The\u00a0Enteric Bacteria\r\nIntroduction\u00a0to the Family Enterobacteriaceae\r\nEnterobacteriaceae are Gram-negative,\u00a0oxidase-negative, rod-shaped bacteria, 0.3-1.0 x 1.0-6.0 um. Typically, they\u00a0are motile by peritrichous flagella. They are facultative anaerobes,\u00a0being\u00a0 chemoorganotrophs that exhibit both respiratory and fermentative\u00a0metabolism. Most grow well between 22 and 35\u00b0C on media containing peptone\u00a0 or beef\u00a0extract. They also grow on MacConkey's agar which may be used for their\u00a0selective isolation. Most grow on glucose as a sole carbon source, although\u00a0some require vitamins and\/or amino acids for growth. Thet produce mixed acids\u00a0and often gas from fermentation of sugars. With very few exceptions they are\u00a0catalase-positive, and most strains reduce nitrate to nitrite. \u00a0Escherichia coli is the type species.\u00a0E. coli is considered the most thoroughly studied of all species of\u00a0bacteria, and the family Enterobacteriaceae, as a whole, is the best\u00a0studied group of microorganisms. Among the reasons for their popularity are\u00a0their medical and economic importance, ease of isolation and cultivation, rapid\u00a0generation time, and their ability to be genetically manipulated. \u00a0Enterobacteriaceae are distributed\u00a0worldwide. They are found in water and soil and as normal intestinal flora in\u00a0humans and many animals. They live as .saprophytically, as symbionts,\u00a0epiphytes, and parasites. Their host range includes animals ranging from\u00a0insects to humans, as well as fruits, vegetables, grains, flowering plants, and\u00a0trees. \r\nEconomic\u00a0and Medical Importance\r\nAs stated above, one\u00a0of the reasons that the enterobacteriaceae have ben so widely studied is due to\u00a0their obvious impact on human and animal health and on agricultural practice.\u00a0The enterobacteriaceae include agents of food poisoning and gastroenteritis,\u00a0hospital-acquired infections, enteric fevers (e.g. typhoid fever) and plague.\u00a0They also cause infections in domestic, farm and zoo animals and include an\u00a0important group of plant pathogens. Some of these bacteria are discussed below.\r\nPlant Pathogens\r\nMany species of Enterobacteriaceae\u00a0are responsible for significant economic losses in agriculture.\u00a0 Erwinia\u00a0species cause blight, wilt, or soft-rot in numerous trees, flowers, and crops,\u00a0often destroying substantial amounts of crops.\u00a0 Among the plants affected\u00a0are walnut and oak trees, rose, orchid and chrysanthemum flowers, and crops\u00a0such as\u00a0 corn, wheat, potato, carrot, sugar beet, sugar cane and\u00a0pineapple. \u00a0Animal Pathogens\u00a0Enterobacteriaceae cause disease in all\u00a0sorts of animals, ranging from nematodes and insects through primates.\u00a0Salmonella alone has been associated with disease in more than 125 species.\u00a0Infections frequently cause problems in zoos, often in snakes and lizards. In\u00a0regional primate centers in the United States, the most frequently diagnosed\u00a0diarrheal diseases were caused by Enterobacteriaceae, most often by Shigella,\u00a0E. coli and Salmonella. Klebsiella pneumoniae is a\u00a0frequent cause of respiratory disease in primates, and Yersinia\u00a0pseudotuberculosis is associated with enterocolitis and peritonitis. \u00a0Pets and farm animals\u00a0are affected by a variety of enterobacterial diseases. Cats and dogs are\u00a0susceptible to cystitis and other urogenital infections caused by E. coli.\u00a0Proteus species cause other diseases in cats and dogs, and these animals\u00a0can be carriers of Salmonella. Salmonellae, especially S.\u00a0typhimurium, S. newport, and S. anatum, cause enteritis with high\u00a0fatality and septic abortion in horses, and K. pneumoniae causes\u00a0metritis in mares and pneumonia in foals. \u00a0Septicemia caused by E.\u00a0coli is an important cause of death in chickens. Serotypes of Salmonella\u00a0enterica are pathogenic and highly fatal for turkeys and other poultry,\u00a0causing a characteristic diarrheal syndrome. Pullorum disease, caused by Salmonella\u00a0pullorum, is highly fatal to eggs and chicks. Fowl typhoid, a septicemic\u00a0disease of poultry, especially chickens, is caused by Salmonella gallinarum.\u00a0Both pullorum disease and fowl typhoid can be largely eradicated if\u00a0infected adult birds are slaughtered.\u00a0 Nearly 200 Salmonella serotypes\u00a0had been isolated from fowl in the United States. The distribution of\u00a0salmonellosis in poultry is worldwide.\u00a0 As in human disease, certain\u00a0serotypes are prevalent in some regions and absent in others. A mortality rate\u00a0of 10-20% is normal in young birds, mostly in the first two weeks after\u00a0hatching. \u00a0Sheep suffer from a\u00a0variety of illnesses caused by Enterobacteriaceae. Infant diarrhea in\u00a0lambs, is usually caused by strains of E. coli producing a heat-stable\u00a0enterotoxin. Most of these strains also contain the K-99 fimbrial adhesin. Salmonella\u00a0abortion is usually caused by Salmonella abortusovis, S. typhimurium, or S.\u00a0dublin, which also cause stillbirths and wool damage. \u00a0Calves are\u00a0susceptible to both systemic colibacillosis and neonatal diarrhea (calf\u00a0scours), which are usually fatal if not promptly treated. Specific heat-stable\u00a0enterotoxigenic E. coli serotypes containing K99 fimbrial adhesin are\u00a0the causative agents. Bovine mastitis has become a very prevalent disease since\u00a0the advent of antibiotics. The most prevalent causative agents are E. coli\u00a0and Serratia species, and less often, Klebsiella species and Citrobacter\u00a0freundii. Salmonellosis is frequent in cattle. Most cases are due to Salmonella\u00a0dublin and S. typhimurium, although more than 100 serotypes have\u00a0been isolated. As with other animal infections, Salmonella is frequently\u00a0introduced through contaminated feed. \u00a0Swine are subject to\u00a0infection with several species of Enterobacteriaceae. E. coli infection\u00a0may present as diarrhea in piglets, or as edema preceded by mild diarrhea. Both\u00a0forms are acute and highly fatal. As in sheep and cows, the causative strains\u00a0produce a heat-stable enterotoxin, but they may also produce a heat-labile\u00a0enterotoxin. Swine strains\u00a0 usually possess a K88 fimbrial adhesin, which\u00a0is antigenically distict from K99. Sows are susceptible to mastitis and\u00a0metritis caused by K. pneumoniae, and to enteritis and lymphadenitis\u00a0caused by Yersinia enterocolitica. More than 100 Salmonella\u00a0serotypes have been isolated from pigs.\u00a0 However, only two serotypes, S.\u00a0choleraesuis and S. typhisuis, have pigs as their primary host. S.\u00a0choleraesuis has a wide host range, including humans, but S. typhisuis\u00a0is rarely pathogenic to animals other than pigs. Salmonella typhimurium and\u00a0S. derby are also frequently isolated from porcine salmonellosis. \u00a0Substantial losses in\u00a0fishing industries are caused by enterobacterial diseases. Yersinia ruckeri\u00a0is the cause of outbreaks of redmouth disease in salmon and trout hatcheries. Edwardsiella\u00a0tarda is pathogenic for eels, catfish, and goldfish, and Edwardsiella\u00a0ictaluri is pathogenic for catfish. \u00a0The host range for\u00a0species of Enterobacteriaceae varies greatly. For example Proteus\u00a0myxofaciens has been isolated only from larvae of gypsy moths and Escherichia\u00a0blattae has been isolated only from the hindgut of cockroaches.\u00a0Shigellae are seen only in primates. Others, including E. coli, many\u00a0salmonellae, and yersiniae, infect or are carried by hosts ranging from insects\u00a0to humans. \u00a0Human Pathogens\u00a0Enterobacteriaceae as a group were\u00a0originally divided into pathogens and nonpathogens based on their ability to\u00a0cause diarrheal disease of humans. The pathogenic genera were Salmonella and\u00a0Shigella. However, it is now known that E. coli causes at least\u00a0five types of gastrointestinal disease in humans. Pathogenicity in E. coli\u00a0strains is due to the presence of one or more virulence factors, including\u00a0invasiveness factors (invasins), heat-labile and heat-stable enterotoxins,\u00a0verotoxins, and colonization factors or adhesins. Pathogenic strains are\u00a0usually identified by detection of a specific virulence factor or of a serotype\u00a0associated with a virulence factor.\u00a0 The most recently identified E.\u00a0coli disease is hemorrhagic colitis caused by strains of serotype 0157:H7.\u00a0The disease, characterized by painful abdominal cramping and bloody diarrhea,\u00a0is caused by strains that produce verotoxin, and the same strains are\u00a0associated with hemolytic uremic syndrome (HUS). \u00a0Yersinia\u00a0enterocolitica\u00a0causes diarrhea, probably by a combination of invasiveness and the presence of\u00a0a heat-stable enterotoxin. Strains of Klebsiella pneumoniae and Enterobacter\u00a0cloacae isolated from patients with tropical sprue contained a heat-stable\u00a0enterotoxin. Edwardsiella tarda and Citrobacter strains are\u00a0occasionally associated with diarrhea and have been shown to produce\u00a0heat-stable or heat-labile enterotoxin.\u00a0Foodborne and\u00a0waterborne disease outbreaks in the U.S. are frequently associated with Enterobacteriaceae. \u00a0According to the Centers for Disease Control (CDC), 40-45% of such outbreaks\u00a0are caused by Enterobacteriaceae, the overwhelming majority by Salmonella.\u00a0Meats, milk and milk products, and eggs are the most common vehicles of\u00a0transmission.\u00a0 Such figures represent only a small fraction of total\u00a0foodborne disease, since the etiologic agent is identified in only about\u00a0one-third of the outbreaks, and many outbreaks are undetected or are not\u00a0reported to the Centers for Disease Control. For Salmonella, it is\u00a0estimated that each reported case represents about 100 total cases. The largest\u00a0outbreak of salmonellosis in the United States occurred in 1985 in Illinois and\u00a0Wisconsin, where an estimated 170,000 to almost 200,000 persons were infected\u00a0with Salmonella typhimurium transmitted in pasteurized milk from a\u00a0single dairy plant. \r\n\u00a0The incidence and\u00a0recognition of rheumatoid disease occurring secondary to foodborne and\u00a0waterborne diarrheal disease have also increased. These diseases include\u00a0reactive arthritis, Reiter's syndrome, ankylosing spondylitis, septic and\u00a0aseptic arthritis, ulcerative colitis, Crohn's disease, and Whipple's disease. Y.\u00a0enterocolitica, Y. pseudotuberculosis, Shigella flexneri, Shigella dysenteriae,\u00a0various salmonellae, E. coli, and K. pneumoniae have been\u00a0associated with these chronic conditions. \u00a0Waterborne disease\u00a0outbreaks due to Enterobacteriaceae are usually due to contaminated\u00a0wells. Cases of shigellosis due to a contaminated wells have been reported;\u00a0even typhoid fever has occurred fairly recently in community water systems\u00a0contaminated with human sewage. \r\n\u00a0Enterobacteriaceae\u00a0not normally associated with the GI tract or diarrheal disease may still be\u00a0pathogens of humans. Most notably, Yersinia pestis, which does not have\u00a0an intestinal habitat, is the etiologic agent of plague a highly fatal disease\u00a0that has dessimated whole populations of individuals at several times in the\u00a0history of civilization. Furthermore, most, if not all, Enterobacteriaceae\u00a0are opportunistic pathogens. Once established, they can cause a variety of\u00a0infections, including urinary tract disease, pneumonia, septicemia, meningitis,\u00a0and wound infection. \u00a0According to the CDC,\r\nEnterobacteriaceae are responsible for 40-50% of nosocomial infections\u00a0occurring in the United States. E. coli is the worst offender, followed\u00a0by Klebsiella, Proteus-Providencia-Morganella, Serratia, and\u00a0Citrobacter. The compromised host is particularly susceptible to nosocomial\u00a0infections. Catheterized patients, patients on immunosuppressants, burn\u00a0patients, cancer patients, and elderly patients are all especially vulnerable\u00a0to opportunistic pathogens. To make matters worse, many of these organisms\u00a0acquired in the hospital setting are multiply drug resistant. \r\nTaxonmy\u00a0and Classification of Enteric Bacteria\r\nIn artificial\u00a0classification schemes (e.g.\u00a0 Bergey's Manual of Systematic\u00a0Bacteriology, 1st edition, 1986) Enterobacteriaceae is a\u00a0family of bacteria in Section\u00a0 8 - Gram-negative facultatively\u00a0anaerobic rods. Because of the large number and broad range of phenotypic\u00a0properties that solidifiy the group, these traits being a reflection of their\u00a0genetic relatedness, these bacteria have remained unified in modern\u00a0phylogenetic schemes based on 16S ribosomal RNA comparison. Thus, Citrobacter,\u00a0Edwardsiella, Enterobacter, Erwinia, Escherichia, Klebsiella, Proteus,\u00a0Providencia, Salmonella, Serratia, Shigella, and Yersinia (along\u00a0with several other genera, including Hafnia, Morganella, Photorhabdus,and\u00a0Xenorhabdus) are\u00a0 presently classified in the subclass Gammaproteobacteria,\u00a0order Enterobacteriales, family Enterobacteriaceae .\r\nThe classic\u00a0definition of an enteric bacterium is one that is found in the\u00a0intestinal tract of warm-blooded animals in health and disease, but\u00a0bacteriologists reserve the term for reference to E. coli and its\u00a0relatives, even though some of the relatives of E. coli rarely or never\u00a0are found growing in the GI tract. But in the end, this is one of the most\u00a0close-related and cohesive groups of bacteria that can be brought together for\u00a0discussion. \r\nThe\u00a0Genus Escherichia\r\nTheodor Escherich\u00a0first described E. coli in 1885, as Bacterium coli commune, which\u00a0he isolated from the feces of neonates. It was later renamed Escherichia\u00a0coli, and for many years the bacterium was simply considered to be a\u00a0commensal organism of the large intestine. It was not until 1935 that a strain\u00a0of E. coli was shown to be the cause of an outbreak of diarrhea among\u00a0neonates. \r\nMost investigations\u00a0of enteric organisms at the turn of the 20th century were concerned with the\u00a0problems of being able to distinguish the "typhoid bacillus" and\u00a0other types of Salmonella from non-Salmonella organisms. Early\u00a0workers also demonstrated that there were a number of types and subtypes of\u00a0these organisms, which could easily be distinguished from the typhoid bacillus\u00a0and E. coli. Thus, the biochemical techniques that have become the basis\u00a0for most taxonomic studies came into being during the early 1900s. These\u00a0studies led to the modern taxonomy of the group, which in principle is still\u00a0valid today. \u00a0Initially, the family\u00a0Enterobacteriaceae was created by Rahn in 1937, for the genus Enterobacter,\u00a0and despite some debate about nomenclature among bacteriologists, the family\u00a0name was maintained with the type genus becoming Escherichia.\u00a0 The\u00a0family currently comprises Gram-negative, nonsporeforming, rod-shaped bacteria\u00a0that are often motile by means of peritrichous flagella. The majority of\u00a0strains grow well on the usual laboratory media in both the presence and\u00a0absence of oxygen, and metabolism can be either respiratory or fermentative.\u00a0The fermentation products of glucose and other carbohydrate substrates include\u00a0mixed acids and (usually) detectable gas. Most strains are oxidase-negative and\u00a0are able to reduce nitrate to nitrite. \r\nThe taxonomic\u00a0distinctiveness of Escherichia has been confirmed by rRNA-DNA\u00a0heteroduplex studies. On the basis of DNA-DNA relatedness studies, the genera\u00a0of enteric bacteria are placed into a series of groupings, with Escherichia\u00a0and Shigella forming a close group distinct from their nearest group,\u00a0which includes the genera\u00a0 Citrobacter, Enterobacter, Klebsiella, and\u00a0Salmonella.\u00a0\u00a0Although most\u00a0investigations of the genus Escherichia have centered on various aspects\u00a0of the E. coli species, it should not be forgotten that a number of\u00a0other species have been described, including E. blattae, E. fergusonii, E.\u00a0hermanii, and E. vulneris. These species can be differentiated on the basis\u00a0of a large battery of biochemical tests. \u00a0For many years it has\u00a0been realized that there exists a close relationship between two genera of\u00a0enterics,\u00a0 Escherichia and Shigella. This is true for their\u00a0biochemical characteristics as well as various other phenotypic traits. Also,\u00a0studies of certain E. coli antigens have shown a close relationship\u00a0("cross reactivity") with Shigella antigens. The "O"\u00a0antigens of virtually all serotypes of Shigella are either identical\u00a0with or closely related to those of E. coli.\u00a0 The discovery that\u00a0the characteristic \u201cinvasiveness\u201d of Shigella strains is also possessed\u00a0by certain types of E. coli, which have become known as enteroinvasive E.\u00a0coli (EIEC), also suggests a close relationship.\u00a0 EIEC can cause\u00a0dysentery-like symptoms clinically indistinguishable from those caused by\u00a0strains of Shigella. Furthermore, antigens of E. coli strain O124\u00a0are shown to have a very close relationship to Shigella dysenteriae type\u00a03 antigens, and a serological relationship between E. coli strain O129\u00a0and S. flexneri type 5 is also known. Finally, enterohemorrhagic strains\u00a0of E. coli\u00a0 (EHEC), specifically E. coli O157:H7 produce the\u00a0shiga (vero) toxin which is identical to the toxin produced by Shigella\u00a0dysenteriae.The point being that it has become apparent that the line\u00a0dividing these two genera of enteric bacteria is exceedingly thin, and it\u00a0should be remembered that on the basis of\u00a0 DNA relatedness alone, Shigella\u00a0and E. coli have been considered one genus. \r\nDetection\u00a0and Isolation of Escherichia coli\r\nE. coli as an Indicator of\r\nFecal Pollution\r\n\r\nFor most of the 20th\r\ncentury, E. coli has been used as the principal indicator of fecal\r\npollution in both tropical and temperate countries. E. coli comprises\r\nabout 1% of the total fecal bacterial flora of humans and most warm-blooded\r\nanimals. Sewage is always likely to contain E. coli in relatively large\r\nnumbers. In addition, E. coli, being a typical member of the Enterobacteriaceae,\r\nis presumed to have survival characteristics very similar to those of the\r\nwell-known pathogenic members of the family, Salmonella and Shigella.\r\nThus, E. coli has been used world-wide as an indicator of fecal\r\nmicrobiological contamination. As such an indicator organism, its value is\r\nsignificantly enhanced by the ease with which it can be detected. and cultured.\r\n\r\nTests to identify\r\nisolates as E. coli have, of necessity, been simple tests designed\r\npredominantly to differentiate them from organisms normally associated with\r\nuncontaminated water. Since full biochemical analyses are not generally\r\nperformed, the term\u00a0 "coliform" has been coined to describe E.\r\ncoli-like organisms that satisfy these limited tests. As a result,\r\nregulations are promulgated throughout the world defining standards for water\r\nbased on the so-called "coliform count." For example, in the U.S.,\r\naccording to a regulation published in the Federal Register (1986), there is a\r\nrequirement that there be 0 coliforms\/100 ml drinking water, as determined by\r\nany method for any sampling frequency. Since not all organisms which meet the\r\ncriteria of a coliform are associated with the intestinal tract (some may be\r\nsaprophytic), a further distinction must be made between "fecal\r\ncoliforms" (E coli) and "nonfecal coliforms" (e.g. Klebsiella\r\nand Enterobacter). \r\nEarly attempts to\r\ndistinguish strains of E. coli from other related Enterobacteriaceae\r\ncentered on being able to distinguish them from the various pathogenic groups,\r\nsince E. coli was initially not considered to be a pathogen. When E.\r\ncoli was recognized to be a useful marker for fecal pollution, it similarly\r\nbecame important to distinguish it from related species likely to be found\r\nnaturally in the environment. The realization that strains of E. coli\r\ngenerally ferment lactose, while those of Salmonella and Shigella do\r\nnot, led to an early method of preliminary differentiation. The IMViC tests\r\nwere developed in order to distinguish strains of E. coli from related\r\nspecies that also produced acid and gas from the fermentation of lactose. IMViC\r\nis an acronym in which the capital letters stand for Indole, Methyl\r\nred, Voges-Proskauer, and Citrate.) The IMViC set of tests\r\nexamines: the ability of an organism to (1) produce Indole; (2) produce\r\nsufficient acid to change the color of Methyl red indicator; (3) produce\r\nacetoin, an intermediate in ther butanediol fermentation pathway (a positive\r\nresult of the Voges-Proskauer test); and (4) the ability to grow on Citrate\r\nas the sole source of carbon. Lactose fermenters are considered E. coli\r\nif they are positive in the first two tests and negative in the second two. \r\nDetection of E.\r\ncoli in Food\r\n\r\nThe International\r\nCommission on Microbiological Specifications for Foods (ICMSF, 1978) has\r\nadopted a set of standard techniques for the enumeration of E. coli in\r\nfood products, accepted by the International Standards Organization (ISO,\r\n1984). This method employs the use of lauryl sulfate tryptose broth at 35 or 37\u00b0C as a mildly selective-enrichment\r\nmedium. This is followed by growth in EC broth containing 0.15%\u00a0 bile\r\nsalts at 45\u00b0C\r\nas a\u00a0 second selective step. The ability to produce indole from tryptophan\r\n(in tryptone broth) at 45\u00b0C\r\ndefines the strains as E. coli. These tests\u00a0 miss some types of E.\r\ncoli, such as those most closely related to the Shigella group, but\r\nit is the detection of possible fecal contamination that is important in these\r\ntests rather than the presence of specific types. \r\nDetection of E.\r\ncoli in Water\r\n\r\nThere is no method\r\nfor the detection of E. coli in water that is accepted throughout the\r\nworld. In the US, a standard method using membrane filter enumeration for both\r\ntotal and thermotolerant coliforms has been established (American Public Health\r\nAssociation (1986). Further IMViC tests on selected isolates can then be\r\nperformed. \r\nIn the UK, the\r\ndefinition of E. coli in water microbiology is also based on the ability\r\nto produce gas from lactose and produce indole from tryptophan at 44\u00b0C. A method for enumeration\r\nemploys a standard multiple tube test with a modified glutamate synthetic\r\nmedium at 37\u00b0C\r\nas a first selective step, followed by further cultivation in standard media at\r\n44\u00b0C. \r\nDetection of E.\r\ncoli in Clinical Specimens\r\nWhile large numbers\r\nof E. coli will be found in fecal specimens or specimens contaminated\r\nwith feces or intestinal contents, most other clinical specimens are usually\r\nnot contaminated with E. coli. The major exception is urine, which\r\nrequires special attention in the clinical situation. From those specimens in\r\nwhich E. coli is likely to be present in large numbers, direct plating\r\non media such as MacConkey agar or Eosin Methylene Blue (EMB) agar is\r\nsufficient. If the number of E. coli is likely to be very low or the\r\namount of specimen is limited, enrichment in a rich nutrient medium such as\r\nbrain heart infusion broth may be used. A number of different commercially\r\navailable kits are generally used to identify the isolates as E. coli. \r\nFrom specimens likely\r\nto contain only a few viable E. coli cells, such as blood from patients\r\nsuspected of having E. coli bacteremia, various enrichment procedures\r\nare used.\u00a0 Identification follows standard bacteriological techniques. \r\nLeft: Escherichia\r\ncoli microcolony. Right: E.coli colonies on EMB Agar.\r\nRapid Methods for\r\nDetecting E. coli\r\nA fluorogenic\r\ndetection method has been developed based on the cleavage of\r\nmethylumbelliferyl-D-glucuronide (MUG) to the free methylumbelliferyl moiety,\r\nwhich fluoresces a blue color after irradiation with long-wave ultraviolet radiation.\r\nAlthough strains of E. coli are generally positive in this test, some\r\nstrains of Salmonella, Shigella, and Yersinia are also capable of\r\nsplitting MUG; the latter two genera are usually not present in food. A\r\ndisadvantage is that enterohemorhagic E. coli (EHEC) strains are\r\ngenerally negative in this test.\u00a0 MUG can be added to various selective\r\nmedia, so there is a great potential in its use for detecting E. coli. \r\nAutomated or\r\nsemi-automated systems are also being used for the detection of E. coli\r\nas part of the detection methods for Enterobacteriaceae. Techniques\r\ninvolving impedance measurements have shown promise. Other techniques such as\r\nimmunoassays and nucleic acid hybridization studies can also be used to\r\nenumerate E. coli, and DNA probes directed at a number of genes have\r\nalso been developed. \r\nPhysiology\r\nof E. coli\r\n\r\nPhysiologically, E.\r\ncoli is versatile and well-adapted to its characteristic habitats.\u00a0 In\r\nthe laboratory it can grow in media with glucose as the sole organic\r\nconstituent. Wild-type E. coli has no growth factor requirements, and\r\nmetabolically it can transform glucose into all of the macromolecular\r\ncomponents that make up the cell. The bacterium can grow in the presence or\r\nabsence of O2. Under anaerobic conditions it will grow by means of\r\nfermentation, producing characteristic "mixed acids and gas" as end\r\nproducts. However, it can also grow by means of anaerobic respiration, since it\r\nis able to utilize NO3 or fumarate as final electron acceptors for\r\nrespiratory electron transport processes. In part, this adapts E. coli\r\nto its intestinal (anaerobic) and its extraintestinal (aerobic or anaerobic)\r\nhabitats. \r\nIn the ecological\r\nniches that E. coli occupies, its abilities to grow both aerobically and\r\nanaerobically are important.\u00a0 E. coli is\u00a0 well adapted to its\r\nintestinal environment as it is able to survive on a relatively limited number\r\nof low-molecular weight substances, which may only be available transiently and\r\nat relatively low concentrations. The generation time for E. coli in the\r\nintestine is thought to be about 12 hours. The type of nutrients available\r\nthere to E. coli\u00a0 consist of mucus, desquamated cells, intestinal\r\nenzyme secretions, and incompletely digested food.\u00a0 Given the absorption\r\ncapacity and efficiency of the intestine, there are probably only small amounts\r\nfree carbohydrates or other easily absorbable forms of nutrients, and there is\r\ncompetition from hundreds of other types pf bacteria. A similar situation\r\nprobably also applies to sources of nitrogen. \r\nIn its natural\r\nenvironment, as well as the laboratory, E. coli can respond to\r\nenvironmental signals such as chemicals, pH, temperature, osmolarity, etc., in\r\na number of very remarkable ways considering it is a single-celled organism.\r\nFor example, it can sense the presence or absence of chemicals and gases in its\r\nenvironment and swim towards or away from them. Or it can stop swimming and\r\ngrow fimbriae that will specifically attach it to a cell or surface receptor.\r\nIn response to changes in temperature and osmolarity, it can vary the pore\r\ndiameter of its outer membrane porins to accommodate larger molecules\r\n(nutrients) or to exclude inhibitory substances (e.g. bile salts). With its\r\ncomplex mechanisms for regulation of metabolism the bacterium can survey the\r\nchemical content its environment in advance of synthesizing any enzymes\r\nnecessary to use these compounds. It does not wastefully produce enzymes for\r\ndegradation of carbon sources unless they are available, and it does not\r\nproduce enzymes for synthesis of metabolites if they are available as nutrients\r\nor growth factors in the environment. \r\nEscherichia\r\ncoli in the Gastrointestinal Tract\r\nThe commensal E.\r\ncoli strains that inhabit the large intestine of all humans and\r\nwarm-blooded animals comprise about 1% of the total\u00a0 bacterial biomass.\r\nThis E. coli flora is in constant flux. One study on the distribution of\r\ndifferent E. coli strains colonizing the large intestine of women during\r\na one year period (in a hospital setting) showed that 52.1% yielded one\r\nserogroup, 34.9% yielded two, 4.4% yielded three, and 0.6% yielded four.\r\nThe most likely source of new serotypes of E. coli is acquisition by the\r\noral route. To study oral acquisition, the carriage rate of E. coli carrying\r\nantibiotic-resistance (R) plasmids was examined among vegetarians, babies, and\r\nnonvegetarians. It was assumed that nonvegetarians might carry more E. coli\r\nwith R factors due to their presumed high incidence in animals treated with\r\ngrowth-promoting antimicrobial agents. However, omnivores had no higher an\r\nincidence of R-factor-containing E. coli than vegetarians, and babies\r\nhad more resistant E. coli in their feces than nonvegetarians. No\r\nsuitable explanation could be offered for these findings.\u00a0 Besides,\r\ninvestigation of the microbial flora of the uninhabited Krakatoa archipelago\r\nhas shown the presence of antibiotic-resistant E. coli associated with\r\nplants. \r\nInfections\r\nCaused by Pathogenic E. coli\r\nE. coli is responsible\r\nprimarily for three types of infections in humans: urinary tract infections,\r\nneonatal meningitis, and intestinal diseases. These condituions\r\ndepend on a specific array of pathogenic (virulence) determinants possessed by\r\nthe organism.\u00a0 Pathogenic E. coli are discussed elsewhere in the\r\ntext in more detail at Pathogenic E.\r\ncoli:\u00a0 Gastroenteritis, Urinary tract Infections and Neonatal Meningitis.\r\nUrinary Tract\r\nInfections\r\n\r\nUropathogenic E.\r\ncoli cause 90% of the urinary tract infections (UTI) in\r\nanatomically-normal, unobstructed urinary tracts. The bacteria colonize from\r\nthe feces or perineal region and ascend the urinary tract to the bladder.\r\nBladder infections are 14-times more common in females than males by virtue of\r\nthe shortened urethra. The typical patient with uncomplicated cystitis is a\r\nsexually-active female who was first colonized in the intestine with a\r\nuropathogenic E. coli strain. The organisms are propelled into the\r\nbladder from the periurethral region during sexual intercourse. With the aid of\r\nspecific adhesins they are able to colonize the bladder. \r\nThe adhesin that has\r\nbeen most closely associated with uropathogenic E. coli is the P fimbria\r\n(or pyelonephritis-associated pili [PAP] pili). The letter designation is\r\nderived from the ability of P fimbriae to bind specifically to the P blood\r\ngroup antigen which contains a D-galactose-D-galactose residue. The fimbriae\r\nbind not only to red cells but to a specific galactose dissaccharide that is\r\nfound on the surfaces uroepithelial cells in approximately 99% of the\r\npopulation. \r\nThe frequency of the\r\ndistribution of this host cell receptor plays a role in susceptibility and\r\nexplains why certain individuals have repeated UTI caused by E. coli.\r\nUncomplicated E. coli UTI virtually never occurs in individuals lacking\r\nthe receptors. \r\nUropathogenic strains\r\nof E. coli possess other determinants of virulence in addition to P\r\nfimbriae. E. coli with P fimbriae also possess the gene for Type 1\r\nfimbriae, and there is evidence that P fimbriae are derived from Type 1\r\nfimbriae by insertion of a new fimbrial tip protein to replace the\r\nmannose-binding domain of Type 1 fimbria. In any case, Type 1 fimbriae could\r\nprovide a supplementary mechanism of adherence or play a role in aggregating\r\nthe bacteria to a specific manosyl-glycoprotein that occurs in urine. \r\nUropathogenic strains\r\nof E. coli usually produce siderophores that probably play an essential\r\nrole in iron acquisition for the bacteria during or after colonization. They\r\nalso produce hemolysins which are cytotoxic due to formation of transmembranous\r\npores in host cells. One strategy for obtaining iron and other nutrients for\r\nbacterial growth may involve the lysis of host cells to release these\r\nsubstances. The activity of hemolysins is not limited to red cells since the\r\nalpha-hemolysins of E. coli also lyse lymphocytes, and the\r\nbeta-hemolysins inhibit phagocytosis and chemotaxis of neutrophils. \r\nAnother factor\r\nthought to be involved in the pathogenicity of the uropathogenic strains of E.\r\ncoli is their resistance to the complement-dependent bactericidal effect of\r\nserum. The presence of K antigens is associated with upper urinary tract\r\ninfections, and antibody to the K antigen has been shown to afford some degree\r\nof protection in experimental infections. The K antigens of E. coli are\r\n"capsular" antigens that may be composed of proteinaceous organelles\r\nassociated with colonization (e.g., CFA antigens), or made of polysaccharides.\r\nRegardless of their chemistry, these capsules may be able to promote bacterial\r\nvirulence by decreasing the ability of antibodies and\/or complement to bind to\r\nthe bacterial surface, and the ability of phagocytes to recognize and engulf\r\nthe bacterial cells. The best studied K antigen, K-1, is composed of a polymer\r\nof N-acetyl neuraminic acid (sialic acid), which besides being antiphagocytic, has\r\nthe additional property of being an antigenic disguise. \r\nNeonatal meningitis\r\n\u00a0Neonatal\r\nmeningitis affects1\/2,000-4,000 infants. Eighty percent of E. coli\r\nstrains involved synthesize K-1 capsular antigens (K-1 is only present 20-40%\r\nof the time in intestinal isolates). \r\nE. coli strains invade the\r\nblood stream of infants from the nasopharynx or GI tract and are carried to the\r\nmeninges. \r\nThe K-1 antigen is\r\nconsidered the major determinant of virulence among strains of E. coli\r\nthat cause neonatal meningitis. K-1 is a homopolymer of sialic acid. It\r\ninhibits phagocytosis, complement, and responses from the host's immunological\r\nmechanisms. K-1 may not be the only determinant of virulence, however, as\r\nsiderophore production and endotoxin are also likely to be involved. \r\nEpidemiologic studies\r\nhave shown that pregnancy is associated with increased rates of colonization by\r\nK-1 strains and that these strains become involved in the subsequent cases of\r\nmeningitis in the newborn. Probably, the infant GI tract is the portal of entry\r\ninto the bloodstream. Fortunately, although colonization is fairly common,\r\ninvasion and the catastrophic sequelae are rare. \r\nNeonatal meningitis\r\nrequires antibiotic therapy that usually includes ampicillin and a\r\nthird-generation cephalosporin. \r\nIntestinal Diseases\r\nAs a pathogen, E.\r\ncoli, of course, is best known for its ability to cause intestinal\r\ndiseases. Five classes (virotypes) of E. coli that cause diarrheal\r\ndiseases are now recognized: enterotoxigenic E. coli (ETEC),\r\nenteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC),\r\nenteropathogenic E. coli (EPEC), and enteroaggregative E. coli\r\n(EAggEC). Each class falls within a serological subgroup and manifests distinct\r\nfeatures in pathogenesis. \r\nEnterotoxigenic E.\r\ncoli (ETEC)\r\nETEC are an important\r\ncause of diarrhea in infants and travelers in underdeveloped countries or\r\nregions of poor sanitation. The diseases vary from minor discomfort to a severe\r\ncholera-like syndrome. ETEC are acquired by ingestion of contaminated food and\r\nwater, and adults in endemic areas evidently develop immunity. The disease\r\nrequires colonization and elaboration of one or more enterotoxins. Both traits\r\nare plasmid-encoded. \r\nETEC adhesins are\r\nfimbriae which are species-specific. For example, the K-88 fimbrial Ag is found\r\non strains from piglets; K-99 Ag is found on strains from calves and lambs; CFA\r\nI, and CFA II, are found on strains from humans. These fimbrial adhesins adhere\r\nto specific receptors on enterocytes of the proximal small intestine. \r\nEnterotoxins produced\r\nby ETEC include the LT (heat-labile) toxin and\/or the ST (heat-stable) toxin,\r\nthe genes for which may occur on the same or separate plasmids. The LT\r\nenterotoxin is very similar to cholera toxin in both structure and mode of\r\naction. It is an 86kDa protein composed of an enzymatically active (A) subunit\r\nsurrounded by 5 identical binding (B) subunits. It binds to the same identical\r\nganglioside receptors that are recognized by the cholera toxin (i.e., GM1), and\r\nits enzymatic activity is identical to that of the cholera toxin. \r\nThe ST enterotoxin is\r\nactually a family of toxins which are peptides of molecular weight about 2,000\r\ndaltons. Their small size explains why they are not inactivated by heat. ST\r\ncauses an increase in cyclic GMP in host cell cytoplasm leading to the same\r\neffects as an increase in cAMP. STa is known to act by binding to a guanylate\r\ncyclase that is located on the apical membranes of host cells, thereby\r\nactivating the enzyme. This leads to secretion of fluid and electrolytes\r\nresulting in diarrhea. \r\nSymptoms ETEC\r\ninfections include diarrhea without fever. The bacteria colonize the GI tract\r\nby means of a fimbrial adhesin, e.g. CFA I and CFA II, and are noninvasive, but\r\nproduce either the LT or ST toxin. \r\nEnteroivasive E.\r\ncoli (EIEC)\r\nEIEC closely resemble\r\nShigella in their pathogenic mechanisms and the kind of clinical illness\r\nthey produce. EIEC penetrate and multiply within epithelial cells of the colon\r\ncausing widespread cell destruction. The clinical syndrome is identical to Shigella\r\ndysentery and includes a dysentery-like diarrhea with fever. EIEC apparently\r\nlack fimbrial adhesins but do possess a specific adhesin that, as in Shigella,\r\nis thought to be an outer membrane protein. Also, likeShigella, EIEC are\r\ninvasive organisms. They do not produce LT or ST toxin and, unlike Shigella,\r\nthey do not produce the shiga toxin. \r\nEnteropathogenic E.\r\ncoli (EPEC)\r\nEPEC induce a watery\r\ndiarrhea similar to ETEC, but they do not possess the same colonization factors\r\nand do not produce ST or LT toxins. They produce a non fimbrial adhesin\r\ndesignated intimin, an outer membrane protein, that mediates the final stages\r\nof adherence. Although they do not produce LT or ST toxins, there are reports\r\nthat they produce an enterotoxin similar to that of Shigella. Other\r\nvirulence factors may be related to those in Shigella. \r\nAdherence of EPEC\r\nstrains to the intestinal mucosa is a very complicated process and produces\r\ndramatic effects in the ultrastructure of the cells resulting in rearrangements\r\nof actin in the vicinity of adherent bacteria. The phenomenon is sometimes\r\ncalled "attaching and effacing" of cells. EPEC strains are said to be\r\n"moderately-invasive" meaning they are not as invasive as Shigella,\r\nand unlike ETEC or EAggEC, they cause an inflammatory response. The diarrhea\r\nand other symptoms of EPEC infections probably are caused by bacterial invasion\r\nof host cells and interference with normal cellular signal transduction, rather\r\nthan by production of toxins. \r\nSome types of EPEC\r\nare referred to as Enteroadherent E. coli (EAEC), based on specific\r\npatterns of adherence. They are an important cause of traveler's diarrhea in\r\nMexico and in North Africa. \r\nEnteroaggregative E.\r\ncoli (EAggEC)\r\nThe distinguishing\r\nfeature of EAggEC strains is their ability to attach to tissue culture cells in\r\nan aggregative manner. These strains are associated with persistent diarrhea in\r\nyoung children. They resemble ETEC strains in that the bacteria adhere to the\r\nintestinal mucosa and cause non-bloody diarrhea without invading or causing\r\ninflammation. This suggests that the organisms produce a toxin of some sort.\r\nRecently, a distinctive heat-labile plasmid-encoded toxin has been isolated\r\nfrom these strains, called the EAST (EnteroAggregative ST) toxin. They also\r\nproduce a hemolysin related to the hemolysin produced by E. coli strains\r\ninvolved in urinary tract infections. The role of the toxin and the hemolysin\r\nin virulence has not been proven. The significance of EAggEC strains in human\r\ndisease is controversial. \r\nEnterohemorrhagic E.\r\ncoli (EHEC)\r\nEHEC are represented\r\nby a single strain (serotype O157:H7), which causes a diarrheal syndrome\r\ndistinct from EIEC (and Shigella) in that there is copious bloody\r\ndischarge and no fever. A frequent life-threatening situation is its toxic\r\neffects on the kidneys (hemolytic uremia). \r\nEHEC has recently\r\nbeen recognized as a cause of serious disease often associated with ingestion\r\nof inadequately cooked hamburger meat. Pediatric diarrhea caused by this strain\r\ncan be fatal due to acute kidney failure (hemolytic uremic syndrome [HUS]).\r\nEHEC are also considered to be "moderately invasive". Nothing is\r\nknown about the colonization antigens of EHEC but fimbriae are presumed to be\r\ninvolved. The bacteria do not invade mucosal cells as readily as Shigella,\r\nbut EHEC strains produce a toxin that is virtually identical to the Shiga\r\ntoxin. The toxin plays a role in the intense inflammatory response produced by\r\nEHEC strains and may explain the ability of EHEC strains to cause HUS. The\r\ntoxin is phage encoded and its production is enhanced by iron deficiency. \r\nBiotechnological\r\nApplications of E. coli\r\nThe advances in\r\nmolecular biology, genetics and biochemistry during the past four decades have\r\nled to an enormous development in the field of biotechnology. Studies with E.\r\ncoli have played a major role in these developments, and the bacterium has\r\nbeen in the forefront of many technological advances. \r\nIn the early days of\r\nbiotechnology (1960s), emphasis was placed on improvements of established\r\nprocedures of bioprocessing, such as the production of yeasts, vaccines, and\r\nantibiotics.\u00a0 These investigations stimulated genetic research of microbes\r\nto increase their potential to produce a wide variety of products in the\r\nservice of\u00a0 humanity. Although much was being learned about E. coli\r\nand its genetics, the direct use of the bacterium in the industry was limited.\r\nThe industrial production of the amino acid threonine by E. coli\r\nmutants, begun in 1961, is an exception.\r\nAt this time, organisms were generally subjected to mutagenic agents, which\r\nproduced a series of random mutations, from which the specifically required\r\nmutants were selected.\r\nIn the last two\r\ndecades, procedures have evolved which permit the preparation of strains that\r\nhave very specific productive capabilities. As the genetic structure of E.\r\ncoli was well known, and it is an organism which can grow on simple media\r\n(mineral salts and glucose) under aerobic and anaerobic conditions, the\r\nbacterium became the basis for most developments in genetic manipulations\r\nleading to genetic engineering. \r\nThe basic principle\r\nof these genetic manipulations is gene cloning, which enables the isolation and\r\nreplication of individual DNA fragments. This consists of a series of linked\r\nsteps, involving the isolation of the desired gene as double-stranded DNA (dsDNA),\r\ninsertion of the gene into a suitable vector, and using the vector to introduce\r\nthe DNA into a cell which will express the desired genetic information.\r\nIn the case of cloning a gene in E. coli,first the DNA of suitable\r\ncharacter is isolated, then it is joined to the DNA of a suitable vector\r\nproducing a series of recombinant molecules. Then the recombinant molecules are\r\nintroduced into the bacterium in which the target gene becomes established.\r\nRecombinants are selected in various ways with the purpose of expressing the\r\ndesired genetic information. \r\nThe source for DNA\r\ncloning can be genomic DNA fragments, cDNA fragments produced by the action of\r\nreverse transcriptase on mRNA molecules, chemically synthesized\r\noligonucleotides, or amplified DNA from the products of the polymerase chain\r\nreaction (PCR).\u00a0 Plasmids, phages, and cosmids have all been successfully\r\nused as vectors, and transformation, transfection, and transduction have all\r\nbeen used to introduce the foreign DNA into the E. coli cell. Plasmids\r\nare among the most widely used vectors for the insertion of foreign DNA into an\r\nE. coli. Plasmids lend themselves very well as vectors since they are\r\nindependent replicons which are stabily inherited in an extrachromosomal state\r\nand can be made to carry easily identifiable phenotypic markers such as\r\nantibiotic resistance or sugar fermentation. \r\nAn example of the use\r\nof plasmids to introduce a foreign gene into E. coli in order to produce\r\na useful product is illustrated by the use of the E. coli plasmid pBR322\r\nto clone the gene for production of the human growth hormone,\r\nsomatostatin.\u00a0 In this case, the gene for the small polypeptide hormone\r\nwas produced by synthetic means. The double-stranded DNA coding for the 15\r\namino acids of somatostatin was synthesized with the addition of a translation\r\na stop signal at the end. The synthetic gene was then recombined with the\r\nplasmid within the beta-galactosidase structural gene and introduced into E.\r\ncoli. In this way, the production of the somatostatin peptide could be\r\ncontrolled by the lac operon.\u00a0 In a similar manner, the genes for human\r\ninsulin production were inserted into E. coli which was then able to\r\nsynthesize the human hormone. \r\nSuch general\r\ntechniques of molecular biology and bacterial genetics are now being applied\r\nwithin research laboratories and industry to produce a wide variety of strains\r\nof genetically engineered E. coli from which a number of useful products\r\ncan be produced. Likewise, the problems associated with the expression of\r\neukaryotic DNA by a procaryotic promoter in E. coli were solved by\r\nconstruction of a fusion gene. In this system, the control region and the\r\nN-terminal coding sequence of an E. coli gene are ligated to a\r\neukaryotic sequence sothat translation of the chimeric protein can occur. The\r\nonly condition is that the eukaryotic sequence must be in the correct reading\r\nframe. The desired protein is then enzymatically or chemically cleaved from the\r\nE. coli product. \r\nE. coli strains ave been\r\ngenetically engineered to produce a variety of mammalian proteins, especially\r\nproducts of medical or veterinary interest including enzymes and vaccine\r\ncomponents. E. coli has also been used to manufacture other\r\nsubstances\u00a0 including enzymes that are useful in the degradation of\r\ncellulose and aromatic compounds\u00a0 and enzymes for ethanol production.\r\nThere may be no limit to what E. coli can produce through recombinant\r\nDNA technology as long as the substance is a natural product for which a\r\ngenetic sequence can be found. \r\nStaphylococcus\r\nStaphylococcus aureus. Electron micrograph\r\nfrom Visuals Unlimited, with\r\npermission.\r\nThe Staphylococci\r\nStaphylococci are Gram-positive spherical\r\nbacteria that occur in microscopic clusters resembling grapes. Bacteriological\r\nculture of the nose and skin of normal humans invariably yields staphylococci.\r\nIn 1884, Rosenbach described the two pigmented colony types of staphylococci\r\nand proposed the appropriate nomenclature: Staphylococcus aureus\r\n(yellow) and Staphylococcus albus (white). The latter species is now\r\nnamed Staphylococcus epidermidis. Although more than 20 species of Staphylococcus\r\nare described in Bergey's Manual (2001), only Staphylococcus aureus and Staphylococcus\r\nepidermidis are significant in their interactions with humans. S. aureus\r\ncolonizes mainly the nasal passages, but it may be found regularly in most\r\nother anatomical locales. S epidermidis is an inhabitant of the skin. \r\nTaxonomically, the genus Staphylococcus\r\nis in the Bacterial family Staphylococcaceae, which includes\r\nthree lesser known genera, Gamella, Macrococcus and Salinicoccus.\r\nThe best-known of its nearby phylogenetic relatives are the members of the\r\ngenus Bacillus in the family Bacillaceae, which is on the same\r\nlevel as the family Staphylococcaceae. The Listeriaceae are also\r\na nearby family. \r\nStaphylococcus aureus forms a fairly large\r\nyellow colony on rich medium, S. epidermidis has a relatively small\r\nwhite colony. S. aureus is often hemolytic on blood agar; S.\r\nepidermidis is non hemolytic. Staphylococci are facultative anaerobes that\r\ngrow by aerobic respiration or by fermentation that yields principally lactic\r\nacid. The bacteria are catalase-positive and oxidase-negative. S. aureus\r\ncan grow at a temperature range of 15 to 45 degrees and at NaCl concentrations\r\nas high as 15 percent. Nearly all strains of S. aureus produce the\r\nenzyme coagulase: nearly all strains of S. epidermidis lack this enzyme.\r\nS. aureus should always be considered a potential pathogen; most strains\r\nof S. epidermidis are nonpathogenic and may even play a protective role\r\nin their host as normal flora. Staphylococcus epidermidis may be a\r\npathogen in the hospital environment. \r\nStaphylococci are perfectly spherical cells\r\nabout 1 micrometer in diameter. They grow in clusters because staphylococci\r\ndivide in two planes. The configuration of the cocci helps to distinguish\r\nstaphylococci from streptococci, which are slightly oblong cells that usually\r\ngrow in chains (because they divide in one plane only). The catalase test is\r\nimportant in distinguishing streptococci (catalase-negative) from\r\nstaphylococci, which are vigorous catalase-producers. The test is performed by\r\nadding 3% hydrogen peroxide to a colony on an agar plate or slant.\r\nCatalase-positive cultures produce O2 and bubble at once. The test should\r\nnot be done on blood agar because blood itself contains catalase.\r\nTable 1. Important phenotypic characteristics of Staphylococcus aureus\r\nGram-positive, cluster-forming coccus\r\nnonmotile, nonsporeforming facultative anaerobe\r\nfermentation of glucose produces mainly lactic acid\r\nferments mannitol (distinguishes from S. epidermidis)\r\ncatalase positive\r\ncoagulase positive\r\ngolden yellow colony on agar\r\nnormal flora of humans found on nasal passages, skin and mucous membranes\r\npathogen of humans, causes a wide range of suppurative infections, as well\r\nas food poisoning and toxic shock syndrome\u00a0\r\nPathogenesis of S. aureus infections\r\nStaphylococcus aureus causes a variety of\r\nsuppurative (pus-forming) infections and toxinoses in humans. It causes superficial\r\nskin lesions such as boils, styes and furunculosis; more\r\nserious infections such as pneumonia, mastitis, phlebitis,\r\nmeningitis, and urinary tract infections; and deep-seated\r\ninfections, such as osteomyelitis and endocarditis. S. aureus\r\nis a major cause of hospital acquired (nosocomial) infection of surgical\r\nwounds and infections associated with indwelling medical devices. S. aureus\r\ncauses food poisoning by releasing enterotoxins into food, and toxic\r\nshock syndrome by release of superantigens into the blood stream. \r\nS. aureus expresses many potential virulence\r\nfactors: (1) surface proteins that promote colonization of host\r\ntissues; (2) invasins that promote bacterial spread in tissues (leukocidin,\r\nkinases, hyaluronidase); (3) surface factors that inhibit\r\nphagocytic engulfment (capsule, Protein A); (4) biochemical\r\nproperties that enhance their survival in phagocytes (carotenoids, catalase\r\nproduction); (5) immunological disguises (Protein A, coagulase, clotting\r\nfactor); and (6) membrane-damaging toxins that lyse eukaryotic cell\r\nmembranes (hemolysins, leukotoxin, leukocidin; (7)\r\nexotoxins that damage host tissues or otherwise provoke symptoms of disease (SEA-G,\r\nTSST, ET (8) inherent and acquired resistance to antimicrobial\r\nagents.\r\nFor the majority of diseases caused by S.\r\naureus, pathogenesis is multifactorial, so it is difficult to determine\r\nprecisely the role of any given factor. However, there are correlations between\r\nstrains isolated from particular diseases and expression of particular\r\nvirulence determinants, which suggests their role in a particular diseases. The\r\napplication of molecular biology has led to advances in unraveling the\r\npathogenesis of staphylococcal diseases. Genes encoding potential virulence\r\nfactors have been cloned and sequenced, and many protein toxins have been\r\npurified. With some staphylococcal toxins, symptoms of a human disease can be\r\nreproduced in animals with the purified protein toxins, lending an\r\nunderstanding of their mechanism of action. \r\nHuman staphylococcal infections are frequent,\r\nbut usually remain localized at the portal of entry by the normal host\r\ndefenses. The portal may be a hair follicle, but usually it is a break in the\r\nskin which may be a minute needle-stick or a surgical wound. Foreign bodies,\r\nincluding sutures, are readily colonized by staphylococci, which may makes\r\ninfections difficult to control. Another portal of entry is the respiratory\r\ntract. Staphylococcal pneumonia is a frequent complication of influenza. The\r\nlocalized host response to staphylococcal infection is inflammation,\r\ncharacterized by an elevated temperature at the site, swelling, the\r\naccumulation of pus, and necrosis of tissue. Around the inflamed area, a fibrin\r\nclot may form, walling off the bacteria and leukocytes as a characteristic\r\npus-filled boil or abscess. More serious infections of the skin may occur, such\r\nas furuncles or impetigo. Localized infection of the bone is called\r\nosteomyelitis. Serious consequences of staphylococcal infections occur when the\r\nbacteria invade the blood stream. A resulting septicemia may be rapidly fatal;\r\na bacteremia may result in seeding other internal abscesses, other skin\r\nlesions, or infections in the lung, kidney, heart, skeletal muscle or meninges.\r\nAdherence to Host Cell Proteins\r\nS. aureus cells express on their surface\r\nproteins that promote attachment to host proteins such as laminin and\r\nfibronectin that form the extracellular matrix of epithelial and endothelial\r\nsurfaces. In addition, most strains express a fibrin\/fibrinogen binding protein\r\n(clumping factor) which promotes attachment to blood clots and traumatized\r\ntissue. Most strains of S. aureus express both fibronectin and\r\nfibrinogen-binding proteins. In addition, an adhesin that promotes attachment\r\nto collagen has been found in strains that cause osteomyelitis and septic\r\narthritis. Interaction with collagen may also be important in promoting\r\nbacterial attachment to damaged tissue where the underlying layers have been\r\nexposed. \r\nEvidence that staphylococcal matrix-binding\r\nproteins are virulence factors has come from studying defective mutants in\r\nadherence assays. Mutants defective in binding to fibronectin and to fibrinogen\r\nhave reduced virulence in a rat model for endocarditis, and mutants lacking the\r\ncollagen-binding protein have reduced virulence in a mouse model for septic\r\narthritis, suggesting that bacterial colonization is ineffective. Furthermore,\r\nthe isolated ligand-binding domain of the fibrinogen, fibronectin and collagen\r\nreceptors strongly blocks attachment of bacterial cells to the corresponding host\r\nproteins. \r\nInvasion\r\nThe invasion of host tissues by staphylococci\r\napparently involves the production of a huge array of extracellular proteins,\r\nsome of which may occur also as cell-associated proteins. These proteins are\r\ndescribed below with some possible explanations for their role in invasive\r\nprocess. \r\nMembrane-damaging toxins\r\na-toxin (a-hemolysin) The best\r\ncharacterized and most potent membrane-damaging toxin of S. aureus is\r\na-toxin. It is expressed as a monomer that binds to the membrane of susceptible\r\ncells. Subunits then oligomerize to form heptameric rings with a central pore\r\nthrough which cellular contents leak. \r\nIn humans, platelets and monocytes are\r\nparticularly sensitive to a-toxin. Susceptible cells have a specific receptor\r\nfor a-toxin which allows the toxin to bind causing small pores through which\r\nmonovalent cations can pass. The mode of action of alpha hemolysin is likely by\r\nosmotic lysis. \r\n\u00df-toxin\u00a0 is a sphingomyelinase which\r\ndamages membranes rich in this lipid. The classical test for \u00df-toxin is lysis\r\nof sheep erythrocytes. The majority of human isolates of S. aureus do\r\nnot express \u00df-toxin. A lysogenic bacteriophage is known to encode the toxin. \r\nd-toxin is a very small peptide toxin\r\nproduced by most strains of S. aureus. It is also produced by S.\r\nepidermidis. The role of d-toxin in disease is unknown. \r\nLeukocidin is a multicomponent protein toxin\r\nproduced as separate components which act together to damage membranes.\r\nLeukocidin forms a hetero-oliogmeric transmembrane pore composed of four LukF\r\nand four LukS subunits, thereby forming an octameric pore in the affected\r\nmwembrane. Leukocidin is hemolytic, but less so than alpha hemolysin. \r\nOnly 2% of all of S. aureus isolates\r\nexpress leukocidin, but nearly 90% of the strains isolated from severe\r\ndermonecrotic lesions express this toxin, which suggests that it is an\r\nimportant factor in necrotizing skin infections. \r\nCoagulase and clumping factor\r\nCoagulase is an extracellular protein which\r\nbinds to prothrombin in the host to form a complex called staphylothrombin. The\r\nprotease activity characteristic of thrombin is activated in the complex,\r\nresulting in the conversion of fibrinogen to fibrin. Coagulase is a traditional\r\nmarker for identifying S aureus in the clinical microbiology laboratory.\r\nHowever, there is no overwhelming evidence that it is a virulence factor,\r\nalthough it is reasonable to speculate that the bacteria could protect\r\nthemselves from phagocytic and immune defenses by causing localized clotting. \r\nThere is some confusion in the literature\r\nconcerning coagulase and clumping factor, the fibrinogen-binding determinant on\r\nthe S. aureus cell surface. Partly the confusion results from the fact\r\nthat a small amount of coagulase is tightly bound on the bacterial cell surface\r\nwhere it can react with prothrombin leading to fibrin clotting. However,\r\ngenetic studies have shown unequivocally that coagulase and clumping factor are\r\ndistinct entities. Specific mutants lacking coagulase retain clumping factor\r\nactivity, while clumping factor mutants express coagulase normally. \r\nStaphylokinase\r\nMany strains of S aureus express a\r\nplasminogen activator called staphylokinase. This factor lyses fibrin.The\r\ngenetic determinant is associated with lysogenic bacteriophages. A complex\r\nformed between staphylokinase and plasminogen activates plasmin-like\r\nproteolytic activity which causes dissolution of fibrin clots. The mechanism is\r\nidentical to streptokinase, which is used in medicine to treat patients\r\nsuffering from coronary thrombosis. As with coagulase, there is no strong\r\nevidence that staphylokinase is a virulence factor, although it seems\r\nreasonable to imagine that localized fibrinolysis might aid in bacterial\r\nspreading. \r\nOther extracellular enzymes\r\nS. aureus can express proteases, a lipase, a\r\ndeoxyribonuclease (DNase) and a fatty acid modifying enzyme (FAME). The first\r\nthree probably provide nutrients for the bacteria, and it is unlikely that they\r\nhave anything but a minor role in pathogenesis. However, the FAME enzyme may be\r\nimportant in abscesses, where it could modify anti-bacterial lipids and prolong\r\nbacterial survival. \r\nAvoidance of Host Defenses\r\nS. aureus expresses a number of factors that\r\nhave the potential to interfere with host defense mechanisms. This includes\r\nboth structural and soluble elements of the bacterium. \r\nCapsular Polysaccharide\r\nThe majority of clinical isolates of S\r\naureus express a surface polysaccharide of either serotype 5 or 8. This has\r\nbeen called a microcapsule because it can be visualized only by electron\r\nmicroscopy unlike the true capsules of some bacteria which are readily\r\nvisualized by light microscopy. S. aureus strains isolated from\r\ninfections express high levels of the polysaccharide but rapidly lose the\r\nability when cultured in the laboratory. The function of the capsule in\r\nvirulence is not entirely clear. Although it does impede phagocytosis in the\r\nabsence of complement, it also impedes colonization of damaged heart valves,\r\nperhaps by masking adhesins. \r\nProtein A\r\nProtein A is a surface protein of S.\r\naureus which binds IgG molecules by their Fc region. In serum, the bacteria\r\nwill bind IgG molecules in the wrong orientation on their surface which\r\ndisrupts opsonization and phagocytosis. Mutants of S. aureus lacking\r\nprotein A are more efficiently phagocytosed in vitro, and mutants in infection\r\nmodels have diminished virulence. \r\nLeukocidin\r\nS. aureus can express a toxin that specifically\r\nacts on polymorphonuclear leukocytes. Phagocytosis is an important defense\r\nagainst staphylococcal infection so leukocidin should be a virulence factor. \r\nExotoxins\r\nS. aureus can express several different types\r\nof protein toxins which are probably responsible for symptoms during\r\ninfections. Those which damage the membranes of cells were discussed above\r\nunder Invasion. Some will lyse erythrocytes, causing hemolysis, but it\r\nis unlikely that hemolysis is a relevant determinant of virulence in vivo.\r\nLeukocidin causes membrane damage to leukocytes, but is not hemolytic. \r\nSystemic release of a-toxin causes septic\r\nshock, while enterotoxins and TSST-1 are superantigens that may cause toxic\r\nshock. Staphylococcal enterotoxins cause emesis (vomiting) when ingested and\r\nthe bacterium is a leading cause of food poisoning. \r\nThe exfoliatin toxin causes the scalded skin\r\nsyndrome in neonates, which results in widespread blistering and loss of the\r\nepidermis. There are two antigenically distinct forms of the toxin, ETA and\r\nETB. The toxins have esterase and protease activity and apparently target a\r\nprotein which is involved in maintaining the integrity of the epidermis.\r\nSuperantigens: enterotoxins and toxic shock\r\nsyndrome toxin\r\n\r\nS. aureus secretes two types of toxin with\r\nsuperantigen activity, enterotoxins, of which there are six antigenic\r\ntypes (named SE-A, B, C, D, E and G), and toxic shock syndrome toxin (TSST-1).\r\nEnterotoxins cause diarrhea and vomiting when ingested and are responsible for\r\nstaphylococcal food poisoning. TSST-1 is expressed systemically and is the\r\ncause of toxic shock syndrome (TSS). When expressed systemically, enterotoxins\r\ncan also cause toxic shock syndrome. In fact, enterotoxins B and C cause 50% of\r\nnon-menstrual cases of TSS. TSST-1 is weakly related to enterotoxins, but it\r\ndoes not have emetic activity. TSST-1 is responsible for 75% of TSS, including\r\nall menstrual cases. TSS can occur as a sequel to any staphylococcal infection\r\nif an enterotoxin or TSST-1 is released systemically and the host lacks\r\nappropriate neutralizing antibodies. \r\nSuperantigens stimulate T cells\r\nnon-specifically without normal antigenic recognition (Figure 4). Up to one in\r\nfive T cells may be activated, whereas only 1 in 10,000 are stimulated\r\nduring a usual antigen presentation. Cytokines are released in large amounts,\r\ncausing the symptoms of TSS. Superantigens bind directly to class II major\r\nhistocompatibility complexes of antigen-presenting cells outside the\r\nconventional antigen-binding grove. This complex recognizes only the Vb element\r\nof the T cell receptor. Thus any T cell with the appropriate Vb element can be\r\nstimulated, whereas normally, antigen specificity is also required in binding.