You are on page 1of 27

- Understanding the physiology and genetics of microorganisms are important to produce therapeutic agents

- Knowledge of mechanisms whereby microorganisms are able to resist antibiotics, colonize medical devices are essential in the production of new drugs and in healthcare practices

Chapter 2: Fundamental features of microbiology

o

MICROORGANISMS

  • - microscopic, living, single-celled organisms

-more versatile than mammals in breaking down foods.

Differ in:

1.Shape

2.Size

3.Genetic Characteristics 4. Metabolic Characteristics

- Major groups:

o

Bacteria

o

Fungi

o

Protozoa

o

Viruses

CLASSIFICATION OF MICROORGANISMS:

o

Viruses

  • - do not have cellular structure

  • - Composition:

Chapter 1: Introduction to Pharmaceutical Microbiology

  • - A modern medicine must be effective, safe, and of good quality

  • - These medicines consists of active

ingredients, stable, and safe during storage

  • - Analytical Chemists and Pharmacists

Pharmaceutical Microbiology

  • - Foundation

  • - Encompasses the subject of sterilization and preservation against microbial spoilage

  • - Pharmacist must be responsible for the safe and hygienic manufacturing of medicines

Antibiotics

  • - Major importance in pharmacy

  • - Naturally occurring substance that would inhibit or kill microorganisms

  • - Microbial metabolite

  • - Synthetic agents that are normally used systematically to treat infection

  • - Antibiotic production began with the

discovery of penicillin in the 1940s

  • - Attack and kill bacteria without harm to the host

Ribotyping

  • - Genetic technique used to identify cross- infection, reduce transmission and optimize

management of hospital-acquired infections

-No nucleic acid -atypical form of mammalian protein

-can interact w/ normal protein molecule and cause it to undergo conformational change and make it into a prion and ceases normal function

-responsible for transmissible spongiform encephalopathies

Ex. Creutzfeldt-jakob disease Bovine spongiform encephalopathy

-simplest and most recognized agents of infectious disese

-extreme resistance to conventionall sterilizing agents (steam,gamma radiation,disinfectant,etc.)

o

Nucleic acid surrounded by proteins

o

Some posses lipid envelope associated w/ glycoprotein

o

Absent:

-recognizable chromosomes -cytoplasm -cell membranes

-incapable of independent replication

  • - intracellular parasites

-reproduced using metabolic capabilities of host cell

-smaller than bacteria

  • - Variation in:

PROKARYOTIC MICROORGANISMS:

-

No true nucleus

-

Usually single chromosomes

-

Haploid

-

Asexual reproduction Examples : Bacteria, Archea

 

o

o

Archea

-no pharmaceutical importance -capable of living in extreme environment -exhibit specialized modes of metabolism

o

Bacteria

-unicellular -posses prokaryotic properties

o

o

Shape (helical, linear, or spherical)

o

Size (20-400nm)

o

Nucleic acid composition (single or double-stranded,linear or circular RNA or DNA)

-Viewed using electron microscope.

Viroids (Virusoids)

-simplier than viruses -infectious particles

-single stranded RNA w/o associated proteins

Ex. Plant phatogens

Prions

-infectious agents

  • - contaminate or cause spoilage of pharmaceutical products

  • - obtain energy by decomposition

of animal and vegetable materials

  • - described as parasites or pathogens

Rickettsia and Chlamydia

  • - obligate intracellular parasites

  • - incapable of growing outside host cell

EUKARYOTIC MICROORGANISMS:

  • - true cell nucleus

-

chromosomes

separated

from

the

cytoplasm by nuclear membrane

  • - diploid

  • - sexual reproduction

Examples: fungi,protozoa,algae,etc.

o

Fungi

  • - structurally more complex and varied appearance than bacteria

  • - non-photosynthizing

  • - some fungi exhibit unicellular(yeast like)

or mycelial (mould-like) upon cultivation

  • - Most are saprophytes w/ few pathogenic potential

  • - ability to make spores that are resistant in drying = pharmaceutical contaminants

-exhibit great diversity in form, habitat, metabolism, pathogenicity

-Bacteria of interest in medicine and pharmacy: Eubacteria

-many bacteria would be described as facultative anaerobes or microaerophils

-most bacteria important in medicine and pharmacy :

 

posses cell wall

grow well at temperatures

between ambient and human body temperature exhibit wide variation in

Examples:

requirement for oxygen Strict aerobes = require atmospheric oxygen Strict anaerobes = oxygen is toxic

Eubacteria

  • - Bacteria of interest in medicine and pharmacy

  • - types:

    • 1. Bacillus- rod shaped

    • 2. Cocci-spherical

    • 3. Curved or spiral cell approximately 0.5 to 5 mm

- Divided into two groups acc. to Christian Gram’s 1884 staining procedure.

  • 1. Gram positive

  • 2. Gram negative

Saprophytes

Microorganisms are more versatile than humans in breaking down food, Many can use alternative methods in breaking down food depending on the environment, and some can obtain energy from carbohydrates, digestion of proteins and other non- carbohydrate materials.

o

Chemoheterotrophs

  • - obtain carbon from nitrogen

  • - gets energy from breaking down organic compounds

  • - organisms of interest in pharmacy in medicine

o

Catabolic Reactions

  • - energy is liberated by digestion of food materials.

o

Anabolic Reactions

  • - use liberated energy to make complex cellular polymers,protein,carbohydrates, and nucleic acids.

o

o

Oxidation

  • - removal or loss of electrons

- when food materials are oxidized energy are released.

  • - does not invariably involve oxygen

  • - when oxidizing molecules accept

electron the other molecule is

reduced; reduction and oxidation is linked and called Redox reaction.

o

Redox Potential

  • - Fungus: covers both terms yeast and moulds

Yeast

 

-

normally unicellular

-

divide by budding

-

larger than bacteria

-

divide by binary fission or budding

Moulds

 

-

imprecise term to describe fungi

that doesn’t form fruiting bodies to

naked eyes

-

grow by extension or branching of

 

hyphae

 

-

excluding toadstool and

mushrooms

-

1 to 50 um wide

-

Differentiated for special

 

function

 

consist of tangled mess or filaments of thread

-

Protozoa

  • - predominantly unicellular

  • - mostly animals than plants

  • - free living motile organisms

  • - occur in water and soil

  • - not normally found as contaminants

-potential to cause disease

Ex. Organism responsible for malaria and ameobic dysentery

*for further comparison of eukaryotes and prokaryotes check Table 2.1 page 11

  • - indicates whether oxidation or reduction will prevail

  • - Anaerobic organism = low redox

potential Aerobes = high redox potential

NAMING OF MICROORGANISMS:

- known by two names genus and species

- written in italic or underlined

MICROBIAL METABOLISM:

Some pathogenic bacteria can ferment:

different sugar, acids, alcohols, acetone, butane, etc.

o

Fundamental Principle of Antibiotic Action

  • - drug must exploit a difference in metabolism between organism to be

killed and the human host.

  • - w/o difference it will be very toxic to the patient.

o

Primary metabolites

  • - metabolic products that arise during period when microbial

culture is growing.

Ex. ethanol, organic acids

o

Secondary metabolites

  • - diverse

  • - have commercial or therapeutic importance

  • - produced after the cell multiplication has stopped.

Ex. Antibiotic,enzymes,toxins, carbohydrates

MICROBIAL CULTIVATION CULTURE MEDIA

  • - Some microorganisms have different sugar fermentation patterns

  • - Sugars in culture media are usually used for identification purposes.

  • - NaCl may be incorporated to adjust osmotic pressure.

o

Oxidizing Agents

  • - molecules that can accept electrons

o

Glycolysis

  • - breakdown of glucose to release energy

  • - metabolic pathway used by pathogenic bacteria and mammals

  • - conversion of glucose to series of reactions to pyruvic acid, oxygen is

not required.

- undertaken by both aerobic and anaerobic

  • - release small amount of energy stored in sugar molecule.

o

Aerobic respiration

- Used by mammals to release more energy in sugarcompared to glycolysis.

- Oxygen in end of sequence of respiratory reaction .

o

Fermentation

  • - an anaerobic process

  • - alternative to respiration

  • - means of releasing more energy from sugar

  • - a process in which in which the final electron acceptor is inorganic molecule

  • - production by yeast of ethanol and carbon dioxide from sugar

  • - many organisms can be used as apart from yeast and is not

restricted to common sugar (sucrose).

-Glucose: readily employed as nutrient

CLASSIFICATION BASED ON CONSISTENCY:

  • 1. Solid

    • - gelled by addition of agar (1-1.5% w/v)

    • - Agar: from seaweeds ; firms at 37C and liquid at 45C.

- For anaerobic organisms contain non- toxic reducing agents

-low redox potential

  • 2. Liquid

    • - contains no agar.

o

Selective or Diagnostic Media

  • - restrict the growth of certain types of microorganisms while

permitting or enhancing the growth of others.

  • - use additives for identification

o

Enrichment Medium

  • - designed to permit a particular

type of organism to grow while others, so the one that grows increase in relative numbers and is

enriched.

CULTIVATION METHODS

o

Binary Fission

  • - division pattern

  • - cell enlarges or elongates then

forms cross wall called septum and

is followed by constriction until finally broken and daughter cells separate.

  • - For yeast and moulds have lower pH (5.5- 6.0)

  • - Lactic acid: used to impart lower pH

CLASSIFICATION BASED ON COMPOSITION:

  • 1. Truly Synthetic Media

    • - chemically defined

    • - for microorganisms that

can synthesize materials needed for growth from simple carbon and nitrogen.

  • 2. Media w/ biochemicals

    • - used for organism that can’t synthesize

    • - some more commonly used

    • - complex w/ precise chemical composition

    • - vary slightly batch to batch

Ex. Routine laboratory media, General Purpose meda, Complex Media - aq. Sol’n of animals and plants extracts that contain:

Hydrolysed protein

-inexpensive sources are meet extracts, milk, and soya -hydrolysed by varying degree to give peptones or amino acids

-trypsin and other proteolytic enzymes preffered to acids

B-group vitamins

-requirement is satisfied by yeast extract

Carbohydrates

-in form of starch and yeast

- Several situations where number of microbial cells in culture, sample or specimen are needed to be measured:

Measuring levels of microbial

contamination in raw material or manufactured medicine Evaluating the effects of

antimicrobial chemical or decontamination process Using microorganisms in

manufacture of therapeutic agents Assessing the nutrient capability of growth medium

-In pyrogen testing and vaccine manufacture both number of dead and living cells are required

o

Total count

–is a counting procedure enumerating both living and dead cells

o

Viable count

-records living cells alone

o

Total Viable Count (TVC)

-used in most pharmacopoeias and by many regulatory agencies -mean a viable count that records all different species or types of microorganism that might be present in sample.

Three Traditional Methods of Viable Counting:

Basis: Living cell will give rise to colonies when introduced w/ suitable medium and incubated.

1.Pour Plating

  • - surface spread method used

2 Miles Misra Method - Surface drop

  • - membrane filter methods used

3. MSP (Most Probable number)

  • - anticipated count is very low

  • - In optimal conditions of laboratory

cultivation of bacteria this division takes place every 25-30 minutes.

- growth continues until one or more nutrient is exhausted or toxic metabolites accumulates

o

Overnight incubation in liquid media

  • - culture media clear but becomes cloudyas concentration increases

  • - indirect means of monitoring culture media.

o

Colony

  • - usually arise on solid media in petri dishes

  • - a collection of cells arising by multiplication of a single original

cell or a cluster

  • - in microscope: hundreds to millions

  • - typically 1-10 mm

  • - Periphery of colony; part that is actively growing

o

Petri dishes incubated in a anaerobic jar

- Where anaerobic microorganisms grows

o

Planktonic Cells

  • - Routinely used to testing procedures designed used to assess the activity

  • - different susceptibilities of lethal agents

  • - Reappraisal appropriate

ENUMERATION OF MICROORGANISMS

  • - fast ,readily automated, and eliminates

long hours of incubation and numerous petri

dishes

  • - not capable of reproducing colonies

  • - more commonly used in water, food, and dairy.

  • - poor accuracy

- last resort

Operating method principles:

*for more information about methods of viable counting check page 18 Table 2.2 and page 19

o

Epifluorescent Techniques

Table 2.3

o

Living cells generate ATP

o

Manometer Techniques

WAYS TO MEASURE BIOMASS:

o

Resistance and capacitance or

impedance of culture media

  • 1. Turbidity Measurements

MICROBIAL GENETICS

  • - Genetic material may be transferred

depending if organism is prokaryote or

eukaryote.

  • - Mutation is very important for resistance of antibiotics

o

Bacteria (Prokaryote)

- genes for growth and metabolism:

chromosomes of double stranded DNA

Bacterial Chromosomes

  • - tightly coiled

  • - 1mm contain 1000-3000 genes

- additional genes for survival advantage under certain circumstances:

Plasmids

Plasmids

  • - smaller and replicate independently

  • - 0.1-1% size of bacterial chromosomes

  • - not essential for normal functioning

  • - replicate independently

-

most common used in estimating the total number of bacteria in sample

-

measured using spectrophotometer or colorimeter

-

not used in fungi

  • 2. Dry Weight Determination

 

-for fungi biomass

3.Direct Microscopic Counting

-

for bacteria, yeast , and fungal spores

-

not for moulds and indirect measure of

biomass

Limitations of traditional method of viable counting:

Relative labour intensive

incubator spaces

Not easy to automate

Slow due to to the need for incubation

May require relative large volumes of

culture media many petri dishes and

Rapid Methods of detecting and counting

microorganisms:

-

enumerate viable organisms(usually

bacteria and yeast)

-

employ various means of indirect

detection of living cells

PHARMACEUTICAL IMPORTANCE OF MAJOR CATEGORIES OF MICROORGANISMS

o

Viruses

  • - importance is based on Pathogenic potential

  • - not susceptible to antibiotics

  • - Hazard Category 4

  • - easy to destroy by heat, radiation, or toxic chemicals

o

Prions

  • - withstand sterilizing conditions

  • - ability to cause incurable and fatal disease

o

Bacteria

- important as pathogens

  • - ability to resist activity of antibiotics and biocides; long

standing notoriety

  • - streptomycetes bacteria produce antibiotics

  • - grow on diverse substrates ensure

potential as agents of spoilage

  • - survive well in drying, dust, and other adverse environments

  • - contaminants

  • - can produce bacterial spores

o

Fungi

  • - survive in drying

  • - produce spores

  • - contaminants

- can be passed to one cell or another by various means

Genes received from other sources:

o

Bacteriophages

  • - Genome of Cell

= Bacterial Chromosomes +

Plasmids + Bacteriophages

o

Eukaryote

  • - nucleus that contain one or more pairs of linear chromosomes

  • - DNA complexed w/ protein

  • - Cells may divide asexually and

undergo mitosis but many have the

potential to undergo sexual reproduction and undergo meiosis

  • - based on RNA instead of DNA

  • - possibility of creating new gene combination

o

Genotype

  • - describes genetic composition

regardless if expressed or not

o

Phenotypic Adaptation

  • - non-genetic adaptation

  • - bacteria adopt a phenotypic change to counter environmental stress.

o

Genetic Adaptation

  • - Acquire new genes either by mutation or conjugation

  • - process of selection ensure that

mutant organisms that are better

suited for new environment becomes numerically dominant.

organisms in order to minimize both formation of damaging ice crystals and osmotic stress that accelerate cell death during freezing and thawing.

Chapter 3: Bacteria

Prokaryotes

 

-

Smallest free – living organism (bacteria and archaea)

 

-

Lack a true nuclear membrane * Eukaryotic cells – presence of a nuclear membrane and internal compartmentalization – Major feature: cytoplasm of membrane- enclosed organelles

Differences (Bacteria and Archaea):

 

-

Cell wall composition (major difference)

Lipid structure making up their cytoplasmic membranes

-

-

Metabolic patterns

 

Bacteria:

Archaea:

 

-

most are anaerobes

-

Vast majority of

-

inhabit extreme

prokaryotes of medical

environments

and pharmaceutical

significance

-

greater stability under

extreme conditions

 

-

no disease-causing

archaea have yet been

identified

Bacteria

 

-

Represent a large diverse group of organism that can exist as single cells or as cell clusters

Have the ability to carry out their life processes of growth, energy generation and

-

reproduction independently of other cells

* Very different from the cells of animals and plants (unable to live alone in nature,

  • - Less degree of resistance than bacteria

  • - little threat to immunocompetent individuals

o

Protozoa

  • - significantly large owning to the pathogenic potential of few species

  • - Do not poses cell wall

  • - Do not survive drying well

  • - Do not display resistance to sterilization to match bacterial spores

- More troublesome in veterinary

PRESERVATION OF MICROORGANISMS

  • - Manufacture of Medicines: microorganisms are employed in variety of test and assays to measure activity of antimicrobial chemicals.

  • - Aim of Culture Preservation: maintain viability of

the highest possible percentage of cells and to

minimize risk of selecting atypical mutants.

  • - Gram positive bacteria tends to survive better than gram negative ones.

Most common procedure for long term storage:

  • 1. Freezing at -80C in refrigerators

  • 2. Storage in liquid nitrogen at -196C special

vessels

  • 3. Lyophilization or freeze-drying

o

Cryoprotectant Chemicals

-compounds like glycerol or dimethylsulphoxide

  • - incorporated at concentrations

10% v/v in liquid culture of

Rarer morphological forms:

a. Actinomycetes – rigid bacteria resembling fungi that may grow as lengthy branched filaments

  • b. Mycoplasmas

– lack a conventional peptidoglycan

(murein) cell wall

– highly pleomorphic organisms of indefinite shape

  • c. Some miscellaneous bacteria – stalked,

sheathed, budded, and slime – producing forms

often associated with aquatic and soil environments

Cellular components

  • - Simple base cell structure compared with eukaryotic cells

Reasons to have a good knowledge of the bacterial cell structures and functions:

  • - Provides an excellent route for probing the

nature of bacterial processes many of which are

shared by multicellular organisms

  • - Normal bacterial processes can be customized to benefit society on a mass scale

  • - To know how to destroy bacterial contaminants and disease-causing organisms (pharmaceutical and

healthcare perspective)

Cell Wall

exist only as a part of a multicellular organism)
exist only as a part of a multicellular
organism)
  • - Capable of growing in a range of different environments

  • - Cannot only cause contamination and

spoilage but also a range of different diseases

Bacterial Diversity and Ubiquity

  • - Bacteria are diverse in shape and sizes (morphology), adaptation to environment,

survival strategies, and metabolic processes

  • - The presence of bacteria may be

considered ubiquitous. There is no natural

environment that is free from bacteria.

Cell Size and Shape

  • - Majority of bacteria are 1-5 um long and 1-2 um in diameter * >5 um (ex: Thiomargarita namibiensis) – extremely rare * bacterial size increased, efficient, and rapid transport and growth rates

  • - Classification of bacteria is made through morphological grounds

  • - Mostly unicellular and possess simple shapes: round (cocci), cylindrical (rod,

bacillus), or ovoid

  • - LPS determines the antigenicity of the bacteria

*extremely toxic to animals

  • - LPS is made up of lipid A, core polysaccharide and O-specific polysaccharide

  • - The cells lose the crystal-violet iodine

complex and are rendered colorless (gram stain)

  • - Appear red under the light microscope

Cytoplasmic membrane

  • - Fragile phospholipid bilayer with protein distributed randomly throughout

  • - Involved in various transport and enzyme functions associated with the membrane

  • - Transports of nutrients, energy generation and electron transports

  • - Selective barrier between the cytoplasm and the cell environment

Cytoplasm

  • - Consists of 80% water and contains enzymes that generate ATP

    • - Compose of the ribosomes, nucleoid and inclusion granules

Nucleoid

  • - Singular, covalently closed circular

molecule of double stranded DNA

Plasmids

  • - Relatively small circular pieces of double stranded extrachromosomal DNA

    • - For autonomous replication

    • - Encode many auxiliary functions that are not usually necessary for bacterial growth

(antibiotic resistance)

  • - Essential for the maintenance of the shape and integrity of the bacterial cell

    • - An obvious target for antibiotics (Cell Lysis)

    • - Provide a strong, rigid structural

component that can withstand the osmotic pressure caused by high chemical concentrations of inorganic ions in the cell

  • - Most bacterial cell walls have

peptidoglycan layer (murein/ glycopeptide) exceptions include the Mycoplasmas, extreme halophiles, and the archaea

  • - Peptidoglycan is composed of N–acetyl muramic acid (NAM) and N–acetyl glucosamine (NAG)

Bacteria can be divided into two large

groups

(on the basis of a differential staining

technique called the Gram stain): Gram-positive,

Gram-negative

Gram-positive Cell Wall

  • - Consist primarily of a single type of molecule

    • - Contains teichoic acids and lipoteichoic acids (negatively charged)

    • - During an infection, lipoteichoic acids

molecules trigger an inflammatory response

  • - Retain the dye (gram stain)

  • - Appear purple under the light microscope

Gram-negative Cell Wall

  • - Multilayered structure, quite complex

  • - Compose of proteins, lipoproteins,

phospholipids and lipopolysaccharide that are unique to gram negative bacteria

- Biofilm formation begins with attaching to surface and form cement cells to protect the bacteria from hazardous materials

BACTERIAL SPORULATION

- Process in which the vegetative cell undergoes a profound biochemical change to give rise to a specialized structure called an endospore or spore - Not part of a reproductive cycle

Spore

Highly resistant

Enables producing organism to survive in

adverse environmental conditions (lack of moisture or essential nutrients, exposure to toxic chemicals, radiation or high temperatures) All sterilization process for pharmaceutical products have been designed to destroy the bacterial spore

Endospore structure

Endospores

Differentiated cells that possess a grossly different structure to that of the parent vegetative cell in which they are formed

Exosporium

Outermost layer

Composed of protein; within are

Cortex

the spore coats (proteinaceous but with a high cysteine content)

Consists of loosely cross-linked peptidoglycan

Central core

Contains the genome

Partially dehydrated (dehydration

shown to increase resistant to both heat and chemicals) Containing only 10-30% of the water content of the vegetative cells

 

transfers, increasing the spread of resistance

-

Ribosomes

 

-

The site of protein synthesis

-

Two subunits: 30S and 50S

Inclusion granules

 

Serves as the storage material for carbon, nitrogen, and sulphur or phosphorus

-

Cell surface components

 

Flagella

-

Bacterial motility

a.

Monotrichous – a single polar flagellum

b.

Lophotrichous – two or more flagella at one pole

of the cell

c.

Amphitrichous – single/tuft of flagella at each

end of the cell

 

d.

Peritrichous – flagella distributed over the

entire cell

Pili and Fimbriae

 

a.

Pili (pilus) – join bacterial cell in preparation of

DNA and to environmental surfaces

– involved in the genetic exchange process of conjugation

 

b.

Fimbriae – for adherence of cells to one another

and to environmental surfaces – responsible for hemaglutination and cell clumping in bacteria

Glycocalyx (Slime Layer and Capsule)

-

General substances that surround cells

-

Gelatinous polymer of polysaccharide, polypeptide, or both

a.

Slime Layer – unorganized and loosely attached

to the cell wall

 

b.

Capsule – substance is organized and firmly

attached to cell wall

 

Biofilms

-

Any surface for microbial habitat

Usually contains more than one species of bacteria which exist and cooperate

-

together

 

Classified as either endotoxin (ex. Cell wall-related, exotoxin, products released extracellularly as the organism grows)

Endotoxin

 

Lipid A component of LPS

Possesses multiple biological

properties including ability to induce fever, initiate the complement and blood cascades Activate B lymphocytes and

stimulate production of tumor necrosis factor Released from lysed or

damaged cels Depyrogenation; process where

Exotoxin

care must be taken to eliminate or exclude such heat- resistant material from parenteral products and their delivery systems.

 

A-B toxins; consists of a B

subunit that binds to a host cell receptor and is also covalently bound to the A subunit that mediates the enzymic activity responsible for toxicity (ex. Diphtheria toxin, cholera toxin) Cytolytic toxins; do not have

seperable A and B portions but work by enzymatically attacking cell constituents, causing lysis (ex. haemolysins and phospholipases) Superantigen toxins; lack an A- B type structure and act by stimulating large numbers of immune response cells to release cytokines, resulting in a massive inflammatory reaction (ex. Staphylococcus aureus)

 

pH; 1 unit lower than the

cytoplasm of the vegetative cell and contains high levels of core- specific proteins that bind tightly to the DNA and protect it from potential damage Core-specific proteins; function as

Spore

an energy source for the outgrowth or germination of a new vegetative cell from the endospore

 

Presence of dipicolinic acid and high levels of calcium ions which complex together

Endospore formation

• Vegetative cell undergoes a complex series of biochemical events in cellular differentiation • Sporulation; accomplished by activation of a

variety of spore-specific genes such as spo and ssp

• Leads to the production of a dry, metabolically inert but extremely resistant endospore Endospore germination • Reversion of endospore back to a vegetative cell • Removal of the stress inducer that initiated sporulation • Germination loss of resistance properties; occurs along with a loss of calcium dipicolinate and cortex components, and degradation of the core- specific proteins

BACTERIAL TOXINS

  • - Organisms, if presented with the correct set of conditions, can cause disease

  • - (ex. Opportunist pathogens; Staphylococcus epidermis,non-pathogenic environmental organism; Ps. Aeruginosa)

Toxins

Products of bacteria that produce immediate host cell damage

Growth and solid surfaces

• Solidified growth media are deployed to separate different types of bacteria and also as an aid to enumerating viable cell numbers in the laboratory

• Agar media are used in the laboratory either poured as a thin layer into a covered dish or contained within a small, capped bottle

• The colour, size, shape and texture of colonies of different species of bacteria very considerably and form a useful diagnostic aid to identification

Growth in liquids

• Growth ceases when the rate of consumption of nutrients exceeds the rate of supply

• Bacteria (being of colloidal dimension and sometimes highly motile, are dispersed evenly through the fluid (nutrients are equally available to all cells)

Liquid batch culture (closed)

Logarithmic growth phase (B); during active growth a logarithmic plot of cell number against time gives a straight line

Lag period (A); the inoculum adapts its physiology to that required for growth on the available nutrients

• Late logarithmic phase; rate of growth

Stationary phase (C); eventual halt

Decline phase (D); starvation, death of some of the cells and adaptation to a dormant state

BACTERIAL REPRODUCTION AND GROWTH KINETICS

- Multiplication and division cycle

Binary fission; process where the majority of cells multiply in number

• Each daughter cell will automatically contain those materials that are dispersed through the mother cell (mRNA, rRNA, ribosomes, enzymes, cytochromes, etc.)

• Bacterial chromosome; circular and attached to the cytoplasmic membrane where it is able to uncoil during DNA replication

• DNA replication if based on the number of base pairs within it and the growth temperature (ex. Escherichia coli; replication of chromosomes will take approximately 45 minutes)

• Gram-negative cells; do not have rigid cell wall, must develop a cross-wall that divides the cell into two equal halves

• Rod-shaped organisms; maintain their diameter during the cell cycle and increase their mass and volume by a process of elongation

• Coccal forms; increase in size by radial expansion Population growth

• Cell numbers will increase exponentially as a function of time

• Generation time; the time interval between one cell division and the next

• A mean generation time is usually calculated when considering a growing culture containing thousands of cells

• F-factor (fertility factor); simplest form of plasmid • F-factor will simply transfer a copy to
• F-factor (fertility factor); simplest form of plasmid • F-factor will simply transfer a copy to
• F-factor (fertility factor); simplest form of plasmid • F-factor will simply transfer a copy to
• F-factor (fertility factor); simplest form of
plasmid
• F-factor will simply transfer a copy to a
recipient cell

ENVIRONMENTAL FACTORS THAT INFLUENCE GROWTH AND SURVIVAL

• Gram-negative bacteria tend to be aquatic • Gram-positive bacteria then to prefer more arid conditions such as the skin Physiochemical factors that affect growth and survival of bacteria Temperature • Permissive temperature; range of temperatures under which bacteria can actively grow and multiply • Chemical and enzymic reactions within the cell proceed more rapidly, and growth becomes faster until an optimal rate is achieved as temperature rises • Beyond such temperature, certain proteins may become irreversibly damaged through the thermal lysis, resulting in a rapid loss of cell viability • pH • PH effects on growth are bell-shaped • Extremes of pH can be lethal • Growth optima of 7.4 and 7.6; microorganisms that have medical or pharmaceutical significance • May dictate the range of microorganisms that could potentially cause its spoilage Water activity/ solutes • Gram-negative cell envelope cannot withstand the high internal osmotic pressures associated with rapid rehydration after desiccation • Water activity (A w ); vapor pressure of water in the space above the material relative to the vapor pressure above pure water, can markedly affect its vulnerability to spoilage contaminants • Availability of oxygen • Oxygen acts as the terminal electron acceptor in respiration and is essential for growth

Growth in open culture

• Bacteria make up >90% of the dry mass of faeces

• In many situations the bacteria become immobilized, as a biofilm, upon a surface and extract nutrients from the bulk fluid phase

Growth and genetic change

Transformation

• Ability of certain types of bacteria to absorb small pieces of naked DNA from the environment that may recombine into recipient chromosome • Means of transferring genes between different types of bacteria Transduction • Bacterial DNA having moved between cells • Temperate phage; rather than enter a replication cycle, the viral DNA becomes incorporated by recombination into the chromosome of the bacterium Conjugation • Thought to have evolved through transduction • Plasmids; DNA strands

concentration, do not discriminate between living and dead cells Microcalorimeters; time taken to detect such heat can be directly related to the numbers of viable cells present

Enrichment culture

• Intended to increase the dominance of a numerically minor component of a mixed culture such that it can be readily detected on an agar plate

• Enrichment media; always liquid, intended to provide conditions that are favourable for the growth of other likely isolates

• MacConkey broth; contains bile salts that will inhibit the growth of non-enteric bacteria and may be used to enrich for Enterobacteriaceae

Selective media

• Solidified enrichment broths, intended to suppress the growth of particular groups of bacteria and to allow the growth of others • Counts of colonies obtained on selective solid media are often documented as presumptive counts Identification media (diagnostic) • Contain nutrients and reagents that indicate, usually through some form of colour formation, the presence of particular organisms

Microscopy

• Simple stains (such as the Gram stain) • Size, shapes, arrangement into clusters, chain and tetrads, specific stains for the presence of endospores, capsules, flagella and inclusion bodies

• Fermentation; carbon substrate is in excess

Nutrition and growth

Chemolithotrops; simple inorganic forms of elements, can utilize atmospheric carbon dioxide and nitrogen as sources of carbon and nitrogen • Diauxic growth; second lag phase during the logarithmic growth period while such adaption takes pace

DETECTION, IDENTIFICATION AND CHARACTERIATION OF ORGANISMS OF PHARMACEUTICAL AND MEDICAL SIGNIFICANCE

Culture technique

- Diluting the sample to varying degrees and inoculate the surface of a predried nutrient agar with known volumes of those dilutions

- Enumeration media Will only ever culture a subset

of cells towards which the medium and incubation conditions are directed Simple salts media with

relatively simple sugars as carbon sources and trace levels of amino acids; often used to enumerate bacteria associated with water Psychrophilic Gram-negative

bacteria; can be a major source of bacterial pyrogen Highly nutritious media are also used as enumeration media (ex. blood agar)

Rapid enumeration techniques

Bioluminescence

Epifluorescence

Impedance techniques

Coulter counters; used to determine bacterial

o

Bread

o

Enzymes

o

Antibiotics

o

Recombinant proteins

 

FUNGI

   
 

O

A

 

B

 

T

U

D

O

S

A

E

S

E

M

C

S

L

T

U

Y

 

O

I

I

O

T

C

M

D

O

M

E

E

 

Y

 

I

M

Y

R

T

C

 

O

Y

C

O

E

E

M

C

E

M

S

T

 

Y

E

T

Y

E

C

T

E

C

S

E

E

S

E

 

T

S

T

E

E

S

S

The Kingdom Fungi can be subdivided into six classes:

Oomycetes

-contains the mildews and water moulds

Ascomycetes

-contains the mildews, some moulds and most yeast species (including Saccharomyces cerevisiae)

Basidiomycetes

-contains the mushrooms and bracket fungi

Teliomycetes

-contains the rust fungi (plant pathogens)

Ustomycetes

-contains the smuts (plant pathogens)

Deuteromycetes

Biochemical testing and rapid identification

• Differing ability of bacteria to ferment sugars, glycosides and polyhydric alcohols (widely used to differentiate the Enterobacteriaceae and in diagnostic bacteriology generally) • Results of oxidase and catalase tests

performed directly on isolated colonies Molecular approaches to identification

• Have not yet become routinely adopted in

the analytical or diagnostic laboratory

• Denaturing gradient gel electrophoresis

(DGGE); isolates and amplifies 16S

ribosomal DNA and, following sequencing

of the bases, compares this with known sequences held in a reference library • Gene probes carrying fluorescent dyes; can be used in hybridization procedures with the collected clinical material

Pharmaceutically and medically relevant microorganisms

• Broadly classified into those organisms that are harmful or problematic, and those that can be used to our advantage

Refer to Table 3.3 of book; examples of some pharmaceutically useful bacteria

Chapter 4: Fungi

Fungi

Eukaryotic organisms

Widely distributed in nature

Extremely important group of microbes in the medical field

Responsible for a number of potentially fatal diseases in humans

Great benefit in humans in terms of:

o

Production of alcoholic beverages

 

Average thickness varies from 100 to 300

 

nm

COMPOSITION OF FUNGAL CELL WALL

     

S

     

P

T

R

Cell Wall

 

R

U

M

A

O

T

G

C

Glucan, the main structural component of

L

N

H

E

fungal cell wall, is a branched polymer of

U

C

T

N

A

I

T

I

I

N

glucose.

C

U

S

 

A

R

N

AND

   

N

A

N

L

 

I

The innermost layer is rich in glucan and chitin

50-

 

L

P

which provides rigidity to the wall and it’s

60%

POLYSACCHARIDES

1-9%

important in regulating cell division.

15-23%

I

 

25

D

 

%

S

Periplasmic space

a thin region that lies directly below the cell wall

contains secreted proteins

location for a number of enzymes required for processing nutrients prior to entry into the cell

Cell membrane / Plasmalemma

located directly below the periplasmic space

a phospholipid bilayer which contains phospholipids, lipids, protein and sterols.

Approximately 10 nm thick

Nucleus

A discrete organelle

-contains species such as Aspergillus, Fusarium and Penicillium

There are four distinct phyla within the fungal kingdom; these are the Chytridiomycota, Zygomycota, Ascomycota and Basidiomycota.

Yeast

Grow as single cells

Reproduce asexually by budding, although a minority of species reproduce by fission

Many yeast species are capable of sexual production and formation of spores

Moulds

Grow as masses of overlapping and interlinking hyphal filaments

Reproduce by producing masses of spores

Structure of the fungal cell

Oval in shape

Surrounded by a rigid cell which contains structural polysaccharides

suppressed as a result of therapy or disease.

The most common fungal pathogens:

o

Yeasts

o

Moulds

o

Dermatophytes

Medically important fungal pathogens of humans

Candida albicans

 

o

Most frequently encountered human fungal pathogen

o

Responsible for a wide range of superficial and systemic infections

o

Opportunistic fungal pathogen which can be present as a normal part of the body’s microflora

o

Exist in two morphologically distinct forms: budding blastospores or hyphae

o

Plays an important role in the degradation of the immunoglobulins IgG and IgA

o

Important in allowing yeast access iron released from ruptured red blood cells

o

Capable of giving rise to a variety of interconvertible phenotypes which can be considered as providing an extra dimension to the existing virulence associated with this yeast.

Aspergillus fumigatus

o

a saprophytic fungus

o

the dominant fungal pulmonary pathogen of humans

Most of the cell’s genome is concentrated in the nucleus

Repository of the DNA

Contains proteins in the form of histones

Mitochondrion

The “powerhouse” of the cell

Possesses its own DNA

Capable of producing its own proteins on its own ribosomes, mitoribosomes

Enzymes of tricarboxylic acid cycle are located in the matrix of the mitochondrion

Electron transport and oxidative phosphorylation occur in the mitochondrial inner membrane

The outer membrane contains enzymes

involved in lipid biosynthesis

Ribosomes

The site of protein biosynthesis

Mediates the export of proteins from the cell

Vacuole

A “storage space”

Nutrients, hydrolytic enzymes or metabolic intermediates are retained until required

Medical significance of fungi

A significant group of pathogens capable of causing a range of diseases

Although majority of fungi appear to be harmless to humans but a normally non- pathogenic fungus can cause a clinically relevant problem if the immune system is

be consumed directly as a dietary supplement

o

No longer regarded as a GRAS (generally regarded as safe) but is now classified as a Biosafety Level 1 pathogen

o

Obstructs capillaries particularly in the brain with concomitant damage to surrounding tissues

Penicillium marneffei

 

o

Very rare and inconsequential cause of disease in humans

o

The most frequent cause of fungal disease in AIDS patients who reside in, or visited, South East Asia

o

An asexual, dimorphic fungus growing as a mycelium at 37°C in tissue and as single cells at 28°C

o

Reproduce by fission

Non-albicans Candida species

o

Candida dubliniensis

-dominant cause of oral candidosis of HIV-positive and –negative populations from many parts of the world

o

Candida krusei

-significant cause of disease in HIV- positive patients, diabetics, and cancer patients

-dominant fungal pathogen in certain classes of diseases

o

Candida glabrata

-a serious cause of disease in neutropenic cancer patients and

o

generally presents a problem in those with pre-existing lung disease or damage

o

frequently encountered growing on decaying vegetation and damp surfaces

Histoplasma capsulatum

 

o

a dimorphic fungus

o

cause of histoplasmosis, the most prevalent fungal pulmonary infection

Cryptococcus neoformans

 

o

an encapsulated yeast

o

most frequently associated with infection in immunocompromised patients

o

meningitis is the most common clinical manifestation

o

a facultative intracellular pathogen that is capable of surviving and replicating within macrophages and withstanding the lytic activity within these cells

Dermatophytes

 

o

group of keratinophilic fungi which can metabolize keratin

o

the principal protein in skin, nails and hair

Emerging fungal pathogens

Saccharomyces cerevisiae

o

Better known as “brewer’s yeast” or “baker’s yeast”

o

Used for the production of bread and alcoholic beverages, and can

Viral nucleic acid:

-

The viral nucleic acid is composed of either DNA or RNA

-

A virus can only have a single genome

Viral capsid (protein core)

-

It protects the viral nucleic acid from detrimental, chemical and physical conditions

-

It is composed of a number of subunits named ‘capsomeres’ genetically encoded by the viral genome

-

Capsomeres give the shape of the capsid, and provide the virus with resistance to physical and chemical agents

Viral envelope

-

Most outer covering of a virus

-

The envelope is added during the replication process

-

It can come from the host cell nuclear membrane, or the cytoplasmic membrane

-

Enveloped viruses are considered to be the most susceptible to chemical and physical conditions

-

They do not survive well on their own outside the host cell, although they can persist longer in organic soil

III. Virus-host cell interactions

Viruses can interact with the host cell in five different ways:

  • 1. Multiplication of the virus and destruction of the host cell upon release of the viral progeny

has been responsible for mortality rates of 5-38%

-Fourth most commonly isolated Candida species

Antibiotic production of fungi

Majority of antibiotics obtained from fungi are produced by fermentation and most are secondary metabolites.

Isolation of Penicillium notatum by Sir Alexander Flemming – Most important discovery regarding the beneficial use of fungi for humans

Antibiotic production can be maximized by optimizing production as a result of random mutagenesis and selection.

Chapter 5: Viruses

I. Introduction

Viruses were first discovered at the end of 19 th century

They were classified as “filterable agents” because they can be retained by filtration

II. General structure of viruses

Viruses are extremely diverse in size and shape

Viruses are much smaller than bacteria

“Intracellular parasites”

Smallest virus: poliovirus -> 28nm in size

Largest virus: mimivirus -> 750nm in size

Viruses with envelope: enveloped nucleocapsid

Viruses with no envelope: naked nucleocapsid Components of a virus:

  • - Envelope fuses from the membrane

  • - It is a process wherein virions can fully enter the membrane

o

Endocytosis: When an envelope nucleocapsid fully enters the host cell, it undergoes endocytosis.

  • - Envelope fuses with the endosome

Uncoating - releases capsin to free nucleic acids (DNA or RNA)

Eclipse stage: replicates and expresses genes

Assembly or maturation - maturation of virus cells; cells become virions again

Release - virions are release outside the host cell, to infect other cells

V. Cultivation of human viruses

1. Cell culture:

May be divided into three types according to their history:

Primary cell lines: The cell lines are derived directly from an intact tissue, ex: human embryo, kidney or monkey kidney

Secondary cell cultures:

derived from primary cultures, usually those arising from embryonic tissue

-

The cells are more homogenous, better

  • 2. Multiplication of the virus and release of the virions without the immediate destruction of the host cell

  • 3. Survival of the virus in a latent stage without noticeable changes to the infected cell

  • 4. Survival of the infected cell in a dramatically altered or transformed state

  • 5. Incorporation of the viral nucleic acid in the host cell genome without noticeable changes to the infected cell

IV. Multiplication of human viruses

Objective of replication cycle: to ensure the multiplication of the virus with the formation of identical viral progeny

The multiplication cycle of human viruses is generally slow, from 4 to more than 40 hours

Bacterial viruses are generally faster and can take a little as 20 minutes to replicate within the bacterial host

There are six distinct phases in a replication cycle:

Adsorption - attachment to the host cell receptor

Penetration - penetrates the virus through the cell membrane. It has three types:

o

Direct injection: type of virus involved -> naked nucleocapsid

-

the virus did not fully enter the host cell

o

Fusion: type of virus involved -> envelope nucleocapsid

  • - The drug used for HIV infections is called

‘antiretrovirals’

  • - Antiretrovirals have considerably prolonged the life expectancy of patients, although not without some side effects

  • - These drugs aim to reduce HIV plasma level as much and as long as possible

a

Herpesvirus infections

  • - Herpesviridae: a family of viruses which include the herpes simplex virus, chickenpox, shingles and cytomegalovirus

  • - Mild herpes simplex virus -> treated with a topical antiviral drug

  • - Primary herpetic gingivostomatitis -> a change of diet & analgesics

  • - Severe infections -> systemic antiviral is used

  • - Antiviral treatments for chickenpox: recommended in patients at risk and in neonates to reduce risks of severe diseases

  • - Antiviral treatments for herpes: is associated with a number of side effects which may vary depending on the drug

  • - Antiviral treatments for cytomegalovirus: usually given to immunocompromised patients and they tend to be more toxic with noticeable nephrotoxicity and a number of side effect

characterized, but might not be as susceptible to viral infection as primary cell lines

Continuous cell lines: usually derived from malignant tissue, and have the capacity to multiply indefinitely in vitro

Cytophatic effect: a characteristic morphological change in the infected cells wherein the cells shrink, or undergo ballooning.

They usually spread to adjacent cells and will result in the formation of a plaque that can easily be identified following staining

Plaques: used for the enumeration of viruses

  • 2. The chick embryo

• Fertile chicken eggs, (9-11 days old) are used to grow a number of human pathogenic viruses.

  • 3. Animal inoculation

• Animals are used to culture certain viruses in order to study antiviral vaccine effectiveness, and also as a source of cell lines for cell cultures

VI. Control of viruses

  • 1. Antiviral chemotherapy - leads to the development of viral resistance, but is still associated with a number of problems a. HIV

    • - The role of antivirals in HIV is to slow or halt disease progression

influenza vaccines rely on chemically inactivated, virus particles or components

Inactivated viruses

Use of viral components

• Hepatitis B: viral DNA encoding for a virus surface antigen expressed in yeasts

• Immunoglobulin: plays a role in the protection of patients with a compromised immunity against viral infections

• IM immunoglobulin used to protect against hepa A virus

  • 1 Viricidal effects of chemical and physical agents on viruses

Viruses are generally transmitted via surface and are often associated with organic materials

In general, viruses are not particularly resistant to chemical or physical agents, although some exceptions exist.

  • 1 Control of viruses in pharmaceutical products

Presence of certain viruses needs to be controlled

Risk of a pharmaceutical product being contaminated by viruses depends on:

The origin of the product component

The history of the donor

The amount of material used

The manufacturing process

Its capacity to remove or destroy contaminants

a

Viral hepatitis

  • - Treatment for acute hepatits B -> interferons

  • - Treatment for chronic hepatitis B -> antivirals

a

Influenza

  • - Two major limitations in the usefulness of the drug

  • - First: the drug needs to be taken within a few hours of the onset of symptoms from mild to severe symptoms reported

  • - Second: the side effects have been very severe

a

Respiratory syncytial virus

  • - RSV is responsible for severe bronchiolitis notably in infants

  • - Treatment can be a monoclonal antibody or an antiviral drug

2.

Vaccination

• the most successful measure against microbial and viral infections

  • - Vaccines are preparations containing antigents that elicit a specific and active immunity against an infecting agent

  • - Vaccines can induce the innate and the adaptive parts of the immune system

  • - Viral vaccines prepare using 3 methods:

Live attenuated viruses: will cause a strong immune response without causing the disease Hepatitis A and

Use of bacteriophages to treat bacterial infection

1

• Introduction of antibiotics in the early 1940’s resulted in the end of phage therapy in the West

o

Natural phages

o

Non-replicating phages

o

Genetically modified phages

• Use of phages for surface disinfection and antisepsis: further work is still needed to develop appropriate phage-based products, especially the effect of the different routes of administration on phage viability and effectiveness

1

Epidemiological uses and diagnosis

• Phage typing: a method that differentiates distinct strains of the same bacterial species on the basis of their susceptibility to phages

VIII. Prions

Prions – devoid of nucleic acid and are extremely resistant to heating and ultraviolet irradiation

They fail to produce an immune response in the host

VII. Viruses as antimicrobials

1.

Bacteriophages

 

Bacteriophages are viruses that infect only bacteria

They were first described at the end of the 19 th century

Size: 20-200nm, and are highly diverse in their structure and host range and it is likely that all bacterial species can be infected by a phage

Phages are extremely specific in their host range and some will only infect a specific bacterial strain

Most studied phages are the complex ones, ‘tadpole-shaped’ which consists of a head that contains the viral genome, and a tail which function is to recognize the host receptor, attach and subsequently serve as a nucleic acid injection device

Two phage replication cycles:

 

Lytic cycle: lysis of bacterial host

Lysogenic cycle: result of a viral nucleic acid being integrated into the host genome

• Infection with lytic phage -> virulent phage, results in the replication of phage within the susceptible bacteria and the release of infectious phage progeny from the host cell following cell lysis

• Lysogenic cycle: viral nucleic acid which has integrated the host genome is called prophage

Host cell that contains the viral genome: lysiogenic