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. coli-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. coli 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. coli 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. coli, 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. coli 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 performed.
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 44°C.
Detection of E. coli 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 coli 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.
Physiology of E. coli
Physiologically, E. coli 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) habitats.
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.
Escherichia coli in the Gastrointestinal Tract
The commensal E. coli 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 plants.
Infections 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 Infections
Uropathogenic E. coli 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 population.
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. coli 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
Neonatal 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 meninges.
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. coli, 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. coli (ETEC)
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. coli (EIEC)
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. coli (EPEC)
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. coli (EAggEC)
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. coli (EHEC)
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.
Biotechnological 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. coli 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. coli 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. coli. 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
Staphylococcus aureus. Electron micrograph from Visuals Unlimited, with permission.
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 epidermidis 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. epidermidis 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 factors: (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 factor); 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 agents.
For the majority of diseases caused by S. aureus, 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 proteins 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 exposed.
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 proteins.
Invasion
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 process.
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. epidermidis. 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.
Staphylokinase
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 spreading.
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 aureus 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. aureus 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.
Leukocidin
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.
Exotoxins
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.
