INTRODUCTIONThe microbiological safety of foods is a major concern to consumers and to the food industry. Despite considerable progress made in technology, consumer education, and regulations, food safety continues to be a major challenge to our public health and economy. During the past two decades, food safety received considerable attention due to the emergence of several new food-borne pathogens and the involvement of foods that traditionally have been considered safe, in many food-borne disease outbreaks. Further, industrialization of the food supply through mass production, distribution, increased globalization, and consumer demands for preservative-free, convenience foods and ready-to-eat meals highlight the significance of the microbial safety of foods. A study published by the U.S. Centers for Disease Control and Prevention (CDC), in 1999, reported an estimated 76 million cases of food-borne illnesses, which resulted in 325,000 hospitalizations and 5,000 deaths in the United States annually.1 Besides the public health impact, outbreaks of food-borne illness impose major economic losses to both the food industry and society. The annual estimated cost of food-borne illnesses caused by the four most common pathogens alone account for approximately $6.9 billion.2 The types of microbiological hazards associated with foods can be classified as bacterial, viral, fungal, and parasitic.
BACTERIAL FOOD-BORNE PATHOGENSBacteria are the major agents of microbial food-borne illnesses, and account for an estimated 4 million food-borne illnesses annually in the United States. Bacterial food-borne diseases can be classified into food-borne infections resulting from ingestion of foods containing viable cells of bacterial pathogens, and food-borne intoxications, which result from consumption of foods containing preformed toxins produced by toxigenic bacteria. The primary bacterial pathogens associated with food-borne diseases are discussed below.
ESCHERICHIA COLI O157:H7Enterohemorrhagic Escherichia coli O157:H7 emerged in 1982 as a food-borne pathogen, and is now recognized as a major public health concern in the United States. Many food-associated outbreaks are reported each year, with 217 confirmed cases reported in 2004.3 Although approximately 50% of the reported outbreaks in the United States have been associated with consumption of undercooked beef burgers, a wide variety of other foods, including raw milk, roast beef, venison jerky, salami, yogurt, lettuce, unpasteurized apple juice, cantaloupe, alfalfa sprouts, and coleslaw, have been implicated as vehicles of E. coli O157:H7 infection.4 In addition, outbreaks involving person-to-person and waterborne transmission have been reported.4 Cattle have been implicated as one of the principal reservoirs of E. coli O157:H7,5–8 with the terminal rectum being a principal site of colonization in adult animals.9 E. coli O157:H7 can survive in bovine feces for many months,10 hence potentially contaminating cattle, food, water, and the environment through contact with manure. Although surveys conducted in the 1980s and 1990s generally showed a low fecal prevalence of E. coli O157:H7 in cattle,8,11,12 later studies using improved enrichment and isolation procedures have revealed that the overall prevalence of E. coli O157:H7 in cattle may be substantially higher than originally found.13–16 A study by Elder et al.13 revealed that, of cattle from 29 feedlots presented for slaughter in the Midwestern United States, 72% had at least one E. coli O157-positive fecal sample and 38% had positive hide samples. The study revealed an overall E. coli O157 prevalence of 28% (91 out of 327) in feces, and 11% (38 out of 355) on the hide. Studies by others revealed that the prevalence of E. coli O157 in feed lots in the United States can be as high as 63%, particularly during the summer, under muddy conditions, or with feeding of barley.17,18 These results are of particular concern, because high fecal shedding and the presence of E. coli O157:H7 on hides can lead to contamination of foods of bovine origin with the pathogen during slaughtering and processing operations.19 In addition, many E. coli O157:H7 outbreaks involving nonbovine foods, such as fruits and vegetables, are often linked to cross contamination of the implicated food with contaminated bovine manure.20–23 Direct zoonotic and environmental transmission is a more recently recognized mode of E. coli O157:H7 spread to humans. Contact with the farm environment, including recreational or occupational visits, has been associated with E. coli O157:H7 infections in humans.24,25 Since reduced fecal shedding of E. coli O157:H7 by cattle would potentially decrease food-borne outbreaks of E. coli O157:H7, a variety of approaches for reducing its carriage in cattle, including vaccination,26 feeding cattle with competitive exclusion bacteria,27 and supplementation of cattle diet with sodium chlorate,28 have been explored.
Acidification is commonly used in food processing to control growth and survival of spoilage-causing and pathogenic microorganisms in foods. The U.S. Food and Drug Administration (FDA) does not regard foods with pH = 4.6 (high-acid foods) to be microbiologically hazardous. However, E. coli O157:H7 has been associated with outbreaks attributed to high-acid foods, including apple juice, mayonnaise, fermented sausage, and yogurt,29 raising concerns about the safety of these foods. Several studies have revealed that many strains of E. coli O157:H7 are highly tolerant to acidic conditions, being able to survive for extended periods of time in synthetic gastric juice and in highly acidic foods.29,30 Further, exposure of E. coli O157:H7 to mild or moderate acidic environments can induce an acid tolerance response, which enables the pathogen to survive extreme acidic conditions. For example, acid-adapted cells of E. coli O157:H7 survived longer in apple cider, fermented sausage, and hydrochloric acid than nonacid-adapted cells.31,32 However, E. coli O157:H7 is not unusually heat resistant33 or salt tolerant34 unless cells are preexposed to acid to become acid adapted. Acid-adapted E. coli O157:H7 cells have been determined to have increased heat tolerance.
In humans, two important manifestations of illness have been reported with E. coli O157:H7 infection. These include hemorrhagic colitis and hemolytic uremic syndrome (HUS).36 Hemorrhagic colitis is characterized by a watery diarrhea that progresses into grossly bloody diarrhea, indicative of significant amounts of gastrointestinal bleeding. Severe abdominal pain is common, but fever is usually not present. The illness typically lasts from 2 to 9 days. HUS is a severe condition, particularly among the very young and the elderly, which involves damage to kidneys, leading to renal failure and death.
Two important factors attributed to the pathogenesis of E. coli O157:H7 include the ability of the pathogen to adhere to the intestinal mucosa of the host, and production of Shiga toxin I or Shiga toxin II.35 Retrospective analysis of foods implicated in outbreaks of E. coli O157:H7 infection suggests a low infectious dose of the pathogen, probably less than 100 cells.36
SALMONELLA SPECIESSalmonella spp. are facultatively anaerobic, gram-negative, rod-shaped bacteria belonging to the family Enterobacteriaceae. Members of the genus Salmonella have an optimum growth temperature of 37°C and utilize glucose with the production of acid and gas.37 Salmonella spp. are widely distributed in nature. They colonize the intestinal tract of humans, animals, birds, and reptiles, and are excreted in feces, which contaminate the environment, water, and foods.38 Many food products, especially foods having contact with animal feces, including beef, pork, poultry, eggs, milk, fruits, and vegetables, have been associated with outbreaks of salmonellosis.39 Salmonella spp. can be divided into host-adapted serovars and those without any host preferences.
The ability of many strains of Salmonella to adapt to extreme environmental conditions emphasizes the potential risk of these microorganisms as food-borne pathogens. Although salmonellae optimally grow at 37°C, the genus Salmonella consists of strains which are capable of growth from 5 to 47°C.40 Salmonella spp. can grow at pH values ranging from 4.5 to 7.0, with optimum growth observed near neutral pH.38 Preexposure of Salmonella to mild acidic environments (pH 5.5 to 6.0) can induce in some strains an acid tolerance response, which enables the bacteria to survive for extended periods of exposure to acidic and other adverse environmental conditions such as heat and low water activity.41,42 However, most Salmonella spp. possess no unusual tolerance to salt and heat. A concentration of 3 to 4% NaCl can inhibit the growth of Salmonella.43 Most salmonellae are sensitive to heat; hence ordinary pasteurization and cooking temperatures are capable of killing the pathogen.44 Most of the food-borne serovars are in the latter group.
The ability of many strains of Salmonella to adapt to extreme environmental conditions emphasizes the potential risk of these microorganisms as food-borne pathogens. Although salmonellae optimally grow at 37°C, the genus Salmonella consists of strains which are capable of growth from 5 to 47°C.40 Salmonella spp. can grow at pH values ranging from 4.5 to 7.0, with optimum growth observed near neutral pH.38 Preexposure of Salmonella to mild acidic environments (pH 5.5 to 6.0) can induce in some strains an acid tolerance response, which enables the bacteria to survive for extended periods of exposure to acidic and other adverse environmental conditions such as heat and low water activity.41,42 However, most Salmonella spp. possess no unusual tolerance to salt and heat. A concentration of 3 to 4% NaCl can inhibit the growth of Salmonella.43 Most salmonellae are sensitive to heat; hence ordinary pasteurization and cooking temperatures are capable of killing the pathogen.44
Salmonellosis is one of the most frequently reported food-borne diseases worldwide.45 The overall incidence of salmonellosis in the United States has been reported to have declined by approximately 8% during the period from 1996 to 2004.46 In the United States, food-associated Salmonella infections are estimated to cost $0.5 to $2.3 billion annually.47 The most common serovars of Salmonella that cause food-borne salmonellosis in humans are Salmonella enterica subsp. enterica serovar Typhimurium and Salmonella enterica subsp. enterica serovar Enteritidis. A wide variety of foods, including beef, pork, milk, chicken, and turkey have been associated with outbreaks caused by S. Typhimurium. Although the incidence of S. Typhimurium in the United States has decreased by approximately 40% during 1996 to 2004,46 the emergence of S. Typhimurium DT 104, a new phage type in the 1990s in the United States and Europe raised a significant public health concern. This is because S. Typhimurium DT 104 is resistant to multiple antibiotics, including ampicillin, chloramphenicol, penicillin, streptomycin, tetracycline, and sulfonamides.48,49 A major risk factor identified in the acquisition of S. Typhimurium DT 104 infection in humans is that the infecting strain is resistant to prior treatment with antimicrobial agents, for the 4 weeks preceding infection.50 CDC reported that 11% of the total Salmonella spp. isolated from humans in 2000 were resistant to at least five different antibiotics, and a few of the multidrug-resistant strains were also resistant to gentamicin and cephalosporins.51 These aforementioned reports underscore the prudent use of antibiotics in human therapy and animal husbandry.
Salmonella Enteritidis outbreaks are most frequently associated with consumption of poultry products, especially undercooked eggs and chicken. Moreover, international travel, especially to developing countries, has been associated with human infections of S. Enteritidis in the United States.52 CDC reported 677 outbreaks of egg-borne S. Enteritidis with 23,366 illnesses, 1,988 hospitalizations, and 33 deaths in the United States during the period of 1990 to 2001.53 Another report estimated 700,000 cases of egg-borne salmonellosis in the United States, accounting for approximately 47% of total food-borne salmonellosis and costing more than $1 billion annually.47 Approximately 65 billion shell eggs are sold annually in the United States,54 with a per capita consumption of approximately 254 eggs per year. Hence, undercooked Salmonella-contaminated eggs are a major hazard to human health. Egg contamination with S. Enteritidis results by penetration through the eggshell from contaminated chicken feces during or after oviposition.55–57 Contamination of egg contents (yolk, albumen, and eggshell membranes) may also occur by transmission of the pathogen from infected ovaries or oviducts by the transovarian route before oviposition.58–60
Salmonella Typhi is the causative agent of typhoid fever, a serious human disease. Typhoid fever has a long incubation period of 7 to 28 days, and is characterized by prolonged and spiking fever, abdominal pain, diarrhea, and headache.37 The disease can be diagnosed by isolating the pathogen from urine, blood, or stool specimens of affected persons. In 2003, 356 cases of typhoid fever were reported in the United States.61 S. Typhi is an uncommon cause of food-borne illness in the United States, with approximately 74% of these cases occurring in persons who traveled internationally, especially to South Asia, 6 weeks preceding the disease appearance.61
CAMPYLOBACTER SPECIESThe genus Campylobacter consists of 14 species; however, C. jejuni subsp. jejuni and C. coli are the dominant food-borne pathogens. C. jejuni is a slender, rod-shaped, microaerophilic bacterium that requires approximately 3 to 6% oxygen for growth. It can be differentiated from C. coli by its ability to hydrolyze hippurate.62 The organism does not survive well in the environment, being sensitive to drying, highly acidic conditions, and freezing. It is also readily killed in foods by adequate cooking.63
C. jejuni is the most commonly reported bacterial cause of food-borne infection in the United States,63–65 with the highest incidence in Hawai.66 Many animals including poultry, swine, cattle, sheep, horses, and domestic pets, harbor C. jejuni in their intestinal tracts, hence serving as sources of human infection. However, chickens serve as the most common reservoir of C. jejuni, where the bacterium primarily colonizes the mucus overlying the epithelial cells in the ceca and small intestine. l-Fucose, the major carbohydrate component present in the mucin of chicken cecal mucus is used by C. jejuni as a sole substrate for growth.67,68 Thus, the cecal environment in chickens is favorable for the survival and proliferation of C. jejuni,67 and selects colonization of C. jejuni in the birds. Although a number of vehicles such as beef, pork, eggs, and untreated water have been implicated in outbreaks of campylobacter enteritis, chicken and unpasteurized milk are reported as the most commonly involved foods.69 Epidemiologic investigations have revealed a significant link between human Campylobacter infection, and handling or consumption of raw or undercooked poultry meat.70–74 Since colonization of broiler chickens by C. jejuni results in horizontal transmission of the pathogen and carcass contamination during slaughter, a variety of approaches for reducing its cecal carriage by chickens has been undertaken. These approaches include competitive exclusion microorganisms,75 feeding birds with bacteriophages,76,77 and acidified feed,78 and vaccination.79,80 In the United States, an increasing number of fluoroquinoloneresistant (e.g., ciprofloxacin) human Campylobacter infections has been reported,81 and this is attributed to the use of this antibiotic in poultry production.82
Usually Campylobacter enteritis in humans is a self-limiting illness characterized by abdominal cramps, diarrhea, headache, and fever lasting up to 4 days. However, severe cases, involving bloody diarrhea and abdominal pain mimicking appendicitis, also occur.62 Guillain-Barre syndrome (GBS) is an infrequent sequela to Campylobacter infection in humans.83 GBS is characterized by acute neuromuscular paralysis63 and is estimated to occur in approximately 1 of every 1000 cases of Campylobacter enteritis.84 A few strains of C. jejuni reportedly produce a heat-labile enterotoxin similar to that produced by Vibrio cholerae and enterotoxigenic E. coli.62 Some strains of C. jejuni and C. coli can also produce a cytolethal distending toxin, which causes a rapid and specific cell cycle arrest in HeLa and Caco-2 cells.85
SHIGELLA SPECIESShigella is a common cause of human diarrhea in the United States. The genus Shigella is divided into four major groups: S. dysenteriae (group A), S. flexneri (group B), S. boydii (group C), and S. sonnei (group D) based on the organism’s somatic (O) antigen. Although all four groups have been involved in human infections, S. sonnei accounts for more than 75% of shigellosis cases in humans,86 and has been linked to persistent infections in community and day-care centers.87–89 Humans are the natural reservoir of Shigella spp. The fecal–oral route is the primary mode of transmission of shigellae and proper personal hygiene and sanitary practices of cooks and food handlers can greatly reduce the occurrence of outbreaks of shigellosis. Most food-borne outbreaks of shigellosis are associated with ingestion of foods such as salads and water contaminated with human feces containing the pathogen. Shigellosis is characterized by diarrhea containing bloody mucus, which lasts 1 to 2 weeks. The infectious dose for Shigella infection is low. The ID50 of S. flexneri and S. sonnei in humans is approximately 5000 microorganisms and that of S. dysenteriae is a few hundred cells, hence secondary transmission of Shigella by person-to-person contact frequently occurs in outbreaks of food-borne illness. A new and emerging serotype of S. boydii, namely serotype 20 has been reported in the United States.90
YERSINIA ENTEROCOLITICAYersinia enterocolitica is a gram-negative, rod-shaped, facultative anaerobic bacterium, which was first isolated and described during the 1930s.91 Swine have been identified as an important reservoir of Yersinia enterocolitica, in which the pathogen colonizes primarily the buccal cavity.92 Although pork and pork products are considered to be the primary vehicles of Y. enterocolitica, a variety of other foods, including milk, beef, lamb, seafood, and vegetables, has been identified as vehicles of Y. enterocolitica infection.93 One of the largest outbreaks of yersiniosis in the United States was associated with milk.94 Water has also been a vehicle of several outbreaks of Y. enterocolitica infection.94 Surveys have revealed that Y. enterocolitica is frequently present in foods, having been isolated from 11% of sandwiches, 15% of chilled foods, and 22% of raw milk in Europe.95 Several serovars of pathogenic Y. enterocolitica have been reported, which include O:3, O:5, O:8, and O:9,96,97 with serovar 0:3 being common in the United States.98–100 In addition to food-borne outbreaks, reports of blood transfusion-associated Y. enterocolitica sepsis indicate another potential mode of transmission of this pathogen.101,102 Among bacteria, Y. enterocolitica has emerged as a significant cause of transfusion-associated bacteremia and mortality (53%), with 49 cases reported since this condition was first documented in 1975.103 A review of these cases revealed that bacteremia may occur in a subpopulation of individuals with Y. enterocolitica gastrointestinal infection.96 The strains of Y. enterocolitica responsible for transfusion acquired yersiniosis are the same serobiotypes as those associated with enteric infections.
An unusual characteristic of Y. enterocolitica that influences food safety is its ability to grow at low temperatures, even as low as –1°C.104 Y. enterocolitica readily withstands freezing and can survive in frozen foods for extended periods, even after repeated freezing and thawing.105 Refrigeration (4°C) is one of the common methods used in food processing to control growth of spoilage and pathogenic microorganisms in foods. However, several studies have revealed growth of Y. enterocolitica in foods stored at refrigeration temperature. Y. enterocolitica grew on pork, chicken, and beef at 0 to 1°C.106,107 The psychrotrophic nature of Y. enterocolitica also poses problems for the blood transfusion industry, mainly because of its ability to proliferate and release endotoxin in blood products stored at 4°C without manifesting any alterations in their physical appearance. The ability of Y. enterocolitica to grow well at refrigeration temperature has been exploited for isolating the pathogen from foods, water, and stool specimens. Such samples are incubated at 4 to 8°C in an enrichment broth for several days to selectively culture Y. enterocolitica based on its psychrotrophic nature.
Y. enterocolitica is primarily an intestinal pathogen with a predilection for extra-intestinal spread under appropriate host conditions such as immunosuppression. In the gastrointestinal tract, Y. enterocolitica can cause acute enteritis, enterocolitis, mesenteric lymphadenitis, and terminal ileitis often mimicking appendicitis.96 Infection with Y. enterocolitica often leads to secondary, immunologically induced sequelae such as arthritis (most common), erythema nodosum, Reiter’s syndrome, glomerulonephritis,
and myocarditis.
VIBRIO SPECIESSeafoods form a vital part of the American diet, and their consumption in the United States has risen steadily over the past few decades from an average of 4.5 kg per person in 1960 to about 7 kg in 2002.108,109 However, according to a recent report published by the Center for Science in the Public Interest, contaminated seafoods have been recognized as a leading known cause of most food-borne illness outbreaks in the United States.110 Vibrios, especially V. parahaemolyticus, V. vulnificus, and V. cholerae, which are commonly associated with estuarine and marine waters, represent the major pathogens resulting in disease outbreaks through consumption of seafoods. V. parahaemolyticus and V. vulnificus are halophilic in nature, requiring the presence of 1 to 3% sodium chloride for optimum growth. V. cholerae can grow in media without added salt, although their growth is stimulated by the presence of sodium ions.
Among the three species of Vibrio, V. parahaemolyticus accounts for the highest number of food-borne diseases outbreaks in the United States. V. parahaemolyticus is present in coastal waters of the United States and throughout the world. V. parahaemolyticus being an obligate halophile, can multiply in substrates with sodium chloride concentrations ranging from 0.5 to 10%, with 3% being the optimal concentration for growth. The ability of V. parahaemolyticus to grow in a wide range of salt concentrations reflects on its existence in aquatic environments with various salinities. V. parahaemolyticus has a remarkable ability for rapid growth, and generation times as short as 12 to 18 min in seafoods have been reported at 30ºC. Growth rates at lower temperatures are slower, but counts were found to increase from 102 to 108 CFU/g after 24 h storage at 25ºC in homogenized shrimp, and from 103 to 108 CFU/g after 7 days of storage at 12ºC in homogenized oysters.111 Because of its rapid growth, proper refrigeration of cooked seafoods to prevent regrowth of the bacterium is critical to product safety. A survey by the U.S. FDA revealed that 86% of 635 seafood samples contained V. parahaemolyticus, being isolated from clams, oysters, lobsters, scallops, shrimp, fish, and shellfish.112 A new serotype of V. parahaemolyticus, O3:K6 that emerged in Southeast Asia in the 1990s, has been implicated in oyster-related outbreaks in the United States in 1997 and 1998.113 An important virulence characteristic of pathogenic strains of V. parahaemolyticus is their ability to produce a thermostable hemolysin (Kanagawa hemolysin).114 Studies in humans on the infectious dose of pathogenic V. parahaemolyticus strains revealed that ingestion of approximately 105 to 107 organisms can cause gastroenteritis.112
V. cholerae serovars O1 and O139, the causative agents of cholera in humans, are a part of the normal estuarine microflora, and foods such as raw fish, mussels, oysters, and clams have been associated with outbreaks of cholera.115 Infected humans can serve as short-term carriers, shedding the pathogen in feces. Cholera is characterized by profuse diarrhea, potentially fatal in severe cases, and often described as “rice water” diarrhea due to the presence of prolific amounts of mucus in the stools. Gastroenteritis caused by non-O1 and non-O139 serovars of V. cholerae is usually mild in nature. During the period from 1996 to 2005, a total of 64 cases of toxigenic V. cholerae O1 were reported in the United States, of which 35 (55%) cases, were acquired during foreign travel and 29 (45%) cases were domestically acquired.116 Seven (24%) of the 29 domestic cases were attributed to consumption of Gulf Coast seafood (crabs, shrimp, or oysters). Moreover, 7 of the 11 domestic cholera cases in 2005 were reported during October to December, after Hurricanes Katrina and Rita, although no evidence suggests increased risk for cholera among Gulf Coast residents or consumers of Gulf Coast seafood after the hurricanes. In 2003, a total of 111,575 cases of cholera worldwide were reported to the World Health Organization from 45 countries.117
V. vulnificus is the most serious of the vibrios and is responsible for most of the seafood-associated deaths in the United States, especially in Florida.112 V. vulnificus results in life threatening bacteremia, septicemia, and necrotizing fasciitis in person with liver disorders and high iron level in blood.118 Although a number of seafoods has been associated with V. vulnificus infection, raw oysters are the most common vehicle associated with cases of illness.119
ENTEROBACTER SAKAZAKIIEnterobacter sakazakii is an emerging food-borne pathogen that causes severe meningitis, meningo-encephalitis, sepsis, and necrotizing enterocolitis in neonates and infants.120–123 The epidemiology and reservoir of this pathogen are still unknown and most strains have been isolated from clinical specimens such as cerebrospinal fluid, blood, skin, wounds, urine, and respiratory and digestive tract samples.124 The bacterium has also been isolated from foods such as cheese, minced beef, sausage, and vegetables.125 Recently, Kandhai et al.126,127 isolated E. sakazakii from household and food production facility environmental samples, such as scrapings from dust, vacuum cleaner bags, and spilled product near equipment, and proposed that the bacterium could be more widespread in the environment than previously thought. Although the environmental source of E. sakazakii has not been identified, epidemiological studies implicate dried infant formula as the route of transmission to preterm infants.123,128–130 The bacterium has been isolated from powdered infant formula by numerous investigators.129,131–133 Muytjens and coworkers133 isolated the pathogen from powdered infant formula from 35 different countries.
E. sakazakii possesses several characteristics that enable it to grow and survive in infant formula. For example, the bacterium can grow at temperatures as low as 5.5°C,134 which is within the temperature range of many home refrigerators.135 A study on the thermal resistance of E. sakazakii in reconstituted infant formula indicated that it is one of most thermotolerant bacteria within the family Enterobacteriaceae.136 A recent study by Breeuwer et al.137 revealed that E. sakazakii also has a high tolerance to osmotic stress and desiccation. In addition, E. sakazakii possesses a short lag time and generation time in reconstituted infant formula,134 whereby improper temperature storage of reconstituted formula may permit its substantial growth. Recently, Iversen and Forsythe138 reported the isolation of E. sakazakii from a variety of foods, including powdered infant formula, dried infant food, and milk powder as well as certain herbs and spices. The first case of neonatal meningitis caused by E. sakazakii was reported in 1958,139 and since then a number of E. sakazakii infections in neonates have been reported worldwide, including the United States. In the United States, an outbreak of E. sakazakii infection involving four preterm infants occurred in the neonatal intensive care unit of a hospital in Memphis, resulting in sepsis, bloody diarrhea, and intestinal colonization. The source of infection was traced to contaminated infant formula that was termperature abused after reconstitution.129 In 2002, Himelright et al. 140 reported a case of fatal neonatal meningitis caused by E. sakazakii in Tennessee, associated with feeding of contaminated infant formula that was temperature abused following reconstitution. The infection occurred in the neonatal intensive care unit of a hospital and surveillance studies identified two more cases of suspected infection with positive stool or urine in seven more infants. There were many recalls of E. sakazakii-contaminated infant formula in the United States. In November 2002, a nationwide recall of more than 1.5 million cans of dry infant formula contaminated with E. sakazakii was reported.141 On April 9, 2002, the FDA issued an alert to U.S. health-care professionals regarding the risk associated with E. sakazakii infections among neonates-fed milk-based, powdered-infant formula. The International Commission on Microbiological Specification for Foods classified E. sakazakii as a “severe hazard for restricted populations, life threatening or substantial chronic sequelae of long duration,” specifically for preterm infants. This places E. sakazakii along with other serious food- and water-borne pathogens such as Listeria monocytogenes, Clostridium botulinum types A and B, and Cryptosporidium parvum.142
The most common clinical manifestations of infections due to E. sakazakii are sepsis and meningitis in neonates. In more than 90% of the cases reported, patients developed meningitis with a very high prevalence for developing brain abscesses, and less frequently ventriculitis and hydrocephalus.143,144 While the reported mortality rates of E. sakazakii infections in neonates has declined over time from 50% or more to less than 20% due to advances in antimicrobial chemotherapy, an increasing incidence of resistance to commonly used antibiotics necessitates a reevaluation of existing treatment strategies.124 Biering et al.131 indicated that besides the high rate of mortality, the central nervous system (CNS) infections due to E. sakazakii often lead to permanent impairment in mental and physical capabilities in surviving patients. In addition to meningitis, E. sakazakii is also reported to cause necrotizing enterocolitis in neonates, and rarely bacteremia, osteomyelitis, and pneumonia in elderly adults.122,123,145,146
AEROMONAS HYDROPHILAAlthough Aeromonas species have been recognized as pathogens of cold-blooded animals, their potential to cause human infections, especially food-borne illness, received attention only recently. A. hydrophila has been isolated from drinking water, fresh and saline waters, and sewage.147 It also has been isolated from a variety of foods such as fish, oyster, shellfish, raw milk, ground beef, chicken, and pork.147 Although A. hydrophila is sensitive to highly acidic conditions and does not possess any unusual thermal resistance, some strains are psychrotrophic and grow at refrigeration temperature.148 A. hydrophila can grow on a variety of refrigerated foods, including pork, asparagus, cauliflower, and broccoli.149,150 However, considering the widespread occurrence of A. hydrophila in water and food and its relatively infrequent association with human illness, it is likely that most strains of this bacterium are not pathogenic for humans. A. hydrophila infection in humans is characterized by watery diarrhea and mild fever. Virulent strains of A. hydrophila produce a 52-kDa polypeptide, which possesses enterotoxic, cytotoxic, and hemolytic activities.151
PLESIOMONAS SHIGELLOIDESP. shigelloides has been implicated in several cases of sporadic and epidemic gastroenteritis.152 The pathogen is present in fresh and estuarine waters, and has been isolated from various aquatic animals.148 Seafoods such as fish, crabs, and oysters have been associated with cases of P. shigelloides infection. The most common symptoms of P. shigelloides infection include abdominal pain, nausea, chills, fever, and diarrhea. Potential virulence factors of P. shigelloides include cytotoxic enterotoxin, invasins, and ß-hemolysin.148 An outbreak of P. shigelloides infection linked to well water and involving 30 persons was reported in New York in 1996.153
LISTERIA MONOCYTOGENESListeria monocytogenes has emerged into a significant food-borne pathogen throughout the world, especially in the United States. There are an estimated 2500 cases of listeriosis annually in the United States, with a mortality rate of ˜25%.1 Further, L. monocytogenes is of economic significance, causing an estimated monetary loss of $2.3 billion annually in the United States.154 A large outbreak of listeriosis involving more than 100 cases and associated with eating contaminated turkey frankfurters occurred during 1998 to 1999.155 During this period of time there were more than 35 recalls of a number of different food products contaminated with listeriae.155 In 2002, a large outbreak of listeriosis in the United States involving 46 people, 7 deaths, and 3 miscarriages, resulted in a recall of 27.4 million pounds of fresh and frozen ready-to-eat chicken and turkey products.156 In 2003, 696 cases of listeriosis were reported in the United States, with more than 50% of the cases occurring in persons above 60 years of age.61
L. monocytogenes is widespread in nature, occurring in soil, vegetation, and untreated water. Humans and a wide variety of farm animals, including cattle, sheep, goat, pig, and poultry, are known sources of L. monocytogenes.157,158 L. monocytogenes also occurs frequently in food processing facilities, especially in moist areas such as floor drains, floors, and processing equipment. 159 L. monocytogenes can also grow in biofilms attached to a variety of processing plant surfaces such as stainless steel, glass, and rubber.160 A wide spectrum of foods, including milk, cheese, beef, pork, chicken, seafoods, fruits, and vegetables, has been identified as vehicles of L. monocytogenes.158 However, ready-to-eat cooked foods such as low-acid soft cheese, pâtes, and cooked poultry meat, which can support the growth of listeriae to large populations (>106 cells/g) when held at refrigeration temperature for several weeks, have been regarded as high-risk foods.161,162 L. monocytogenes possesses several characteristics which enable the pathogen to successfully contaminate, survive, and grow in foods, thereby resulting in outbreaks. These traits include an ability to grow at refrigeration temperature and in a medium with minimal nutrients, to survive in acidic conditions, for example, pH 4.2, to tolerate up to 10% sodium chloride, to survive incomplete cooking or subminimal pasteurization treatments, and to survive in biofilm on equipment in food processing plants and resist superficial cleaning and disinfection treatments.155
Approximately 3 to 10% of humans carry listeriae in their gastrointestinal tract with no symptoms of illness.163 Human listeriosis is an uncommon illness with a high mortality rate. The infection most frequently occurs in people who are older, pregnant, or immune compromised. Clinical manifestations range from mild influenza-like symptoms to meningitis, and meningoencephalitis.Pregnant females infected with the pathogen may not present symptoms of illness or may exhibit only mild influenza-like symptoms. However, spontaneous abortion, premature birth, or stillbirth are frequent sequela to listeriosis in pregnant females.162 Although the infective dose of L. monocytogenes in not known, published reports indicate that it is likely to be more than 100 CFU/g of food.162 However, the infective dose depends on the age, condition of health, and immunological status of the host.
L. monocytogenes crosses the intestinal barrier in hosts infected by the oral route. However, before reaching the intestine, the bacterium must withstand the adverse environment of the stomach. Gastric acidity may destroy a substantial number of L. monocytogenes ingested with contaminated food. The site at which intestinal translocation of L. monocytogenes occurs is not clearly elucidated. However, both epithelial cells and M cells in the Peyer’s patches are believed to be the potential sites of entry.164 The bacteria are then internalized by macrophages where they survive and replicate. This is followed by transport of the pathogen via blood to the mesenteric lymph nodes, spleen, and the liver. The primary site of L. monocytogenes replication in the liver is the hepatocyte. In the initial phase of infection, the infected hepatocytes are the target for neutrophils, and subsequently for mononuclear phagocytes, which aid in the control and resolution of the infection.162 If the immune system fails to contain L. monocytogenes, subsequent propagation of pathogen via blood to the brain or uterus takes place.165 The major virulence factors in L. monocytogenes include hemolysin, phospholipases, metalloprotease, Clp proteases and APTases, internalins, surface protein p104, protein p60, listeriolysin O, and the surface protein ActA.162
STAPHYLOCOCCUS AUREUSA preformed, heat-stable enterotoxin produced by S. aureus that can resist boiling for several minutes is the agent responsible for staphylococcal food poisoning. Humans are the principal reservoir of S. aureus strains involved in outbreaks of food-borne illness.
In addition, a recent study revealed that S. aureus can be transmitted between healthy, lactating mothers without mastitis and their infants by breast-feeding.166 Colonized humans can be long-term carriers of S. aureus, and thereby contaminate foods and other humans.167 The bacterium commonly resides in the throat and nasal cavity, and on the skin, especially in boils and carbuncles.167 Protein-rich foods such as ham, poultry, fish, dairy products, custards, cream-filled bakery products, and salads containing cooked meat, chicken, or potatoes are the vehicles most frequently associated with S. aureus food poisoning.168 S. aureus is usually overgrown by competing bacterial flora in raw foods, hence raw foods are not typical vehicles of staphylococcal food poisoning. Cooking eliminates most of the normal bacterial flora of raw foods thereby enabling the growth of S. aureus, which can be introduced by infected cooks and food handlers into foods after cooking. The incubation period of staphylococcal food poisoning is very short, with symptoms being observed within 2 to 6 h after eating toxin-contaminated food. Symptoms include nausea, vomiting, diarrhea, and abdominal pain.
S. aureus can grow in media within a wide range of pH values from 4 to 9.3, with optimum growth occurring at pH 6 to 7. S. aureus has an exceptional tolerance to sodium chloride being able to grow in foods in the presence of 7 to 10% NaCl, with some strains tolerating up to 20% NaCl.168 S. aureus also has the unique ability to grow at a water activity as low as 0.83 to 0.86.169 S. aureus produces nine different enterotoxins which are quite heat resistant, losing their serological activity at 121°C, but not at 100°C for several minutes.169
Besides being a food-borne pathogen, S. aureus has emerged as an important pathogen in nosocomial infections and community-acquired diseases, because of its toxin-mediated virulence, invasiveness, and antibiotic resistance.170 This is especially significant due to the emergence of methicillin-resistant strains of S. aureus (MRSA), and 50% of health-care-acquired S. aureus isolates in the United States in 1997 were methicillin resistant.171 Although MRSA is commonly linked to nosocomial infections, the first report of MRSA-associated food-borne disease in a community was reported in 2002.171
CLOSTRIDIUM BOTULINUMFood-borne botulism is an intoxication caused by ingestion of foods containing preformed botulinal toxin, which is produced by C. botulinum under anaerobic conditions. Botulinal toxin is a neurotoxin, which causes the neuroparalytic disease called botulism. The toxin binds irreversibly to the presynaptic nerve endings of the nervous system, where it inhibits the release of acetylcholine. Unlike botulism in adults, infant botulism results from the colonization and germination of C. botulinum spores in the infant’s gastrointestinal tract. The disease usually occurs in infants during the second month of age, and is characterized by constipation, poor feeding or sucking, and decreased muscle tone with a “floppy” head.172 Although the source of infection is unknown in most cases, the most commonly suspected food in infant botulism is honey.173
There are seven types of C. botulinum (A, B, C, D, E, F, and G) which are classified on the basis of the antigenic specificity of the neurotoxin they produce.174 The bacterium is present in soil, vegetation, and sedimentation under water. Type A strains are proteolytic, whereas type E strains are nonproteolytic.175 Another classification divides C. botulinum into four groups: group I(type A strains and proteolytic strains of types B and F), group II (type E strains and nonproteolytic strains of B and F), group III (type C and D strains), and group IV (type G strains). Types A, B, E, and F are associated with botulism in humans. Type AC. botulinum occurs frequently in soils of the western United States, whereas type B strains are more often present in the eastern states and in Europe.175 Type E strains are largely associated with aquatic environments and fish. Foods most often associated with cases of botulism include fish, meat, honey, and home-canned vegetables.174 Type A cases of botulism in the United States are frequently associated with temperature-abused, home-prepared canned foods. Proteolytic type A, B, and F strains produce heat-resistant spores, which pose a safety concern in low-acid canned foods. In contrast, nonproteolytic type B, E, and F strains produce heat-labile spores, which are of concern in pasteurized or unheated foods.175 The minimum pH for growth of groups I and II strains is 4.6 and 5, respectively.174 Group I strains can grow at a minimum water activity of 0.94, whereas group II strains do not grow below a water activity of 0.97.176 The proteolytic strains of C. botulinum are generally more resistant to heat than nonproteolytic strains.
CLOSTRIDIUM PERFRINGENSC. perfringens is a major bacterial cause of food-borne disease, with 1062 cases reported in the United States in 2004.3 C. perfringens strains are grouped into five types: A, B, C, D, and E, based on the type(s) of toxin(s) produced. C. perfringens foodborne illness is almost exclusively associated with type A isolates. C. perfringens is commonly present in soil, dust, water, and in the intestinal tract of humans and animals.177 It is frequently present in foods; about 50% of raw or frozen meat and poultry contain C. perfringens.178 Spores produced by C. perfringens are quite heat resistant, and can survive boiling for up to 1 h.178 C. perfringens spores can survive in cooked foods and if not properly cooled before refrigerated storage, the spores will germinate and vegetative cells can grow to large cell numbers during holding at growth temperatures. Large populations of C. perfringens cells (>106/g) ingested with contaminated food will enter the small intestine, multiply and sporulate. During sporulation in the small intestine C. perfringens enterotoxin is produced which induces a diarrheal response. The enterotoxin is a 35-kDa heat-labile polypeptide that damages the epithelial cells of the gastrointestinal tract to cause fluid and electrolyte loss.179,180 Although vegetative cells of C. perfringens are sensitive to cold temperature and freezing, spores tolerate cold temperature well and can survive in refrigerated foods.
BACILLUS CEREUSBacillus cereus is a spore-forming pathogen present in soil and on vegetation. It is responsible for a growing number of foodborne illnesses in the industrial countries,181 with 103 outbreak-associated confirmed cases reported in the United States in 2004.3 It is frequently isolated from foods such as meat, spices, vegetables, dairy products, and cereal grains, especially fried rice.182 There are two types of food-borne illness caused by B. cereus, that is, a diarrheagenic illness and an emetic syndrome.181,183 The diarrheal syndrome, caused by heat-labile enterotoxins, is usually mild in its course and is characterized by abdominal cramps, nausea, and watery stools. Types of foods implicated in outbreaks of diarrheal syndrome include cereal food products containing corn and corn starch, mashed potatoes, vegetables, milk, and cooked meat products. The emetic syndrome, caused by a heat-stable peptide toxin,181 is more severe and acute in its course, characterized by severe vomiting. Refried or rewarmed boiled rice, pasta, noodles, and pastry are frequently implicated vehicles in outbreaks of emetic syndrome.184 The dose of B. cereus required to produce diarrheal illness is estimated at more than 105 cells/g.185
BRUCELLA SPECIESBrucella spp. are pathogens in many animals, causing sterility and abortion. In humans, Brucella is the etiologic agent of undulant fever. The genus Brucella consists of six species, of which those of principal concern are B. abortus, B. suis, and B. melitensis.186 B. abortus causes disease in cattle, B. suis in swine, and B. melitensis is the primary pathogen of sheep. B. melitensis is the most pathogenic species for humans. Human brucellosis is primarily an occupational disease of veterinarians and meat industry workers. Brucellosis can be transmitted by aerosols and dust. Food-borne brucellosis can be transmitted to humans by consumption of meat and milk products from infected farm animals. The most common food vehicle of brucellosis for humans is unpasteurized milk.186 Meat is a less common source of food-borne brucellosis, because the bacteria are destroyed by cooking. Since the National Brucellosis Education program has almost eradicated B. abortus infection from U.S. cattle herds, the risk of food-borne infection of brucellosis through consumption of domestically produced milk and dairy products is minimal.61
HELICOBACTER PYLORIH. pylori is a human pathogen causing chronic gastritis, gastric ulcer, and gastric carcinoma.187,188 Although, humans are the primary host of H. pylori, the bacterium has been isolated from cats.161 H. pylori does not survive well outside its host, but it has been detected in water and vegetables.189,190 A study on the effect of environmental and substrate factors on the growth of H. pylori indicated that the pathogen likely lacks the ability to grow in most foods.191 However, H. pylori may survive for long periods in low-acid environments under refrigerated conditions. H. pylori infections spread primarily by person-to-person transmission, especially among children, and contaminated water and food are considered potential vehicles of the pathogen. In the United States, a significant association between H. pylori infection and iron deficiency/anemia, regardless of the presence or absence of peptic ulcer, has been reported.192,193
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By Kumar S. Venkitanarayanan and Michael P. Doyle in "Handbook of Nutrition and Food" Edited by Carolyn D.Berdanier, Johanna Dwyer and Elaine Feldman, CRC Press USA,2007. Adapted and illustrated to be posted by Leopoldo Costa.