NUTRITION AND GROWTH bacteria vary considerably in their

requirements for nutrients and in their ability to synthesize for themselves various vitamins and growth factors

there is a need for the provision of carbon, nitrogen, water, phosphorus, potassium and sulfur

minor requirement for trace elements such as magne- sium, calcium, iron, etc

chemolithothrops - able to derive much of their nutrition from simple inorganic forms of these elements; can even utilize atmospheric carbon dioxide and nitrogen as sources of carbon and nitrogen

many classes of bacteria are auxotrophic and can grow on simple sugars together with ammonium ions, a source of potassium and trace elements

such bacteria can synthesize for themselves all the amino acids and ancillary factors required for growth and division (pseudomonads and Achromobacter)

a faster rate of growth is often obtained when glucose or succinate is the carbon source rather than lactose or glycerol, and when amino acids are provided as sources of nitrogen rather than ammonium salts

Diauxic growth on a mixture of glucose and lactose.

many pathogens require complex growth media if they are to be cultured in vitro

possible that in the future many disease states currently thought to have no microbiological involvement could be identified as being of bacterial origin (Helicobacter pylori)

DETECTION, IDENTIFICATION AND CHARACTERIZATION OF ORGANISMS OF

PHARMACEUTICAL AND MEDICAL SUBSTANCES

once a microorganism has been isolated in pure culture, usually from a single colony grown on an agar plate, then further characterization may be made by the application of micros- copy together with some relatively simple biochemical tests

characterization of organisms has become simplified by the introduction of rapid identification systems

molecular approaches have enabled identification of organisms without the need to culture them

CULTURE TECHNIQUES majority of samples taken for

examination contain mixtures of different species

simple plating onto an agar surface may fail to detect an organism that is present at <2% of the total viable population

various enrichment culture techniques may therefore be deployed to detect trace numbers of particular pathogens, prior to confirmatory identification

Enumeration simplest way in which to enumerate

the microorganisms that contaminate an object or liquid sample is to dilute that sample to varying degrees and inoculate the surface of a pre-dried nutrient agar with known volumes of those dilutions

number of colonies formed might not relate to the viable number of cells, as clumps of cells will only produce a single colony and they will only detect a particular subset of the viable bacteria present in the sample that can grow under the chosen conditions

number of techniques are currently being developed in order speed up the enumeration process

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 are often used to enumerate bacteria associated with water

highly nutritious media, e.g. blood agar, are also used as enumeration media (Staphylococci)

Sometimes inhibitors of bacterial growth (e.g. Rose Bengal) can be added to a medium in order to select for moulds

some of the rapid methods that have been used for bacteria and other microorganisms, e.g. bioluminescence, epifluorescence and impedance techniques

examination of pharmaceutical waters and aqueous pharmaceutical products electronic particle counters, e.g. Coulter counters, can be used to determine bacterial concentration

similar counters are available that are able to analyze particles found in air

other rapid techniques aim to detect microbial growth rather than to visualize individual cells and colonies

bioluminescence can only detect those organisms that are able to grow in the chosen medium

none of the rapid techniques are able to isolate individual organisms; they do not therefore aid in the characterization or identification of the contaminants

Enrichment Culture Enrichment cultures are 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

always liquid and are intended to provide conditions that are favourable

for the growth of the desired organism and unfavourable for the growth of other likely isolates

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

Selective Media selective media are solidified enrichment

broths, so again they are intended to suppress the growth of particular groups of bacteria and to allow the growth of others

mannitol salts agar will favor the growth of micrococci and staphylococci, and cetrimide agar will favor the growth of pseudomonads

counts of colonies obtained on selective solid media are often documented as presumptive counts

Identification Media (Diagnostic) identification media contain nutrients

and reagents that indicate, usually through some form of color formation, the presence of particular organisms

inclusion of lactose sugar and a pH indicator into McConkey agar facilitates the identification of colonies of bacteria that can ferment lactose

fermentation leads to a reduction in pH within these colonies and can be detected by an acid shift in the pH indicator

the inclusion of egg-yolk lecithin into an agar gives it a cloudy appearance that clears around colonies of organisms that produce lecithinase

MICROSCOPY application of simple stains such as the

Gram stain can divide the various genera of bacteria into two convenient broad groups

size and shapes of individual cells and their arrangement into clusters, chains and tetrads will also guide identification

examination of wet preparations can give an indication as to the motility status of the isolate, and these

procedures all represent an important first stage in the identification process

BIOCHEMICAL TESTING AND RAPID IDENTIFICATION

differing ability of bacteria to ferment sugars, glycosides and polyhydric alcohols is widely used to differentiate the Enterobacteriaceae and in diagnostic bacteriology generally

identification of particular species and genera by such processes is time-consuming, expensive and may require numerous media and reagents

process has become simplified in recent years by the development of rapid identification methods

the latter often use multiwell microtitration plates that can be inoculated in a single operation either with an inoculated wire or with a suspension of a pure culture

simple kits may perform only 8–15 tests, more complex ones are capable of performing 96 simultaneous biochemical evaluations

large diagnostic laboratories and in quality assurance laboratories automated systems are deployed that can inoculate, incubate and analyze hundreds of individual samples at a time

MOLECULAR APPROACHES TO IDENTIFICATION

the need to identify microorganisms rapidly has led to the development of a number of molecular identification and characterization tools

these have not yet become routinely adopted in the analytical or diagnostic laboratory but probably will be in the future

molecular approaches can be of especially useful when attempting to detect a particular species

PHARMACEUTICALLY AND MEDICALLY RELEVANT MICROORGANISMS

microorganisms of medical and pharmaceutical relevance can be broadly classified into those organisms that are harmful or problematic, and those that can be used to our advantage

some microorganisms, depending on the situation, can fall into both categories

microorganisms cause some of the most important diseases of humans and animals and they can also be found as major contaminants of pharmaceutical products

many large-scale industrial processes, e.g. antibiotic production, are based on microorganisms, and selected species can be used to test disinfectant efficacy and to monitor sterilization procedures

FUNGI yeast, such as brewers’ yeast, and

moulds, such as Penicillium chrysogenum which produces the antibiotic penicillin, are classified as fungi

yeast cells tend to grow as single cells which reproduce asexually in a process known as budding

fungi are eukaryotic organisms, i.e. their cells possess a nuclear membrane, consequently there are many similarities between the biochemistry of fungal cells and human cells

medically, fungi are an extremely important group of microbes, being responsible for a number of potentially fatal diseases in humans

significant number of fungi are of great benefit to humanity in terms of the production of alcoholic beverages, bread, enzymes, antibiotics and recombinant proteins

kingdom Fungi can be subdivided into six classes

class Oomycetes contains the mildews and water moulds

class Ascomycetes contains the mildews, some moulds and most yeast species

class Basidiomycetes contains the mushrooms and bracket fungi

class Teliomycetes contains the rust fungi

class Ustomycetes contains the smuts class Deuteromycetes contains species

such as Aspergillus, Fusarium and Penicillium

four distinct phyla within the fungal kingdom; these are the Chytridiomycota,

Zygomycota, Ascomycota and Basidiomycota

STRUCTURE OF FUNGAL CELL typical yeast cell is oval in shape and is

surrounded by a rigid cell wall which contains a number of structural polysaccharides and may account for up to 25% of the dry weight of the cell wall

the thickness of the cell wall may vary during the life of the cell

glucan, the main structural component of the fungal cell wall, is a branched polymer of glucose which exists in three forms in the cell: β-1,6- glucan, β-1,3-glucan and β-1,3,-β-1,6-complexed with chitin

mannan is a polymer of the sugar mannose and is found in the outer layers of the cell wall

chitin, is concentrated in bud scars that are areas of the cell from which a bud has detached

mannoproteins form a fibrillar layer that radiates from an internal skeletal layer that is formed by the polysaccharide component of the cell wall

enzymatic or mechanical removal of the cell wall leaves an osmotically fragile protoplast which will burst if not maintained in an osmotically stabilized environment

periplasmic space is a thin region that lies directly below the cell wall

cell membrane or plasmalemma is located directly below the periplasmic space and is a phospholipid bilayer which contains phospholipids, lipids, protein and sterols

nuclear membrane contains pores to allow communication with the rest of the cell

nucleus is a discrete organelle and, in addition to being the repository of the DNA, also contains proteins in the form of histones

mitochondrion is a semi- independent organelle as it possesses its own DNA and is capable of producing its own proteins on its own ribosomes which are referred to as mitoribosomes

fungal cell contains a vast number of ribosomes which are usually present in the form of polysomes— lines of ribosomes strung together by a strand of mRNA

ribosomes are the site of protein biosynthesis

vacuole is employed as a ‘storage space’ where nutrients, hydrolytic enzymes or metabolic intermediates are retained until required

MEDICAL SIGNIFICANCE OF FUNGI yeast C. albicans is the most frequently

encountered human fungal pathogen, being responsible for a wide range of superficial and systemic infections (oropharyngeal and genital conditions)

mould Aspergillus fumigatus is the dominant fungal pulmonary pathogen of humans and generally presents as a problem in those with pre-existing lung disease or damage

most commonly encountered dermatophytic infections are athlete’s foot and ringworm

ANTIFUNGAL THERAPY choice and dose of an antifungal will

depend upon the nature of the condition, whether there are any under- lying diseases, the health of the patient and whether anti- fungal resistance has been identified as compromising therapy

ideal antifungal drug should target a pathway or process specific to the fungal cell, so reducing the possibility of damaging tissue and inducing unwanted side effects

Polyene Antifungals polyene antifungals are characterized by

having a large macrolide ring of carbon atoms closed by the formation of an internal ester or lactone

principal polyenes are amphotericin B and nystatin

amphotericin B is produced by the bacterium Streptomyces nodosus and its activity is due to the ability to bind ergosterol, which is the dominant sterol in fungal cell membranes, and consequently increases membrane permeability by the formation of pores

nystatin was discovered in 1950 and exhibits the same mode of action as amphotericin B but tends to have lower solubility

Azole Antifungals first generation of azole antifungals

revolutionized the treatment of mucosal and invasive fungal infections

azoles are still the most widely used group of anti- fungal agents

azoles in current clinical use are clot- rimazole, miconazole, econazole and ketoconazole; newer

drugs such as itraconazole, fluconazole and voriconazole have important applications in the treatment of systemic infections

miconazole was the first azole used to treat systemic fungal infections but demonstrated a number of toxic side effects

ketoconazole produced high serum concentrations upon oral administration but had poor activity against aspergillosis

fluconazole has proved highly effective in the treatment of infections caused by C. albicans but shows limited activity against Aspergillus

itraconazole became available for clinical use in the late 1980s and was the first azole with proven efficacy against Aspergillus

novel azole drugs with increased ability to inhibit the fungal 14-α demethylase are also becoming available

agents which include voriconazole, posaconazole and ravuconazole, have a wider spectrum of activity than fluconazole and it has been suggested that some of them show fungicidal effects to some species

voriconazole is one of the newest second-generation triazole antifungal drugs and it shows good activity against pulmonary aspergillosis and cere- bral aspergillosis