Foodborne Gastroenteritis Caused by Salmonella and Shigella 627 Table 26–3 Some Characteristics of All Outbreaks of Salmonella serotype Enteritidis Infection and Outbreaks in Health-Care Facilities, by Year—United States, 1985–1998 (Summarized from Reference 11) All outbreaks Year 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 Total 47 58 48 81 85 74 63 66 51 56a 50 44 47 a Includes Outbreaks in health-care facilities Number of Outbreaks Number ill Deaths Number of Outbreaks Number ill Deaths 26 1,444 2,616 1,201 2,518 2,656 2,461 2,348 2,215 5,492 1,312 1,460 1,098 709 796 1,159 15 11 15 28,689 8 19 12 6 3 79(0.28%) 96 489 227 505 265 118 42 66 32 147 64 13 32 87 55 14 13 4 0 2,151 64(3%) one outbreak associated with a Komodo dragon exhibit at a zoological park frequently isolated serotype since about 1975 The second most common is S Enteritidis, and it is discussed further below For the years 1981–1995, 3,504 (23.8% of all) bacterial foodborne illnesses in Korea were caused by salmonella while in Japan for the same period, there were 101,395 (19.9%) cases.32 Since the late 1970s, S Enteritidis has been the cause of a series of outbreaks in the north-eastern part of the United States and in parts of Europe The U.S outbreaks for 1985–1998 are listed in Table 26–3 The cases and deaths from all outbreaks are compared to those among individuals in health-care facilities Although it is the second most frequently isolated serotype in the United States, it is of significance because of its high association with eggs and its fatality rate It may be noted from Table 26–3 that 796 outbreaks were recorded with 28,689 cases and 79 deaths for the period 1985–1998.11 Raw or undercooked shell eggs accounted for 82% of the illness cases The 79 deaths accounted for 0.27% of all cases but in health-care facilities, the death rate was 2.97% In 1976, the number of cases in the United States per 100,000 population was 0.6 but by 1996 the rate was 3.6 The rate decreased to 2.2/100,000 in 1998.11 Because in the United States S Enteritidis is so highly associated with the consumption of raw or undercooked eggs, the CDCP recommends the following11 : (1) Raw or undercooked eggs should be avoided, especially by the young, elderly, and immunocompromised; (2) when eggs are not properly cooked, pasteurized egg products should be used; (3) eggs should be cooked at ≥145◦ F (63◦ C) for 15 seconds, or until both yolk and white are firm, and then should be eaten promptly; (4) casseroles and other dishes containing raw eggs should be cooked to 160◦ F (71◦ C); and (5) raw eggs should be stored at ≤ 45◦ F (7.2◦ C) at all times 628 Modern Food Microbiology S Enteritidis was the most common serovar in eggs in Spain44 and the most predominant cause of foodborne salmonellosis in England and Wales in 1988, where it was found in both poultry meat and eggs.24 Unlike U.S outbreaks, those in Europe are caused by phage type strains, which are more invasive for young chicks than phage types 7, 8, or 13a 24 Why the increased incidence of S Enteritidis outbreaks are associated with eggs and poultry products is unclear The organism has been found by some investigators inside the eggs and ovaries of laying hens,44 but others have failed to find it in unbroken eggs It was recovered from the ovaries of only of 42 layer flocks located primarily in the southeast United States.2 The S Enteritidis outbreaks have occurred more in July and August than other months, suggesting growth of the organism in or on eggs and other poultry products The strains in question are not heat resistant, and many of the outbreaks have occurred following the consumption of raw or undercooked eggs In one study in which S Enteritidis was inoculated into the yolk of eggs from normal hens, no growth occurred at 7◦ C in 94 days.6 Growth in yolks at 37◦ C was faster from normal hens than in those from hens that were seropositive The possible routes of S Enteritidis to eggs are the following:31 Transovarial Translocation from peritoneum to yolk sac or oviduct Penetration of shell by organisms as eggs pass through the cloaca Egg washing Food handlers Except for the association of S Enteritidis with poultry and egg products, it is difficult to predict the association of most other salmonellae serovars with specific food products The three outbreaks traced to fresh tomatoes in the United States during the years 1990, 1993, and 1997–1998 were caused by three different serotypes (see Table 26–4) S Muenchen was the etiologic agent in an orange juice outbreak in 199913 ; and it was also the cause of an alfalfa sprout outbreak during the same year.45 A multi-state outbreak of S Poona in the United States was traced to fresh cantaloupes from Mexico10 , and a raw-milk outbreak in U S states in 2003 was caused by S Tennessee9 Table 26–4 Synopses of Some Foodborne Outbreaks of Nontyphoidal Salmonellosis (From the Literature) Products/Location/Year(s) Number of Cases Salmonella Serotype Fresh tomatoes, states USA, 1990 Fresh tomatoes, USA, 1993 Fresh tomatoes, states USA, 1998–1999 Alfalfa sprouts, states USA, 1999 Alfalfa sprouts, states USA, 2001 Cantaloupes, 12 states USA, and Canada, 2002 Unpasteurized orange juice, 13 states USA, Canadian Provinces, 1999 Raw/undercooked shell eggs, states USA, 1997–1998 Raw/undercooked ground beef, states USA, 2002 Chocolate; Germany, Denmark, other countries, 2001 Shandong peanuts; Australia, Canada, U.K., 2001 176 100 85 157 31 47 298 241 47 >316 Ca 102 Javiana Montevideo Baidon Muenchen Kottbus Poona Muenchen Enteritidis Newport Oranienberg Stanley Foodborne Gastroenteritis Caused by Salmonella and Shigella 629 Synopses of the three leading serotypes recovered from six different sources are presented below Retail foods in Korea (1993–2001) Of the 1,334 samples tested, 2.2% were salmonellae positive Most common serotypes: Enteritidis, Virginia, Haardt.16 Minced beef and pork in Germany (1996–1997) Of the 1,445 samples, 6.3% were salmonellae positive Most common serotypes: Typhimurium, Derby, Typhimurium var Copenhagen.53 Human food and beverages in Singapore (1998) Of the 2,617 samples, 1.4% were salmonellae positive Most common serotypes: Typhimurium, Agona, and Dumfries and Enteritidis.41 Hen layer feed in Japan (1993–1998) Of 10,418 samples, 0.5% were salmonellae positive Most common: Untypable, Eastbourne, Orion.49 Feedlot cattle feces in the United States (1996) Of the 4,977 samples, 5.5% were salmonellae positive Most common: Anatum, Montevideo, Muenster.20 Beef cow feces in the United States (1998) Three most common serotypes: Oranienburg, Cerro, Anatum.19 Prevention and Control of Salmonellosis The intestinal tract of humans and other animals is the primary reservoir of the etiological agents Animal fecal matter is of greater importance than human, and animal hides may become contaminated from the fecal source Salmonella spp are maintained within an animal population by means of nonsymptomatic animal infections and in animal feeds Both sources serve to keep slaughter animals reinfected in a cyclical manner, although animal feeds seem less important than once believed Secondary contamination is another of the important sources of salmonellae in human infections Their presence in meats, eggs, and even air makes their presence in certain foods inevitable through the agency of handlers and direct contact of noncontaminated foods with contaminated foods.25 In view of the worldwide distribution of salmonellae, the ultimate control of foodborne salmonellosis will be achieved by freeing animals and humans of the organisms This is obviously a difficult task but not impossible; only about 35 of the more than 2,400 serovars account for around 90% of human isolates and approximately 80% of nonhuman isolates.37 At the consumer level, the Salmonella carrier is thought to play a role, but just how important this role may be is not clear Improper preparation and handling of foods in homes and food service establishments continue to be the primary factors in outbreaks With respect to the colonization of chickens by S Enteritidis, one study used a phage type strain administered orally 108 to adult laying hens.31 Within two days, the organism was found throughout the body, including the ovary and oviduct It was detected in some forming eggs, although its incidence was much lower in freshly laid eggs Investigators concluded that forming eggs are subject to descending infection from colonized ovarian tissue, to ascending infections from colonized vaginal and cloacal tissues, and to lateral infections from colonized upper oviduct tissues.31 The hatchery eggs are of critical importance because if they are contaminated, hatchlings may become infected at this early stage Salmonellae rapidly penetrate freshly laid fertile eggs, become entrapped in the membrane, and may be ingested by an embryo as it emerges from the egg Competitive Exclusion to Reduce Salmonellae Carriage in Poultry It is generally agreed that the primary source of salmonellae in poultry products is the gastrointestinal tract, including the ceca If young chicks become colonized with salmonellae, the bacteria may be shed 630 Modern Food Microbiology in feces, through which other birds become contaminated Among the methods that may be employed to reduce or eliminate intestinal carriage is competitive exclusion (the Nurmi concept) Under natural conditions where salmonellae exist when eggs hatch, young chicks develop a gastrointestinal tract flora that consists of these organisms and campylobacters, in addition to a variety of nonpathogens Once the pathogens are established, they may remain and be shed in droppings during the entire lifetime of the bird Competitive exclusion is a phenomenon whereby feces from salmonellae-free birds, or a mixed fecal culture of bacteria, are given to young chicks so that they will colonize the same intestinal sites that salmonellae employ and, thus, exclude the subsequent attachment of salmonellae or other enteropathogens This concept was advanced in the 1970s and has been studied and found to be workable by a number of investigators relative to salmonellae exclusion The enteropathogen-free biota may be administered orally to newly hatched chicks through drinking water or by spray inoculation in the hatchery Protection is established within a few hours and generally persists throughout the life of the fowl or as long as the biota remains undisturbed Older birds can be treated by first administering antibacterial agents to eliminate enteropathogens, and they are then administered the competitive exclusion biota Only viable cells are effective, and both aerobic and anaerobic components of the gut flora seem to be required The crop and ceca appear to be the major adherence sites, with the ceca being higher in germ-free chickens In one study, the protective flora remained attached to cecal walls after four successive washings.52 Partial protection was achieved in 0.5–1.0 hour, but full protection required 6–8 hours after treatment of 1-day-old chicks.50 In a review of the microbiology of competitive exclusion of Salmonella in poultry, the use of undefined cultures afford more protection than the use of defined cultures, especially under laboratory conditions.51 Field trials in several European countries have shown the success of the competitive exclusion treatment in preventing or reducing the entry of salmonellae in broilers and adult breeder birds.39 In chicks pretreated with a cecal culture and later challenged with a Salmonella sp., the latter failed to multiply in the ceca over a 48-hour period, whereas in untreated control birds, more than 106 /g of salmonellae were colonized in the ceca.27 The gist of competitive exclusion is that salmonellae and the native gut biota compete for the same adherence sites on gut walls The precise nature of the bacterial adhesins is not entirely clear, although fimbriae, flagella, and pili have been suggested In regard to the attachment of salmonellae to poultry skin, these bacterial cell structures were found not to be critical.36 Extracellular polysaccharides of a glycocalyx nature may be involved, and if so, treatment of young chicks with this material may be as effective as the use of live cultures Although the competitive exclusion treatment seems quite feasible for large hatcheries, its practicality for small producers seems less likely The sugar mannose is a receptor in the intestinal tract to which bacterial pathogens such as salmonellae bind Since the yeast strain Saccharomyces cerevisiae var boulardii contains mannose in its outer wall, some have suggested that the feeding of this yeast to susceptible poults should reduce the attachment of salmonellae In essence, the yeast cell wall material would outcompete the gastrointestinal tract for the pathogens The possible use of probiotic cultures to exclude some Gram-negative pathogens from the gut biota has been investigated by several groups When a 3-strain mixture of probiotic bacteria (competitive exclusion of E coli strains) was used on weaned calves and challenged with E coli serotypes 0111:NM, 026:H11, and 0157:H7, the probiotic-treated calves showed a significant reduction in the shedding of two of the three pathogens but not E coli serotype 026:H11.55 In another study, a mixed culture of Lactobacillus crispatus and Clostridium lactatifermentans cultured under cecal growth conditions inhibited the growth of S Enteritidis.57 When ca 109 cells/chicken of a chicken isolate of Enterococcus faecium was orally administered to 30-hour-old broiler chicks followed by a challenge with 105 cells Foodborne Gastroenteritis Caused by Salmonella and Shigella 631 of S Pullorum per chick, the chicks survived However, chicks that were infected on the first day and then treated with the lactic culture died four days later.2 These authors concluded that the E faecium strain could prevent newly hatched chicks from S Pullorum infections but it was not a good therapeutic agent SHIGELLOSIS The genus Shigella belongs to the family Enterobacteriaceae, as the salmonellae and escherichiae Only four species are recognized: S dysenteriae,S flexneri,S boydii, and S sonnei S dysenteriae is a primary pathogen that causes classic bacillary dysentery; as few as 10 cfu are known to initiate infection in susceptible individuals By applying data from two cruise ship outbreaks to a mathematical model, it was estimated that the outbreaks could have been due to ingestion of a mean of 344 Shigella cells per meal and 10.5 to 12 cells per glass of water.17 Although this syndrome can be contracted from foods, it is not considered to be a food-poisoning organism in the same sense as the other three species, and it is not discussed further Unlike the salmonellae and escherichiae, the shigellae have no known nonhuman animal reservoirs Some of the many differences among the three genera are noted in Table 26–5 The shigellae are phylogenetically closer to the escherichiae than to the salmonellae The three species of concern as etiological agents of foodborne gastroenteritis are placed in separate serologic groups based on O antigens: S flexneri in group B, S boydii in group C, and S sonnei in group D They are nonmotile, oxidase negative, produce acid only from sugars, not grow on citrate as sole carbon source, not grow on KCN agar, and not produce H2 S In general, their growth on ordinary culture media is not as abundant as that of the escherichiae Of shigellae isolated from humans in the United States in 1984, 64% were S sonnei, 31% S flexneri, 3.2% S boydii, and 1.5% S dysenteriae.14 The Shigella species of concern are typical of most other enteric bacteria in their growth requirements, with growth reported to occur at least as low as 10◦ C and as high as 48◦ C In one study, growth of S flexneri was not observed in brain heart infusion (BHI) broth at 10◦ C.59 It appears that S sonnei can grow at lower temperatures than the other three species Growth at pH 5.0 has been recorded, with best growth occurring in the range of 6–8 With S flexneri, no growth occurred at pH 5.5 at 19◦ C in BHI broth.59 This species has been shown to be inhibited by nitrite as temperature and pH were decreased or as NaCl was increased.58 It is unclear whether they can grow at aw values below those for the salmonellae or escherichiae Their resistance to heat appears to parallel that of E coli strains Table 26–5 A Comparison of Salmonella, Shigella, and Escherichia Genus Escherichia Salmonella Shigella ∗ Usually † Type strains Glucose Motility H2 S Indole Citrate Mol 1% G + C AG AG A +∗ +∗ − − + − +† − − − + − 48–52 50–53 49–53 ... become colonized with salmonellae, the bacteria may be shed 630 Modern Food Microbiology in feces, through which other birds become contaminated Among the methods that may be employed to reduce or... have failed to find it in unbroken eggs It was recovered from the ovaries of only of 42 layer flocks located primarily in the southeast United States.2 The S Enteritidis outbreaks have occurred more... Oranienberg Stanley Foodborne Gastroenteritis Caused by Salmonella and Shigella 629 Synopses of the three leading serotypes recovered from six different sources are presented below Retail foods in Korea