CEPHALOSPORIN

Government College University Faisalabad

COLLEGE OF PHARMACY

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

MICROBIOLOGY II

TO:Assistant Professor HADAYAT RASOOL

CONTENTS

ANTIBIOTICS---introduction

CEPHALOSPORIN Introduction Fermentation

EXTRACTION

LYOPHILIZATION

PACKAGING AND LABELLING

ANTIBIOTICS

INTRODUCTION

ANTIBIOTICSDEFINITION:

Antibiotics are substances that kill or inhibit growth of

microorganisms but causes little or no damage to the host.

ORAntibiotics are antimicrobial agents produced naturally by

other microbes (usually fungi or bacteria).

HOW ANTIBIOTICS COME?

Bacteria cause infections in1. Humans2. Other higher organisms

Point to be noted

How other plants, animals and fungi protect themselves?

HISTORY

The first antibiotic was discovered in 1896 by Ernest Duchesne

and "rediscovered" by

Alexander Flemming in 1928 from the filamentous fungus

Penicilium notatum.

HISTORY

Ernest Duchesne

A French physician

Discovery: thirty-two years before

Alexander Fleming discovery

Entered “The Military Health Service School of Lyon” in 1894

HISTORY

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.

HISTORY

Ernest DuchesneObserve:

Arab stable boys

at the army hospital

kept their saddles in a dark and damp room

to encourage mold to grow on them

HISTORY

Ernest Duchesne

Asked , why ?

To heal the saddle sores on

the horses

HISTORY

Ernest DuchesneAchievement no. 1:

Prepared a solution of the mold

Injected it into a series of diseased guinea pigs.

All recovered

HISTORY

Ernest DuchesneAchievement no. 2:

Made interaction between

bacteria (Escherichia coli) and fungus (Penicillium glaucum)

Fungus eliminated bacteria from medium

HISTORY

Ernest DuchesneBad Luck:

1. He was 23 and unknown

2. His army service after getting his degree prevented him from doing any further work

3. Failed to report a connection between the fungus and a substance that had antibacterial properties

4. died at the age of 37

HISTORY

Alexander Flemming

in 1928 Accidentally Discovered penicillin

from the filamentous fungus

Penicilium notatum.

HISTORY

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.

HISTORY

Alexander Flemming and Florey and Chain

Share Nobel prize

In 1945

CEPHALOSPORIN

INTRODUCTION, CLASSIFICATION, PROPERTIES AND PRODUCTION

INTRODUCTION

INTRODUCTION

DEFINITION:

Any of various broad-spectrum B-Lactam antibiotics, closely

related to the penicillins, that were originally derived from the

fungus Cephalosporium acremonium.

INTRODUCTION

HISTORYCephalosporin compounds were

first isolated from cultures of

Cephalosporium acremonium

in 1948 by Italian scientist Giuseppe Brotzu

INTRODUCTION

HISTORY

He noticed that these cultures produced substances that were effective against

Salmonella typhi, the cause of typhoid fever,

which had beta-lactamase.

INTRODUCTION

ACTION:

Inhibition of cell wall synthesis

INTRODUCTION

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

INTRODUCTION

Common Use:

In surgical procedures

to reduce the risk of post operative infections

CLASSIFICATION4 GENERATIONS

CLASSIFICATION

Based Upon:

The spectrum of antimicrobial activity

Grouped w.r.t increased gram –ive and decreased gram +ive

activity,as

CLASSIFICATION

First Generation Examples:

Cefazolin Cephalexin

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

Use: S. aureus infection, surgical prophylaxis.

CLASSIFICATION

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

CLASSIFICATION

Third Generation Examples:

Ceftriaxone Cefotaxime

Spectrum: enhanced G- activity

Use: Meningitis highly resistant and multi drug resistant

strep pneumo along with vancomycin.

CLASSIFICATION

Fourth GenerationExamples:

CefepimeSpectrum:

Active against Strep, Staph aerobic gram negatives

Cephalosporium acremoniumINTRODUCTION

Cephalosporium acremonium

Important To Learn:

IsolationCharacteristics

Colonial characters Microscopic characters

▪ Without staining▪ With staining

ISOLATION

SAMPLECephalosporium acremonium

Most common in 1. Soil2. Plant debris3. Rotting mushrooms

Found in1. Europe2. Asia3. Egypt4. North and Central America

CHARACTERISTICS

Colonial: Colour: pink Moist Gradually become more hyphal

Microscopic: Narrow hyphae Phialides Conidia

▪ Narrow▪ Cylindrical

Arthrospores

PRODUCTIONINDUSTRIAL SCALE

PRODUCTION

STEPS:

1. Fermentation2. Extraction3. Lyophilization4. Packaging and Labelling

SHAKE FLASK CULTURE METHOD

METHOD OF FERMENTATION

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.

FERMENTATION

STEPS1. Isolation of microbe2. Characterization of microbe3. Purification of culture4. Preparation of inoculum5. Quality control6. Lab scale fermentation7. Quality control8. Seed cultivation9. Quality control

ISOLATION

SAMPLECephalosporium acremonium

Most common in 1. Soil2. Plant debris3. Rotting mushroomsFound in4. Europe5. Asia6. Egypt7. North and Central America

ISOLATION

SABOURAUD'S AGAR1. Glucose-------------

40g

2. Peptone-------------10g

3. Agar-----------------15g

4. H2O (dist)-q.s—1000ml

Filter sterilization

Autoclave

sterilization

ISOLATION

SAMPLE

STERILIZED MEDIUM

CONDITIONS1. TEMP= 25-28oC2. 7 Days3. Humidity

FERMENTATION

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

CHARACTERISTICS

Colonial: Colour: pink Moist Gradually become more hyphal

Microscopic: Narrow hyphae Phialides Conidia

▪ Narrow▪ Cylindrical

Arthrospores

FERMENTATION

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

PURIFICATION

Malt extract agar (Blakeslee's formula)

1. Malt extract...............20.0 g2. Glucose.....................20.0 g3. Peptone......................1.0 g4. Agar........................20.0 g5. Distilled water..............1.0 L

Autoclave at 121C for 15 minutes Add glucose separately sterilized

MacCorteny bottle

PURIFICATION

Other media used for cephalosporins are1. Potato dextrose agar2. Potato carrot agar

Conditions:1. TEMP= 25-28oC

2. 7 Days3. Humidity

Repeated growth on these media leads to purified culture.

FERMENTATION

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

CHARACTERISTICS

Colonial: Colour: pink Moist Gradually become more hyphal

Microscopic: Narrow hyphae Phialides Conidia

▪ Narrow▪ Cylindrical

Arthrospores

PREPARATION OF INOCULUM

5ml water + glass beeds

Pour in

MacCorteny Bottle

ShakePour out

Inoculum

FERMENTATION

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

QUALITY CONTROL

Check No. of spores in inoculum

FERMENTATION

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

SHAKE FLASK CULTIVATION

"SEED STAGE" MEDIUM(per flask)

1. Corn steep liquor 0.5% nitrogen Lactose ……...……………46 g/l

2. Glucose …………………………………….2 g/l 3. Methionine ……………………………..2.3 g/l 4. Phenyl acetyl ethanbolamine …….1.5 g/l 5. Calcium carbonate ……………………16 g/l 6. Urea ………………………………………0.8 g/l 7. Ammonium sulphate ………………..3.4 g/l 8. Maize oil ……………………………….6 drops 9. Salts solution…………………………… 1 ml/l10. Distilled water ………..q.s…………….1000L

SHAKE FLASK CULTIVATION

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° C2. 250 rpm3. ph 6.54. 72 hours

FERMENTATION

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

Quality control

Sampling After every 8 hours1. Contents examination

Morphological properties of biomass Average no of spores Occurrence of pellet

2. pH 3. Nutrients concentration

Production Analysis

Qualitative Test

Take a strip

Dip in 0.2% bromophenol

2% of sample

dried

Production Analysis

Loopful of beta-lactam resistant strain

Beta-lactamase

Denaturation of lactam ring

Blue to yellow

PRODUCTION ANALYSIS

QUANTITATIVE TESTDisc soaked in cephalosporin sample

and standard solution

Placed on Mueller Hinton Agar

Diameter of inhibition zone of resistant strain

calculation

PRODUCTION ANALYSIS

Mueller Hinton Agar1. 30.0% beef infusion2. 1.75% casein hydrolysate3. 0.15% starch4. 1.7% agar

pH adjusted to neutral at 25 °C.

FERMENTATION

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 cultivation

In huge fermentors of capacity 10L to 1000L

FERMENTATION

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

Quality control

Sampling After every 8 hours1. Contents examination

Morphological properties of biomass Average no of spores Occurrence of pellet

2. pH 3. Nutrients concentration

Production Analysis

Qualitative Test

Take a strip

Dip in 0.2% bromophenol

2% of sample

dried

Production Analysis

Loopful of beta-lactam resistant strain

Beta-lactamase

Denaturation of lactam ring

Blue to yellow

PRODUCTION ANALYSIS

QUANTITATIVE TESTDisc soaked in cephalosporin sample

and standard solution

Placed on Mueller Hinton Agar

Diameter of inhibition zone of resistant strain

calculation

PRODUCTION ANALYSIS

Mueller Hinton Agar1. 30.0% beef infusion2. 1.75% casein hydrolysate3. 0.15% starch4. 1.7% agar

pH adjusted to neutral at 25 °C.