Etiket Arşivleri: Lecture Notes

Industrial Fermentation Process ( SAIFUL IRWAN ZUBAIRI )



  1. Eng. (Chemical-Bioprocess) (Hons.), UTM

  2. Eng. (Bioprocess), UTM


TEL. (OFF.): 03-89215828,




28 MAY 2008


Cooling and Freezing process.

Concentration & Dehydration: Concentration, Spray Drying & Vacuum Drying process.

Industrial Fermentation process.

Irradiation Process.

Homogenization Process.

Wet and Dry Mixing process.

Transport and Conveying process.

Industrial Fermentation

Fermentation has many important uses in industry. Though the word fermentation can have stricter definitions, when speaking of it in industrial fermentation it more loosely refers to the breakdown of organic substances and re-assembly into other substances.

Somewhat ironically, fermenter culture in industrial capacity often refers to highly oxygenated and aerobic growth conditions, whereas fermentation in the biochemical context is a strictly anaerobic process

Food Fermentation

Ancient fermented food processes, such as making bread, wine, cheese and etc., can be dated to more than 6,000 years ago.

They were developed long before man had any knowledge of the existence of the microorganisms involved.

Fermentation is a powerful economic incentive for semi-industrialized countries, in their willingness to produce bio-ethanol.

Pharmaceuticals and the biotechnology industry

There are 5 major groups of commercially important fermentation:

Microbial cells or biomass as the product, e.g.: bakers yeast, lactobacillus, etc.

Microbial enzymes: catalase, amylase, protease, pectinase, glucose isomerase, cellulase, hemicellulase, lipase, lactase, streptokinase, etc.

Microbial metabolites:

Primary metabolites: ethanol, citric acid, glutamic acid, lysine, vitamins, polysaccharides etc.

Secondary metabolites: all antibiotic fermentation

Recombinant products: insulin, HBV, interferon, GCSF, streptokinase.

Biotransformation: phenyl acetyl carbinol, steroid biotransformation, etc.

Nutrient sources for industrial fermentation

Growth media are required for industrial fermentation, since any microbe requires water, oxygen, an energy source, a carbon source, a nitrogen source and micronutrients for growth.

Carbon & energy source + nitrogen source + O2 + other requirements → Biomass + Product + byproducts + CO2 + H2O + heat (aerobic fermentation)

Nutrient sources for industrial fermentation

Nutrient sources for industrial fermentation

Trace elements: Fe, Zn, Cu, Mn, Mo, Co.

Antifoaming agents: Esters, fatty acids, fats, silicones, sulphonates, polypropylene.

Buffers: Calcium carbonate, phosphates.

Growth factors: Some microorganisms cannot synthesize the required cell components themselves and need to be supplemented, e.g.: with thiamine, biotin, calcium pentothenate.

Nutrient sources for industrial fermentation

Precursors: Directly incorporated into the desired product: Phenyl ethylamine into Benzyl penicillin, Phenyl acetic acid into Penicillin G (for the making of Penicillin).

Inhibitors: To get the specific products: e.g.: sodium barbital for rifamycin.

Inducers: The majority of the enzymes used in industrial fermentation are inducible and are synthesized in response of inducers: e.g. starch for amylases, maltose for pollulanase, pectin for pectinase,olive oil and tween are also used at times.

Chelators: Chelators are the chemicals used to avoid the precipitation of metal ions. Chelators like EDTA, citric acid, polyphosphates are used in low concentrations.

EXAMPLE: Fermentation (wine)

The process of fermentation in wine is the catalyst function that turns grape juice into an alcoholic beverage.

During fermentation = yeast interact with sugars in the juice to create ethanol, commonly known as ethyl alcohol, and carbon dioxide (as a by-product).

In winemaking the temperature and speed of fermentation is an important consideration as well as the levels of oxygen present in the must at the start of the fermentation (aerobic fermentation).

The risk of immovable fermentation and the development of several wine faults can also occur during this stage which can last anywhere from 5 to 14 days for primary fermentation and potentially another 5 to 10 days for a secondary fermentation.

Fermentation may be done in stainless steel tanks, which is common with many white wines in an open wooden container, inside a wine barrel and inside the wine bottle itself like in the production of many sparkling wines

Winemaking considerations

During fermentation there are several factors that winemakers take into consideration.

The most notable is that of the internal temperature of the medium.

The biochemical process of fermentation itself creates a lot of residual heat which can take the medium out of the ideal temperature range for the wine.

Typically white wine is fermented between 64-68 °F (18-20 °C) though a wine maker may choose to use a higher temperature to bring out some of the complexity of the wine.

Red wine is typically fermented at higher temperatures up to 85 °F (29 °C).

Fermentation at higher temperatures may have adverse effect on the wine in stunning the yeast to inactivity and even “boiling off” some of the flavors of the wines.

Winemaking considerations

Some winemakers may ferment their red wines at cooler temperatures more typical of white wines in order to bring out more fruit flavors.

To control the heat generated during fermentation the winemaker has to choose a suitable vessel size or to use cooling devices of various sorts from the ancient Bordeaux traditions of placing the fermentation container on top of blocks of ice to today’s modern use of sophisticated fermentation tanks with built in cooling rings.

A risk factor involved with fermentation is the development of chemical residue and spoilage which can be corrected with the addition of sulfur dioxide (SO2), although excess SO2 can lead to a wine fault.

A winemaker who wishes to make a wine with high levels of residual sugar (like a dessert wine) may stop fermentation early either by dropping the temperature of the medium to stun the yeast or by adding a high level of alcohol (like brandy) to the medium to kill off the yeast and create a fortified wine.

Other types of fermentation

In winemaking there are different processes that fall under the title of “Fermentation” but might not follow the same procedure commonly associated with wine fermentation.

  (1) Bottle fermentation:

Bottle fermentation is a method of sparkling wine production originating in the Champagne region where after the medium has gone through a primary yeast fermentation – the wine is then bottled and goes through a secondary fermentation where sugar and additional yeast is added to the wine.

This secondary fermentation is what creates the carbon dioxide bubbles that sparkling wine is known for.

Other types of fermentation

  (2) Carbonic maceration (soaking): [anaerobic fermentation]

The process of carbonic maceration is also known as whole grape fermentation where instead of yeast being added to grape, the medium fermentation is encouraged to take place inside the individual grape berries (enzymes inside the grapes).

This method is involving with whole clusters of grapes being stored in a closed container with the oxygen in the container being replaced with carbon dioxide.

Unlike normal fermentation where yeast converts sugar into alcohol, carbonic maceration works by enzymes within the grape by breaking down the cellular matter to form ethanol and other chemical properties.

The resulting wines are typically soft and fruity.

Other types of fermentation

  (3) Malolactic fermentation:

Instead of yeast, bacteria plays a fundamental role in malolactic fermentation which is essentially the conversion of malic acid into lactic acid.

This has the benefit of reducing some of the bitterness (acidity) and making the resulting wine taste softer.

Depending on the style of wine that the winemaker is trying to produce, malolactic fermentation may take place at the same time as the yeast fermentation.

“Carbon dioxide is visible during the fermentation process in the form of bubbles in the fermented medium”

Fermentation Reactor


Mass transfer is the net movement of a component in a mixture from one location to another where the component exists at a different concentration. Often, the transfer takes place between 2 phases across interface.

Lecture Notes

The Enteric Bacteria

Introduction to the Family Enterobacteriaceae

Enterobacteriaceae are Gram-negative, oxidase-negative, rod-shaped bacteria, 0.3-1.0 x 1.0-6.0 um. Typically, they are motile by peritrichous flagella. They are facultative anaerobes, being  chemoorganotrophs that exhibit both respiratory and fermentative metabolism. Most grow well between 22 and 35°C on media containing peptone  or beef extract. They also grow on MacConkey’s agar which may be used for their selective isolation. Most grow on glucose as a sole carbon source, although some require vitamins and/or amino acids for growth. Thet produce mixed acids and often gas from fermentation of sugars. With very few exceptions they are catalase-positive, and most strains reduce nitrate to nitrite.  Escherichia coli is the type species. E. coli is considered the most thoroughly studied of all species of bacteria, and the family Enterobacteriaceae, as a whole, is the best studied group of microorganisms. Among the reasons for their popularity are their medical and economic importance, ease of isolation and cultivation, rapid generation time, and their ability to be genetically manipulated.  Enterobacteriaceae are distributed worldwide. They are found in water and soil and as normal intestinal flora in humans and many animals. They live as .saprophytically, as symbionts, epiphytes, and parasites. Their host range includes animals ranging from insects to humans, as well as fruits, vegetables, grains, flowering plants, and trees.

Economic and Medical Importance

As stated above, one of the reasons that the enterobacteriaceae have ben so widely studied is due to their obvious impact on human and animal health and on agricultural practice. The enterobacteriaceae include agents of food poisoning and gastroenteritis, hospital-acquired infections, enteric fevers (e.g. typhoid fever) and plague. They also cause infections in domestic, farm and zoo animals and include an important group of plant pathogens. Some of these bacteria are discussed below.

Plant Pathogens

Many species of Enterobacteriaceae are responsible for significant economic losses in agriculture.  Erwinia species cause blight, wilt, or soft-rot in numerous trees, flowers, and crops, often destroying substantial amounts of crops.  Among the plants affected are walnut and oak trees, rose, orchid and chrysanthemum flowers, and crops such as  corn, wheat, potato, carrot, sugar beet, sugar cane and pineapple.  Animal Pathogens Enterobacteriaceae cause disease in all sorts of animals, ranging from nematodes and insects through primates. Salmonella alone has been associated with disease in more than 125 species. Infections frequently cause problems in zoos, often in snakes and lizards. In regional primate centers in the United States, the most frequently diagnosed diarrheal diseases were caused by Enterobacteriaceae, most often by Shigella, E. coli and Salmonella. Klebsiella pneumoniae is a frequent cause of respiratory disease in primates, and Yersinia pseudotuberculosis is associated with enterocolitis and peritonitis.  Pets and farm animals are affected by a variety of enterobacterial diseases. Cats and dogs are susceptible to cystitis and other urogenital infections caused by E. coli. Proteus species cause other diseases in cats and dogs, and these animals can be carriers of Salmonella. Salmonellae, especially S. typhimurium, S. newport, and S. anatum, cause enteritis with high fatality and septic abortion in horses, and K. pneumoniae causes metritis in mares and pneumonia in foals.  Septicemia caused by E. coli is an important cause of death in chickens. Serotypes of Salmonella enterica are pathogenic and highly fatal for turkeys and other poultry, causing a characteristic diarrheal syndrome. Pullorum disease, caused by Salmonella pullorum, is highly fatal to eggs and chicks. Fowl typhoid, a septicemic disease of poultry, especially chickens, is caused by Salmonella gallinarum. Both pullorum disease and fowl typhoid can be largely eradicated if infected adult birds are slaughtered.  Nearly 200 Salmonella serotypes had been isolated from fowl in the United States. The distribution of salmonellosis in poultry is worldwide.  As in human disease, certain serotypes are prevalent in some regions and absent in others. A mortality rate of 10-20% is normal in young birds, mostly in the first two weeks after hatching.  Sheep suffer from a variety of illnesses caused by Enterobacteriaceae. Infant diarrhea in lambs, is usually caused by strains of E. coli producing a heat-stable enterotoxin. Most of these strains also contain the K-99 fimbrial adhesin. Salmonella abortion is usually caused by Salmonella abortusovis, S. typhimurium, or S. dublin, which also cause stillbirths and wool damage.  Calves are susceptible to both systemic colibacillosis and neonatal diarrhea (calf scours), which are usually fatal if not promptly treated. Specific heat-stable enterotoxigenic E. coli serotypes containing K99 fimbrial adhesin are the causative agents. Bovine mastitis has become a very prevalent disease since the advent of antibiotics. The most prevalent causative agents are E. coli and Serratia species, and less often, Klebsiella species and Citrobacter freundii. Salmonellosis is frequent in cattle. Most cases are due to Salmonella dublin and S. typhimurium, although more than 100 serotypes have been isolated. As with other animal infections, Salmonella is frequently introduced through contaminated feed.  Swine are subject to infection with several species of Enterobacteriaceae. E. coli infection may present as diarrhea in piglets, or as edema preceded by mild diarrhea. Both forms are acute and highly fatal. As in sheep and cows, the causative strains produce a heat-stable enterotoxin, but they may also produce a heat-labile enterotoxin. Swine strains  usually possess a K88 fimbrial adhesin, which is antigenically distict from K99. Sows are susceptible to mastitis and metritis caused by K. pneumoniae, and to enteritis and lymphadenitis caused by Yersinia enterocolitica. More than 100 Salmonella serotypes have been isolated from pigs.  However, only two serotypes, S. choleraesuis and S. typhisuis, have pigs as their primary host. S. choleraesuis has a wide host range, including humans, but S. typhisuis is rarely pathogenic to animals other than pigs. Salmonella typhimurium and S. derby are also frequently isolated from porcine salmonellosis.  Substantial losses in fishing industries are caused by enterobacterial diseases. Yersinia ruckeri is the cause of outbreaks of redmouth disease in salmon and trout hatcheries. Edwardsiella tarda is pathogenic for eels, catfish, and goldfish, and Edwardsiella ictaluri is pathogenic for catfish.  The host range for species of Enterobacteriaceae varies greatly. For example Proteus myxofaciens has been isolated only from larvae of gypsy moths and Escherichia blattae has been isolated only from the hindgut of cockroaches. Shigellae are seen only in primates. Others, including E. coli, many salmonellae, and yersiniae, infect or are carried by hosts ranging from insects to humans.  Human Pathogens Enterobacteriaceae as a group were originally divided into pathogens and nonpathogens based on their ability to cause diarrheal disease of humans. The pathogenic genera were Salmonella and Shigella. However, it is now known that E. coli causes at least five types of gastrointestinal disease in humans. Pathogenicity in E. coli strains is due to the presence of one or more virulence factors, including invasiveness factors (invasins), heat-labile and heat-stable enterotoxins, verotoxins, and colonization factors or adhesins. Pathogenic strains are usually identified by detection of a specific virulence factor or of a serotype associated with a virulence factor.  The most recently identified E. coli disease is hemorrhagic colitis caused by strains of serotype 0157:H7. The disease, characterized by painful abdominal cramping and bloody diarrhea, is caused by strains that produce verotoxin, and the same strains are associated with hemolytic uremic syndrome (HUS).  Yersinia enterocolitica causes diarrhea, probably by a combination of invasiveness and the presence of a heat-stable enterotoxin. Strains of Klebsiella pneumoniae and Enterobacter cloacae isolated from patients with tropical sprue contained a heat-stable enterotoxin. Edwardsiella tarda and Citrobacter strains are occasionally associated with diarrhea and have been shown to produce heat-stable or heat-labile enterotoxin. Foodborne and waterborne disease outbreaks in the U.S. are frequently associated with Enterobacteriaceae.  According to the Centers for Disease Control (CDC), 40-45% of such outbreaks are caused by Enterobacteriaceae, the overwhelming majority by Salmonella. Meats, milk and milk products, and eggs are the most common vehicles of transmission.  Such figures represent only a small fraction of total foodborne disease, since the etiologic agent is identified in only about one-third of the outbreaks, and many outbreaks are undetected or are not reported to the Centers for Disease Control. For Salmonella, it is estimated that each reported case represents about 100 total cases. The largest outbreak of salmonellosis in the United States occurred in 1985 in Illinois and Wisconsin, where an estimated 170,000 to almost 200,000 persons were infected with Salmonella typhimurium transmitted in pasteurized milk from a single dairy plant.

 The incidence and recognition of rheumatoid disease occurring secondary to foodborne and waterborne diarrheal disease have also increased. These diseases include reactive arthritis, Reiter’s syndrome, ankylosing spondylitis, septic and aseptic arthritis, ulcerative colitis, Crohn’s disease, and Whipple’s disease. Y. enterocolitica, Y. pseudotuberculosis, Shigella flexneri, Shigella dysenteriae, various salmonellae, E. coli, and K. pneumoniae have been associated with these chronic conditions.  Waterborne disease outbreaks due to Enterobacteriaceae are usually due to contaminated wells. Cases of shigellosis due to a contaminated wells have been reported; even typhoid fever has occurred fairly recently in community water systems contaminated with human sewage.

 Enterobacteriaceae not normally associated with the GI tract or diarrheal disease may still be pathogens of humans. Most notably, Yersinia pestis, which does not have an intestinal habitat, is the etiologic agent of plague a highly fatal disease that has dessimated whole populations of individuals at several times in the history of civilization. Furthermore, most, if not all, Enterobacteriaceae are opportunistic pathogens. Once established, they can cause a variety of infections, including urinary tract disease, pneumonia, septicemia, meningitis, and wound infection.  According to the CDC,
Enterobacteriaceae are responsible for 40-50% of nosocomial infections occurring in the United States. E. coli is the worst offender, followed by Klebsiella, Proteus-Providencia-Morganella, Serratia, and Citrobacter. The compromised host is particularly susceptible to nosocomial infections. Catheterized patients, patients on immunosuppressants, burn patients, cancer patients, and elderly patients are all especially vulnerable to opportunistic pathogens. To make matters worse, many of these organisms acquired in the hospital setting are multiply drug resistant.

Taxonmy and Classification of Enteric Bacteria

In artificial classification schemes (e.g.  Bergey’s Manual of Systematic Bacteriology, 1st edition, 1986) Enterobacteriaceae is a family of bacteria in Section  8 – Gram-negative facultatively anaerobic rods. Because of the large number and broad range of phenotypic properties that solidifiy the group, these traits being a reflection of their genetic relatedness, these bacteria have remained unified in modern phylogenetic schemes based on 16S ribosomal RNA comparison. Thus, Citrobacter, Edwardsiella, Enterobacter, Erwinia, Escherichia, Klebsiella, Proteus, Providencia, Salmonella, Serratia, Shigella, and Yersinia (along with several other genera, including Hafnia, Morganella, Photorhabdus,and Xenorhabdus) are  presently classified in the subclass Gammaproteobacteria, order Enterobacteriales, family Enterobacteriaceae .

The classic definition of an enteric bacterium is one that is found in the intestinal tract of warm-blooded animals in health and disease, but bacteriologists reserve the term for reference to E. coli and its relatives, even though some of the relatives of E. coli rarely or never are found growing in the GI tract. But in the end, this is one of the most close-related and cohesive groups of bacteria that can be brought together for discussion.

The Genus Escherichia

Theodor Escherich first described E. coli in 1885, as Bacterium coli commune, which he isolated from the feces of neonates. It was later renamed Escherichia coli, and for many years the bacterium was simply considered to be a commensal organism of the large intestine. It was not until 1935 that a strain of E. coli was shown to be the cause of an outbreak of diarrhea among neonates.

Most investigations of enteric organisms at the turn of the 20th century were concerned with the problems of being able to distinguish the “typhoid bacillus” and other types of Salmonella from non-Salmonella organisms. Early workers also demonstrated that there were a number of types and subtypes of these organisms, which could easily be distinguished from the typhoid bacillus and E. coli. Thus, the biochemical techniques that have become the basis for most taxonomic studies came into being during the early 1900s. These studies led to the modern taxonomy of the group, which in principle is still valid today.  Initially, the family Enterobacteriaceae was created by Rahn in 1937, for the genus Enterobacter, and despite some debate about nomenclature among bacteriologists, the family name was maintained with the type genus becoming Escherichia.  The family currently comprises Gram-negative, nonsporeforming, rod-shaped bacteria that are often motile by means of peritrichous flagella. The majority of strains grow well on the usual laboratory media in both the presence and absence of oxygen, and metabolism can be either respiratory or fermentative. The fermentation products of glucose and other carbohydrate substrates include mixed acids and (usually) detectable gas. Most strains are oxidase-negative and are able to reduce nitrate to nitrite.

The taxonomic distinctiveness of Escherichia has been confirmed by rRNA-DNA heteroduplex studies. On the basis of DNA-DNA relatedness studies, the genera of enteric bacteria are placed into a series of groupings, with Escherichia and Shigella forming a close group distinct from their nearest group, which includes the genera  Citrobacter, Enterobacter, Klebsiella, and Salmonella.  Although most investigations of the genus Escherichia have centered on various aspects of the E. coli species, it should not be forgotten that a number of other species have been described, including E. blattae, E. fergusonii, E. hermanii, and E. vulneris. These species can be differentiated on the basis of a large battery of biochemical tests.  For many years it has been realized that there exists a close relationship between two genera of enterics,  Escherichia and Shigella. This is true for their biochemical characteristics as well as various other phenotypic traits. Also, studies of certain E. coli antigens have shown a close relationship (“cross reactivity”) with Shigella antigens. The “O” antigens of virtually all serotypes of Shigella are either identical with or closely related to those of E. coli.  The discovery that the characteristic “invasiveness” of Shigella strains is also possessed by certain types of E. coli, which have become known as enteroinvasive E. coli (EIEC), also suggests a close relationship.  EIEC can cause dysentery-like symptoms clinically indistinguishable from those caused by strains of Shigella. Furthermore, antigens of E. coli strain O124 are shown to have a very close relationship to Shigella dysenteriae type 3 antigens, and a serological relationship between E. coli strain O129 and S. flexneri type 5 is also known. Finally, enterohemorrhagic strains of E. coli  (EHEC), specifically E. coli O157:H7 produce the shiga (vero) toxin which is identical to the toxin produced by Shigella dysenteriae.The point being that it has become apparent that the line dividing these two genera of enteric bacteria is exceedingly thin, and it should be remembered that on the basis of  DNA relatedness alone, Shigella and E. coli have been considered one genus.

Detection and Isolation of Escherichia coli

E. coli as an Indicator of
Fecal Pollution

For most of the 20th
century, E. coli has been used as the principal indicator of fecal
pollution in both tropical and temperate countries. E. coli comprises
about 1% of the total fecal bacterial flora of humans and most warm-blooded
animals. Sewage is always likely to contain E. coli in relatively large
numbers. In addition, E. coli, being a typical member of the Enterobacteriaceae,
is presumed to have survival characteristics very similar to those of the
well-known pathogenic members of the family, Salmonella and Shigella.
Thus, E. coli has been used world-wide as an indicator of fecal
microbiological contamination. As such an indicator organism, its value is
significantly enhanced by the ease with which it can be detected. and cultured.

Tests to identify
isolates as E. coli have, of necessity, been simple tests designed
predominantly to differentiate them from organisms normally associated with
uncontaminated water. Since full biochemical analyses are not generally
performed, the term  “coliform” has been coined to describe E.
like organisms that satisfy these limited tests. As a result,
regulations are promulgated throughout the world defining standards for water
based on the so-called “coliform count.” For example, in the U.S.,
according to a regulation published in the Federal Register (1986), there is a
requirement that there be 0 coliforms/100 ml drinking water, as determined by
any method for any sampling frequency. Since not all organisms which meet the
criteria of a coliform are associated with the intestinal tract (some may be
saprophytic), a further distinction must be made between “fecal
coliforms” (E coli) and “nonfecal coliforms” (e.g. Klebsiella
and Enterobacter).

Early attempts to
distinguish strains of E. coli from other related Enterobacteriaceae
centered on being able to distinguish them from the various pathogenic groups,
since E. coli was initially not considered to be a pathogen. When E.
was recognized to be a useful marker for fecal pollution, it similarly
became important to distinguish it from related species likely to be found
naturally in the environment. The realization that strains of E. coli
generally ferment lactose, while those of Salmonella and Shigella do
not, led to an early method of preliminary differentiation. The IMViC tests
were developed in order to distinguish strains of E. coli from related
species that also produced acid and gas from the fermentation of lactose. IMViC
is an acronym in which the capital letters stand for Indole, Methyl
red, Voges-Proskauer, and Citrate.) The IMViC set of tests
examines: the ability of an organism to (1) produce Indole; (2) produce
sufficient acid to change the color of Methyl red indicator; (3) produce
acetoin, an intermediate in ther butanediol fermentation pathway (a positive
result of the Voges-Proskauer test); and (4) the ability to grow on Citrate
as the sole source of carbon. Lactose fermenters are considered E. coli
if they are positive in the first two tests and negative in the second two.

Detection of E.
in Food

The International
Commission on Microbiological Specifications for Foods (ICMSF, 1978) has
adopted a set of standard techniques for the enumeration of E. coli in
food products, accepted by the International Standards Organization (ISO,
1984). This method employs the use of lauryl sulfate tryptose broth at 35 or 37°C as a mildly selective-enrichment
medium. This is followed by growth in EC broth containing 0.15%  bile
salts at 45°C
as a  second selective step. The ability to produce indole from tryptophan
(in tryptone broth) at 45°C
defines the strains as E. coli. These tests  miss some types of E.
such as those most closely related to the Shigella group, but
it is the detection of possible fecal contamination that is important in these
tests rather than the presence of specific types.

Detection of E.
in Water

There is no method
for the detection of E. coli in water that is accepted throughout the
world. In the US, a standard method using membrane filter enumeration for both
total and thermotolerant coliforms has been established (American Public Health
Association (1986). Further IMViC tests on selected isolates can then be

In the UK, the
definition of E. coli in water microbiology is also based on the ability
to produce gas from lactose and produce indole from tryptophan at 44°C. A method for enumeration
employs a standard multiple tube test with a modified glutamate synthetic
medium at 37°C
as a first selective step, followed by further cultivation in standard media at

Detection of E.
in Clinical Specimens

While large numbers
of E. coli will be found in fecal specimens or specimens contaminated
with feces or intestinal contents, most other clinical specimens are usually
not contaminated with E. coli. The major exception is urine, which
requires special attention in the clinical situation. From those specimens in
which E. coli is likely to be present in large numbers, direct plating
on media such as MacConkey agar or Eosin Methylene Blue (EMB) agar is
sufficient. If the number of E. coli is likely to be very low or the
amount of specimen is limited, enrichment in a rich nutrient medium such as
brain heart infusion broth may be used. A number of different commercially
available kits are generally used to identify the isolates as E. coli.

From specimens likely
to contain only a few viable E. coli cells, such as blood from patients
suspected of having E. coli bacteremia, various enrichment procedures
are used.  Identification follows standard bacteriological techniques.

Left: Escherichia
microcolony. Right: E.coli colonies on EMB Agar.

Rapid Methods for
Detecting E. coli

A fluorogenic
detection method has been developed based on the cleavage of
methylumbelliferyl-D-glucuronide (MUG) to the free methylumbelliferyl moiety,
which fluoresces a blue color after irradiation with long-wave ultraviolet radiation.
Although strains of E. coli are generally positive in this test, some
strains of Salmonella, Shigella, and Yersinia are also capable of
splitting MUG; the latter two genera are usually not present in food. A
disadvantage is that enterohemorhagic E. coli (EHEC) strains are
generally negative in this test.  MUG can be added to various selective
media, so there is a great potential in its use for detecting E. coli.

Automated or
semi-automated systems are also being used for the detection of E. coli
as part of the detection methods for Enterobacteriaceae. Techniques
involving impedance measurements have shown promise. Other techniques such as
immunoassays and nucleic acid hybridization studies can also be used to
enumerate E. coli, and DNA probes directed at a number of genes have
also been developed.

of E. coli

Physiologically, E.
is versatile and well-adapted to its characteristic habitats.  In
the laboratory it can grow in media with glucose as the sole organic
constituent. Wild-type E. coli has no growth factor requirements, and
metabolically it can transform glucose into all of the macromolecular
components that make up the cell. The bacterium can grow in the presence or
absence of O2. Under anaerobic conditions it will grow by means of
fermentation, producing characteristic “mixed acids and gas” as end
products. However, it can also grow by means of anaerobic respiration, since it
is able to utilize NO3 or fumarate as final electron acceptors for
respiratory electron transport processes. In part, this adapts E. coli
to its intestinal (anaerobic) and its extraintestinal (aerobic or anaerobic)

In the ecological
niches that E. coli occupies, its abilities to grow both aerobically and
anaerobically are important.  E. coli is  well adapted to its
intestinal environment as it is able to survive on a relatively limited number
of low-molecular weight substances, which may only be available transiently and
at relatively low concentrations. The generation time for E. coli in the
intestine is thought to be about 12 hours. The type of nutrients available
there to E. coli  consist of mucus, desquamated cells, intestinal
enzyme secretions, and incompletely digested food.  Given the absorption
capacity and efficiency of the intestine, there are probably only small amounts
free carbohydrates or other easily absorbable forms of nutrients, and there is
competition from hundreds of other types pf bacteria. A similar situation
probably also applies to sources of nitrogen.

In its natural
environment, as well as the laboratory, E. coli can respond to
environmental signals such as chemicals, pH, temperature, osmolarity, etc., in
a number of very remarkable ways considering it is a single-celled organism.
For example, it can sense the presence or absence of chemicals and gases in its
environment and swim towards or away from them. Or it can stop swimming and
grow fimbriae that will specifically attach it to a cell or surface receptor.
In response to changes in temperature and osmolarity, it can vary the pore
diameter of its outer membrane porins to accommodate larger molecules
(nutrients) or to exclude inhibitory substances (e.g. bile salts). With its
complex mechanisms for regulation of metabolism the bacterium can survey the
chemical content its environment in advance of synthesizing any enzymes
necessary to use these compounds. It does not wastefully produce enzymes for
degradation of carbon sources unless they are available, and it does not
produce enzymes for synthesis of metabolites if they are available as nutrients
or growth factors in the environment.

in the Gastrointestinal Tract

The commensal E.
strains that inhabit the large intestine of all humans and
warm-blooded animals comprise about 1% of the total  bacterial biomass.
This E. coli flora is in constant flux. One study on the distribution of
different E. coli strains colonizing the large intestine of women during
a one year period (in a hospital setting) showed that 52.1% yielded one
serogroup, 34.9% yielded two, 4.4% yielded three, and 0.6% yielded four.
The most likely source of new serotypes of E. coli is acquisition by the
oral route. To study oral acquisition, the carriage rate of E. coli carrying
antibiotic-resistance (R) plasmids was examined among vegetarians, babies, and
nonvegetarians. It was assumed that nonvegetarians might carry more E. coli
with R factors due to their presumed high incidence in animals treated with
growth-promoting antimicrobial agents. However, omnivores had no higher an
incidence of R-factor-containing E. coli than vegetarians, and babies
had more resistant E. coli in their feces than nonvegetarians. No
suitable explanation could be offered for these findings.  Besides,
investigation of the microbial flora of the uninhabited Krakatoa archipelago
has shown the presence of antibiotic-resistant E. coli associated with

Caused by Pathogenic E. coli

E. coli is responsible
primarily for three types of infections in humans: urinary tract infections,
neonatal meningitis, and intestinal diseases. These condituions
depend on a specific array of pathogenic (virulence) determinants possessed by
the organism.  Pathogenic E. coli are discussed elsewhere in the
text in more detail at Pathogenic E.
coli:  Gastroenteritis, Urinary tract Infections and Neonatal Meningitis.

Urinary Tract

Uropathogenic E.
cause 90% of the urinary tract infections (UTI) in
anatomically-normal, unobstructed urinary tracts. The bacteria colonize from
the feces or perineal region and ascend the urinary tract to the bladder.
Bladder infections are 14-times more common in females than males by virtue of
the shortened urethra. The typical patient with uncomplicated cystitis is a
sexually-active female who was first colonized in the intestine with a
uropathogenic E. coli strain. The organisms are propelled into the
bladder from the periurethral region during sexual intercourse. With the aid of
specific adhesins they are able to colonize the bladder.

The adhesin that has
been most closely associated with uropathogenic E. coli is the P fimbria
(or pyelonephritis-associated pili [PAP] pili). The letter designation is
derived from the ability of P fimbriae to bind specifically to the P blood
group antigen which contains a D-galactose-D-galactose residue. The fimbriae
bind not only to red cells but to a specific galactose dissaccharide that is
found on the surfaces uroepithelial cells in approximately 99% of the

The frequency of the
distribution of this host cell receptor plays a role in susceptibility and
explains why certain individuals have repeated UTI caused by E. coli.
Uncomplicated E. coli UTI virtually never occurs in individuals lacking
the receptors.

Uropathogenic strains
of E. coli possess other determinants of virulence in addition to P
fimbriae. E. coli with P fimbriae also possess the gene for Type 1
fimbriae, and there is evidence that P fimbriae are derived from Type 1
fimbriae by insertion of a new fimbrial tip protein to replace the
mannose-binding domain of Type 1 fimbria. In any case, Type 1 fimbriae could
provide a supplementary mechanism of adherence or play a role in aggregating
the bacteria to a specific manosyl-glycoprotein that occurs in urine.

Uropathogenic strains
of E. coli usually produce siderophores that probably play an essential
role in iron acquisition for the bacteria during or after colonization. They
also produce hemolysins which are cytotoxic due to formation of transmembranous
pores in host cells. One strategy for obtaining iron and other nutrients for
bacterial growth may involve the lysis of host cells to release these
substances. The activity of hemolysins is not limited to red cells since the
alpha-hemolysins of E. coli also lyse lymphocytes, and the
beta-hemolysins inhibit phagocytosis and chemotaxis of neutrophils.

Another factor
thought to be involved in the pathogenicity of the uropathogenic strains of E.
is their resistance to the complement-dependent bactericidal effect of
serum. The presence of K antigens is associated with upper urinary tract
infections, and antibody to the K antigen has been shown to afford some degree
of protection in experimental infections. The K antigens of E. coli are
“capsular” antigens that may be composed of proteinaceous organelles
associated with colonization (e.g., CFA antigens), or made of polysaccharides.
Regardless of their chemistry, these capsules may be able to promote bacterial
virulence by decreasing the ability of antibodies and/or complement to bind to
the bacterial surface, and the ability of phagocytes to recognize and engulf
the bacterial cells. The best studied K antigen, K-1, is composed of a polymer
of N-acetyl neuraminic acid (sialic acid), which besides being antiphagocytic, has
the additional property of being an antigenic disguise.

Neonatal meningitis

meningitis affects1/2,000-4,000 infants. Eighty percent of E. coli
strains involved synthesize K-1 capsular antigens (K-1 is only present 20-40%
of the time in intestinal isolates).

E. coli strains invade the
blood stream of infants from the nasopharynx or GI tract and are carried to the

The K-1 antigen is
considered the major determinant of virulence among strains of E. coli
that cause neonatal meningitis. K-1 is a homopolymer of sialic acid. It
inhibits phagocytosis, complement, and responses from the host’s immunological
mechanisms. K-1 may not be the only determinant of virulence, however, as
siderophore production and endotoxin are also likely to be involved.

Epidemiologic studies
have shown that pregnancy is associated with increased rates of colonization by
K-1 strains and that these strains become involved in the subsequent cases of
meningitis in the newborn. Probably, the infant GI tract is the portal of entry
into the bloodstream. Fortunately, although colonization is fairly common,
invasion and the catastrophic sequelae are rare.

Neonatal meningitis
requires antibiotic therapy that usually includes ampicillin and a
third-generation cephalosporin.

Intestinal Diseases

As a pathogen, E.
, of course, is best known for its ability to cause intestinal
diseases. Five classes (virotypes) of E. coli that cause diarrheal
diseases are now recognized: enterotoxigenic E. coli (ETEC),
enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC),
enteropathogenic E. coli (EPEC), and enteroaggregative E. coli
(EAggEC). Each class falls within a serological subgroup and manifests distinct
features in pathogenesis.

Enterotoxigenic E.

ETEC are an important
cause of diarrhea in infants and travelers in underdeveloped countries or
regions of poor sanitation. The diseases vary from minor discomfort to a severe
cholera-like syndrome. ETEC are acquired by ingestion of contaminated food and
water, and adults in endemic areas evidently develop immunity. The disease
requires colonization and elaboration of one or more enterotoxins. Both traits
are plasmid-encoded.

ETEC adhesins are
fimbriae which are species-specific. For example, the K-88 fimbrial Ag is found
on strains from piglets; K-99 Ag is found on strains from calves and lambs; CFA
I, and CFA II, are found on strains from humans. These fimbrial adhesins adhere
to specific receptors on enterocytes of the proximal small intestine.

Enterotoxins produced
by ETEC include the LT (heat-labile) toxin and/or the ST (heat-stable) toxin,
the genes for which may occur on the same or separate plasmids. The LT
enterotoxin is very similar to cholera toxin in both structure and mode of
action. It is an 86kDa protein composed of an enzymatically active (A) subunit
surrounded by 5 identical binding (B) subunits. It binds to the same identical
ganglioside receptors that are recognized by the cholera toxin (i.e., GM1), and
its enzymatic activity is identical to that of the cholera toxin.

The ST enterotoxin is
actually a family of toxins which are peptides of molecular weight about 2,000
daltons. Their small size explains why they are not inactivated by heat. ST
causes an increase in cyclic GMP in host cell cytoplasm leading to the same
effects as an increase in cAMP. STa is known to act by binding to a guanylate
cyclase that is located on the apical membranes of host cells, thereby
activating the enzyme. This leads to secretion of fluid and electrolytes
resulting in diarrhea.

Symptoms ETEC
infections include diarrhea without fever. The bacteria colonize the GI tract
by means of a fimbrial adhesin, e.g. CFA I and CFA II, and are noninvasive, but
produce either the LT or ST toxin.

Enteroivasive E.

EIEC closely resemble
Shigella in their pathogenic mechanisms and the kind of clinical illness
they produce. EIEC penetrate and multiply within epithelial cells of the colon
causing widespread cell destruction. The clinical syndrome is identical to Shigella
dysentery and includes a dysentery-like diarrhea with fever. EIEC apparently
lack fimbrial adhesins but do possess a specific adhesin that, as in Shigella,
is thought to be an outer membrane protein. Also, likeShigella, EIEC are
invasive organisms. They do not produce LT or ST toxin and, unlike Shigella,
they do not produce the shiga toxin.

Enteropathogenic E.

EPEC induce a watery
diarrhea similar to ETEC, but they do not possess the same colonization factors
and do not produce ST or LT toxins. They produce a non fimbrial adhesin
designated intimin, an outer membrane protein, that mediates the final stages
of adherence. Although they do not produce LT or ST toxins, there are reports
that they produce an enterotoxin similar to that of Shigella. Other
virulence factors may be related to those in Shigella.

Adherence of EPEC
strains to the intestinal mucosa is a very complicated process and produces
dramatic effects in the ultrastructure of the cells resulting in rearrangements
of actin in the vicinity of adherent bacteria. The phenomenon is sometimes
called “attaching and effacing” of cells. EPEC strains are said to be
“moderately-invasive” meaning they are not as invasive as Shigella,
and unlike ETEC or EAggEC, they cause an inflammatory response. The diarrhea
and other symptoms of EPEC infections probably are caused by bacterial invasion
of host cells and interference with normal cellular signal transduction, rather
than by production of toxins.

Some types of EPEC
are referred to as Enteroadherent E. coli (EAEC), based on specific
patterns of adherence. They are an important cause of traveler’s diarrhea in
Mexico and in North Africa.

Enteroaggregative E.

The distinguishing
feature of EAggEC strains is their ability to attach to tissue culture cells in
an aggregative manner. These strains are associated with persistent diarrhea in
young children. They resemble ETEC strains in that the bacteria adhere to the
intestinal mucosa and cause non-bloody diarrhea without invading or causing
inflammation. This suggests that the organisms produce a toxin of some sort.
Recently, a distinctive heat-labile plasmid-encoded toxin has been isolated
from these strains, called the EAST (EnteroAggregative ST) toxin. They also
produce a hemolysin related to the hemolysin produced by E. coli strains
involved in urinary tract infections. The role of the toxin and the hemolysin
in virulence has not been proven. The significance of EAggEC strains in human
disease is controversial.

Enterohemorrhagic E.

EHEC are represented
by a single strain (serotype O157:H7), which causes a diarrheal syndrome
distinct from EIEC (and Shigella) in that there is copious bloody
discharge and no fever. A frequent life-threatening situation is its toxic
effects on the kidneys (hemolytic uremia).

EHEC has recently
been recognized as a cause of serious disease often associated with ingestion
of inadequately cooked hamburger meat. Pediatric diarrhea caused by this strain
can be fatal due to acute kidney failure (hemolytic uremic syndrome [HUS]).
EHEC are also considered to be “moderately invasive”. Nothing is
known about the colonization antigens of EHEC but fimbriae are presumed to be
involved. The bacteria do not invade mucosal cells as readily as Shigella,
but EHEC strains produce a toxin that is virtually identical to the Shiga
toxin. The toxin plays a role in the intense inflammatory response produced by
EHEC strains and may explain the ability of EHEC strains to cause HUS. The
toxin is phage encoded and its production is enhanced by iron deficiency.

Applications of E. coli

The advances in
molecular biology, genetics and biochemistry during the past four decades have
led to an enormous development in the field of biotechnology. Studies with E.
have played a major role in these developments, and the bacterium has
been in the forefront of many technological advances.

In the early days of
biotechnology (1960s), emphasis was placed on improvements of established
procedures of bioprocessing, such as the production of yeasts, vaccines, and
antibiotics.  These investigations stimulated genetic research of microbes
to increase their potential to produce a wide variety of products in the
service of  humanity. Although much was being learned about E. coli
and its genetics, the direct use of the bacterium in the industry was limited.
The industrial production of the amino acid threonine by E. coli
mutants, begun in 1961, is an exception.

At this time, organisms were generally subjected to mutagenic agents, which
produced a series of random mutations, from which the specifically required
mutants were selected.

In the last two
decades, procedures have evolved which permit the preparation of strains that
have very specific productive capabilities. As the genetic structure of E.
was well known, and it is an organism which can grow on simple media
(mineral salts and glucose) under aerobic and anaerobic conditions, the
bacterium became the basis for most developments in genetic manipulations
leading to genetic engineering.

The basic principle
of these genetic manipulations is gene cloning, which enables the isolation and
replication of individual DNA fragments. This consists of a series of linked
steps, involving the isolation of the desired gene as double-stranded DNA (dsDNA),
insertion of the gene into a suitable vector, and using the vector to introduce
the DNA into a cell which will express the desired genetic information.
In the case of cloning a gene in E. coli,first the DNA of suitable
character is isolated, then it is joined to the DNA of a suitable vector
producing a series of recombinant molecules. Then the recombinant molecules are
introduced into the bacterium in which the target gene becomes established.
Recombinants are selected in various ways with the purpose of expressing the
desired genetic information.

The source for DNA
cloning can be genomic DNA fragments, cDNA fragments produced by the action of
reverse transcriptase on mRNA molecules, chemically synthesized
oligonucleotides, or amplified DNA from the products of the polymerase chain
reaction (PCR).  Plasmids, phages, and cosmids have all been successfully
used as vectors, and transformation, transfection, and transduction have all
been used to introduce the foreign DNA into the E. coli cell. Plasmids
are among the most widely used vectors for the insertion of foreign DNA into an
E. coli. Plasmids lend themselves very well as vectors since they are
independent replicons which are stabily inherited in an extrachromosomal state
and can be made to carry easily identifiable phenotypic markers such as
antibiotic resistance or sugar fermentation.

An example of the use
of plasmids to introduce a foreign gene into E. coli in order to produce
a useful product is illustrated by the use of the E. coli plasmid pBR322
to clone the gene for production of the human growth hormone,
somatostatin.  In this case, the gene for the small polypeptide hormone
was produced by synthetic means. The double-stranded DNA coding for the 15
amino acids of somatostatin was synthesized with the addition of a translation
a stop signal at the end. The synthetic gene was then recombined with the
plasmid within the beta-galactosidase structural gene and introduced into E.
In this way, the production of the somatostatin peptide could be
controlled by the lac operon.  In a similar manner, the genes for human
insulin production were inserted into E. coli which was then able to
synthesize the human hormone.

Such general
techniques of molecular biology and bacterial genetics are now being applied
within research laboratories and industry to produce a wide variety of strains
of genetically engineered E. coli from which a number of useful products
can be produced. Likewise, the problems associated with the expression of
eukaryotic DNA by a procaryotic promoter in E. coli were solved by
construction of a fusion gene. In this system, the control region and the
N-terminal coding sequence of an E. coli gene are ligated to a
eukaryotic sequence sothat translation of the chimeric protein can occur. The
only condition is that the eukaryotic sequence must be in the correct reading
frame. The desired protein is then enzymatically or chemically cleaved from the
E. coli product.

E. coli strains ave been
genetically engineered to produce a variety of mammalian proteins, especially
products of medical or veterinary interest including enzymes and vaccine
components. E. coli has also been used to manufacture other
substances  including enzymes that are useful in the degradation of
cellulose and aromatic compounds  and enzymes for ethanol production.
There may be no limit to what E. coli can produce through recombinant
DNA technology as long as the substance is a natural product for which a
genetic sequence can be found.


Staphylococcus aureus. Electron micrograph
from Visuals Unlimited, with

The Staphylococci

Staphylococci are Gram-positive spherical
bacteria that occur in microscopic clusters resembling grapes. Bacteriological
culture of the nose and skin of normal humans invariably yields staphylococci.
In 1884, Rosenbach described the two pigmented colony types of staphylococci
and proposed the appropriate nomenclature: Staphylococcus aureus
(yellow) and Staphylococcus albus (white). The latter species is now
named Staphylococcus epidermidis. Although more than 20 species of Staphylococcus
are described in Bergey’s Manual (2001), only Staphylococcus aureus and Staphylococcus
are significant in their interactions with humans. S. aureus
colonizes mainly the nasal passages, but it may be found regularly in most
other anatomical locales. S epidermidis is an inhabitant of the skin.

Taxonomically, the genus Staphylococcus
is in the Bacterial family Staphylococcaceae, which includes
three lesser known genera, Gamella, Macrococcus and Salinicoccus.
The best-known of its nearby phylogenetic relatives are the members of the
genus Bacillus in the family Bacillaceae, which is on the same
level as the family Staphylococcaceae. The Listeriaceae are also
a nearby family.

Staphylococcus aureus forms a fairly large
yellow colony on rich medium, S. epidermidis has a relatively small
white colony. S. aureus is often hemolytic on blood agar; S.
is non hemolytic. Staphylococci are facultative anaerobes that
grow by aerobic respiration or by fermentation that yields principally lactic
acid. The bacteria are catalase-positive and oxidase-negative. S. aureus
can grow at a temperature range of 15 to 45 degrees and at NaCl concentrations
as high as 15 percent. Nearly all strains of S. aureus produce the
enzyme coagulase: nearly all strains of S. epidermidis lack this enzyme.
S. aureus should always be considered a potential pathogen; most strains
of S. epidermidis are nonpathogenic and may even play a protective role
in their host as normal flora. Staphylococcus epidermidis may be a
pathogen in the hospital environment.

Staphylococci are perfectly spherical cells
about 1 micrometer in diameter. They grow in clusters because staphylococci
divide in two planes. The configuration of the cocci helps to distinguish
staphylococci from streptococci, which are slightly oblong cells that usually
grow in chains (because they divide in one plane only). The catalase test is
important in distinguishing streptococci (catalase-negative) from
staphylococci, which are vigorous catalase-producers. The test is performed by
adding 3% hydrogen peroxide to a colony on an agar plate or slant.
Catalase-positive cultures produce O2 and bubble at once. The test should
not be done on blood agar because blood itself contains catalase.

Table 1. Important phenotypic characteristics of Staphylococcus aureus

Gram-positive, cluster-forming coccus

nonmotile, nonsporeforming facultative anaerobe

fermentation of glucose produces mainly lactic acid

ferments mannitol (distinguishes from S. epidermidis)

catalase positive

coagulase positive

golden yellow colony on agar

normal flora of humans found on nasal passages, skin and mucous membranes

pathogen of humans, causes a wide range of suppurative infections, as well
as food poisoning and toxic shock syndrome 

Pathogenesis of S. aureus infections

Staphylococcus aureus causes a variety of
suppurative (pus-forming) infections and toxinoses in humans. It causes superficial
skin lesions such as boils, styes and furunculosis; more
serious infections such as pneumonia, mastitis, phlebitis,
meningitis, and urinary tract infections; and deep-seated
infections, such as osteomyelitis and endocarditis. S. aureus
is a major cause of hospital acquired (nosocomial) infection of surgical
wounds and infections associated with indwelling medical devices. S. aureus
causes food poisoning by releasing enterotoxins into food, and toxic
shock syndrome
by release of superantigens into the blood stream.

S. aureus expresses many potential virulence
: (1) surface proteins that promote colonization of host
tissues; (2) invasins that promote bacterial spread in tissues (leukocidin,
kinases, hyaluronidase); (3) surface factors that inhibit
phagocytic engulfment (capsule, Protein A); (4) biochemical
properties that enhance their survival in phagocytes (carotenoids, catalase
production); (5) immunological disguises (Protein A, coagulase, clotting
); and (6) membrane-damaging toxins that lyse eukaryotic cell
membranes (hemolysins, leukotoxin, leukocidin; (7)
exotoxins that damage host tissues or otherwise provoke symptoms of disease (SEA-G,
TSST, ET (8) inherent and acquired resistance to antimicrobial

For the majority of diseases caused by S.
, pathogenesis is multifactorial, so it is difficult to determine
precisely the role of any given factor. However, there are correlations between
strains isolated from particular diseases and expression of particular
virulence determinants, which suggests their role in a particular diseases. The
application of molecular biology has led to advances in unraveling the
pathogenesis of staphylococcal diseases. Genes encoding potential virulence
factors have been cloned and sequenced, and many protein toxins have been
purified. With some staphylococcal toxins, symptoms of a human disease can be
reproduced in animals with the purified protein toxins, lending an
understanding of their mechanism of action.

Human staphylococcal infections are frequent,
but usually remain localized at the portal of entry by the normal host
defenses. The portal may be a hair follicle, but usually it is a break in the
skin which may be a minute needle-stick or a surgical wound. Foreign bodies,
including sutures, are readily colonized by staphylococci, which may makes
infections difficult to control. Another portal of entry is the respiratory
tract. Staphylococcal pneumonia is a frequent complication of influenza. The
localized host response to staphylococcal infection is inflammation,
characterized by an elevated temperature at the site, swelling, the
accumulation of pus, and necrosis of tissue. Around the inflamed area, a fibrin
clot may form, walling off the bacteria and leukocytes as a characteristic
pus-filled boil or abscess. More serious infections of the skin may occur, such
as furuncles or impetigo. Localized infection of the bone is called
osteomyelitis. Serious consequences of staphylococcal infections occur when the
bacteria invade the blood stream. A resulting septicemia may be rapidly fatal;
a bacteremia may result in seeding other internal abscesses, other skin
lesions, or infections in the lung, kidney, heart, skeletal muscle or meninges.

Adherence to Host Cell Proteins

S. aureus cells express on their surface
that promote attachment to host proteins such as laminin and
fibronectin that form the extracellular matrix of epithelial and endothelial
surfaces. In addition, most strains express a fibrin/fibrinogen binding protein
(clumping factor) which promotes attachment to blood clots and traumatized
tissue. Most strains of S. aureus express both fibronectin and
fibrinogen-binding proteins. In addition, an adhesin that promotes attachment
to collagen has been found in strains that cause osteomyelitis and septic
arthritis. Interaction with collagen may also be important in promoting
bacterial attachment to damaged tissue where the underlying layers have been

Evidence that staphylococcal matrix-binding
proteins are virulence factors has come from studying defective mutants in
adherence assays. Mutants defective in binding to fibronectin and to fibrinogen
have reduced virulence in a rat model for endocarditis, and mutants lacking the
collagen-binding protein have reduced virulence in a mouse model for septic
arthritis, suggesting that bacterial colonization is ineffective. Furthermore,
the isolated ligand-binding domain of the fibrinogen, fibronectin and collagen
receptors strongly blocks attachment of bacterial cells to the corresponding host


The invasion of host tissues by staphylococci
apparently involves the production of a huge array of extracellular proteins,
some of which may occur also as cell-associated proteins. These proteins are
described below with some possible explanations for their role in invasive

Membrane-damaging toxins

a-toxin (a-hemolysin) The best
characterized and most potent membrane-damaging toxin of S. aureus is
a-toxin. It is expressed as a monomer that binds to the membrane of susceptible
cells. Subunits then oligomerize to form heptameric rings with a central pore
through which cellular contents leak.

In humans, platelets and monocytes are
particularly sensitive to a-toxin. Susceptible cells have a specific receptor
for a-toxin which allows the toxin to bind causing small pores through which
monovalent cations can pass. The mode of action of alpha hemolysin is likely by
osmotic lysis.

ß-toxin  is a sphingomyelinase which
damages membranes rich in this lipid. The classical test for ß-toxin is lysis
of sheep erythrocytes. The majority of human isolates of S. aureus do
not express ß-toxin. A lysogenic bacteriophage is known to encode the toxin.

d-toxin is a very small peptide toxin
produced by most strains of S. aureus. It is also produced by S.
. The role of d-toxin in disease is unknown.

Leukocidin is a multicomponent protein toxin
produced as separate components which act together to damage membranes.
Leukocidin forms a hetero-oliogmeric transmembrane pore composed of four LukF
and four LukS subunits, thereby forming an octameric pore in the affected
mwembrane. Leukocidin is hemolytic, but less so than alpha hemolysin.

Only 2% of all of S. aureus isolates
express leukocidin, but nearly 90% of the strains isolated from severe
dermonecrotic lesions express this toxin, which suggests that it is an
important factor in necrotizing skin infections.

Coagulase and clumping factor

Coagulase is an extracellular protein which
binds to prothrombin in the host to form a complex called staphylothrombin. The
protease activity characteristic of thrombin is activated in the complex,
resulting in the conversion of fibrinogen to fibrin. Coagulase is a traditional
marker for identifying S aureus in the clinical microbiology laboratory.
However, there is no overwhelming evidence that it is a virulence factor,
although it is reasonable to speculate that the bacteria could protect
themselves from phagocytic and immune defenses by causing localized clotting.

There is some confusion in the literature
concerning coagulase and clumping factor, the fibrinogen-binding determinant on
the S. aureus cell surface. Partly the confusion results from the fact
that a small amount of coagulase is tightly bound on the bacterial cell surface
where it can react with prothrombin leading to fibrin clotting. However,
genetic studies have shown unequivocally that coagulase and clumping factor are
distinct entities. Specific mutants lacking coagulase retain clumping factor
activity, while clumping factor mutants express coagulase normally.


Many strains of S aureus express a
plasminogen activator called staphylokinase. This factor lyses fibrin.The
genetic determinant is associated with lysogenic bacteriophages. A complex
formed between staphylokinase and plasminogen activates plasmin-like
proteolytic activity which causes dissolution of fibrin clots. The mechanism is
identical to streptokinase, which is used in medicine to treat patients
suffering from coronary thrombosis. As with coagulase, there is no strong
evidence that staphylokinase is a virulence factor, although it seems
reasonable to imagine that localized fibrinolysis might aid in bacterial

Other extracellular enzymes

S. aureus can express proteases, a lipase, a
deoxyribonuclease (DNase) and a fatty acid modifying enzyme (FAME). The first
three probably provide nutrients for the bacteria, and it is unlikely that they
have anything but a minor role in pathogenesis. However, the FAME enzyme may be
important in abscesses, where it could modify anti-bacterial lipids and prolong
bacterial survival.

Avoidance of Host Defenses

S. aureus expresses a number of factors that
have the potential to interfere with host defense mechanisms. This includes
both structural and soluble elements of the bacterium.

Capsular Polysaccharide

The majority of clinical isolates of S
express a surface polysaccharide of either serotype 5 or 8. This has
been called a microcapsule because it can be visualized only by electron
microscopy unlike the true capsules of some bacteria which are readily
visualized by light microscopy. S. aureus strains isolated from
infections express high levels of the polysaccharide but rapidly lose the
ability when cultured in the laboratory. The function of the capsule in
virulence is not entirely clear. Although it does impede phagocytosis in the
absence of complement, it also impedes colonization of damaged heart valves,
perhaps by masking adhesins.

Protein A

Protein A is a surface protein of S.
which binds IgG molecules by their Fc region. In serum, the bacteria
will bind IgG molecules in the wrong orientation on their surface which
disrupts opsonization and phagocytosis. Mutants of S. aureus lacking
protein A are more efficiently phagocytosed in vitro, and mutants in infection
models have diminished virulence.


S. aureus can express a toxin that specifically
acts on polymorphonuclear leukocytes. Phagocytosis is an important defense
against staphylococcal infection so leukocidin should be a virulence factor.


S. aureus can express several different types
of protein toxins which are probably responsible for symptoms during
infections. Those which damage the membranes of cells were discussed above
under Invasion. Some will lyse erythrocytes, causing hemolysis, but it
is unlikely that hemolysis is a relevant determinant of virulence in vivo.
Leukocidin causes membrane damage to leukocytes, but is not hemolytic.

Systemic release of a-toxin causes septic
shock, while enterotoxins and TSST-1 are superantigens that may cause toxic
shock. Staphylococcal enterotoxins cause emesis (vomiting) when ingested and
the bacterium is a leading cause of food poisoning.

The exfoliatin toxin causes the scalded skin
syndrome in neonates, which results in widespread blistering and loss of the
epidermis. There are two antigenically distinct forms of the toxin, ETA and
ETB. The toxins have esterase and protease activity and apparently target a
protein which is involved in maintaining the integrity of the epidermis.

Superantigens: enterotoxins and toxic shock
syndrome toxin

S. aureus secretes two types of toxin with
superantigen activity, enterotoxins, of which there are six antigenic
types (named SE-A, B, C, D, E and G), and toxic shock syndrome toxin (TSST-1).
Enterotoxins cause diarrhea and vomiting when ingested and are responsible for
staphylococcal food poisoning. TSST-1 is expressed systemically and is the
cause of toxic shock syndrome (TSS). When expressed systemically, enterotoxins
can also cause toxic shock syndrome. In fact, enterotoxins B and C cause 50% of
non-menstrual cases of TSS. TSST-1 is weakly related to enterotoxins, but it
does not have emetic activity. TSST-1 is responsible for 75% of TSS, including
all menstrual cases. TSS can occur as a sequel to any staphylococcal infection
if an enterotoxin or TSST-1 is released systemically and the host lacks
appropriate neutralizing antibodies.

Superantigens stimulate T cells
non-specifically without normal antigenic recognition (Figure 4). Up to one in
five T cells may be activated, whereas only 1 in 10,000 are stimulated
during a usual antigen presentation. Cytokines are released in large amounts,
causing the symptoms of TSS. Superantigens bind directly to class II major
histocompatibility complexes of antigen-presenting cells outside the
conventional antigen-binding grove. This complex recognizes only the Vb element
of the T cell receptor. Thus any T cell with the appropriate Vb element can be
stimulated, whereas normally, antigen specificity is also required in binding.