

2. 3. 4. 5. 6. 7. 8. 9. 10.

Corn steep liquor 0.5% nitrogen Lactose ...46 g/l Glucose .2 g/l Methionine ..2.3 g/l Phenyl acetyl ethanbolamine .1.5 g/l Calcium carbonate 16 g/l Urea 0.8 g/l Ammonium sulphate ..3.4 g/l Maize oil .6 drops Salts solution 1 ml/l Distilled water ..q.s.1000L

 Salt solution: 1. Fe (NH 4 ) 2 (SO 4 ) 2 .6H 2 O ......15 g/100 ml 2. Mn SO 4 .4H 2 O ........................3 g/100 ml 3. Zn SO 4 .7H 2 O .........................3 g/100 ml 4. Cu SO 4 .5H 2 O ......................0.8 g/100 ml Conditions: 1. 25 C 2. 250 rpm 3. ph 6.5 4. 72 hours

STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum Quality control Lab scale fermentation Quality control Seed cultivation Quality control

1.

Sampling After every 8 hours Contents examination

Morphological properties of biomass Average no of spores Occurrence of pellet

2. 3.

pH Nutrients concentration

Qualitative Test Take a strip Dip in 0.2% bromophenol 2% of sample dried

Loopful of beta-lactam resistant strain Beta-lactamase Denaturation of lactam ring Blue to yellow

QUANTITATIVE TEST Disc soaked in cephalosporin sample and standard solution Placed on Mueller Hinton Agar Diameter of inhibition zone of resistant strain calculation

1. 2. 3. 4.

Mueller Hinton Agar 30.0% beef infusion 1.75% casein hydrolysate 0.15% starch 1.7% agar

pH adjusted to neutral at 25 C.

STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum Quality control Lab scale fermentation Quality control Seed cultivation Quality control

In huge fermentors of capacity 10L to 1000L

STEPS Isolation of microbe Characterization of microbe Purification of culture Preparation of inoculum Quality control Lab scale fermentation Quality control Seed cultivation Quality control

1.

Sampling After every 8 hours Contents examination

Morphological properties of biomass Average no of spores Occurrence of pellet

2. 3.

pH Nutrients concentration

Qualitative Test Take a strip Dip in 0.2% bromophenol 2% of sample dried

Loopful of beta-lactam resistant strain Beta-lactamase Denaturation of lactam ring Blue to yellow

QUANTITATIVE TEST Disc soaked in cephalosporin sample and standard solution Placed on Mueller Hinton Agar Diameter of inhibition zone of resistant strain calculation

1. 2. 3. 4.

Mueller Hinton Agar 30.0% beef infusion 1.75% casein hydrolysate 0.15% starch 1.7% agar

pH adjusted to neutral at 25 C.