Enteric Bacilli
Stool cultures incubated under aerobic conditions yield primarily members of the family Enterobacteriaceae. They are gram-negative non-sporing facultative anaerobes They are found mostly in the vertebrate intestine as normal flora or pathogens. E. Coli is the predominant facultative organism in stool. Its presence, along with other coliforms (Klebsiella, Enterobacter, and Citrobacter) is used by Public Health Departments as presumptive faecal contamination of water. The enterobacteriacae, together with the gram-negative vibrios, are also frequently referred to as enteric bacilli or enterics. The term enterics has sometimes been extended loosely to include other aerobic gram-negative bacilli occasionally found in the gut, such as pseudomonas.
Classification
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Genera
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Pathogenicity |
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Escherichia |
Certain types product diarrhoea or invasive dysenteric dieases
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Salmonella
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Gastroenteritis, septicaemia, enteric fever |
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Shigella
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Bacilliary dysentery |
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Edwardsiella |
Can cause gastroenteritis and other salmonellosis-like diseases
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Yersinia
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Plague, enterocolitis, mesenteric lymphadenitis |
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Proteus |
Pathogenic only under special circumstances |
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Klebsiella |
“ “ “ “ “ |
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Enterobacter |
“ “ “ “ “ |
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Hafnia |
“ “ “ “ “ |
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Serratia |
“ “ “ “ “ |
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Providencia |
“ “ “ “ “ |
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Erwinia |
Plant pathogens or saprophytes |
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Pectobacterium |
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The several groups of facultative and aerobic gram-negative rods are differentiated by a variety of criteria. In general, the enterobacteraicaeae are a family of gram-negative, facultatively anaerobic bacilli that ferment glucose with the production of acid, reduce nitrates to nitrites, and do not produce cytochrome oxidase. Except for Shigella and Klebsiella, they are motile. In addition to flagella, many species produce fimbriae (pili) and/or capsules, which are often important virulence determinants. Members of the family can be found in a variety of hosts, including vertebrates, invertebrates, and higher plants. Those of medical importance usually reside in the lower GI tract of humans and animals.
A variety of special differential and selective culture media are used in the isolation of enteric bacteria from clinical specimens. Typically, such media contain inhibitors of gram-positive organisms (bacteriostatic dyes such as brilliant green or surface-active compounds such as bile salts), an acid-base indicator (eg. Neutral red), and one or more fermentable carbohydrates. Examples of selective agar include MacConkey agar and deoxycholate agar). Enterobacteriaceae are still identified by and large on the basis of a series of biochemical tests. It is important to note that 80-95% of the isolates seen in hospitals belong to three species: E. Coli, Klebsiella pneumoniae, and Proteus Mirabilis.
Escherichia Coli
The alimentary tract of most warm-blooded animals is colonized within a few hours of birth by E. Coli from ingested food or water. E. coli is normally the most common facultative anaerobe in the large bowel. E. coli has O, K, and H antigens and theoretically there are 106 O/H/K combinations although not all are found. The correlation between antigenicity and pathogenicity is high and serotyping has been of great importance in distinguishing the small number of strains that cause disease.
Pathogenesis
The three principal kinds of disease are urinary tract infections, neonatal meningitis, and diarrhoeal diseases.
1. Urinary tract infections - E. coli is responsible for 90% of infections in anatomically normal unobstructed urinary tracts. The uropathogenic strains are present in the stool and subsequently colonize the vaginal and periurethral region. The bacteria may then ascend into the bladder. Females are more prone to UTIs than males because of their shorter urethra.
2. Neonatal meningitis - E. coli is the most common cause of neonatal meningitis, affecting 1 in 2000 to 4000 infants. Approximately 80% of the isolates synthesize the K1 capsular polysaccharide. E. coli meningitis is best treated with a beta-lactam antibiotic.
3. Intestinal diseases
The majority of E. coli in the GI tract are harmless unless they are displaced to other parts of the body such as the urinary tract or meninges. The pathogenic, or enterovirulent E. coli are divided into 4 groups according to their action in the body:
Enteropathogenic (EPEC)
Enteropathogenic (EPEC) is responsible for severe infantile diarrhoea. They cause outbreaks of diarrhoea in infant nurseries and rarely adult diarrhoea. The most common O serotypes involved are O55, O111, O119, O127, and O128. EPEC cause either watery or bloody diarrhoea, the former is due to the attachment to and physical alteration of the integrity of the intestine. Bloody diarrhoea is associated with attachment and an acute tissue-destructive process, caused by a toxin similar to that of Sh. dysenterie, also called verotoxin. Occasionally, the diarrhoea is prolonged, leading to dehydration, electrolyte imbalance and death. EPEC is highly infectious for infants and the dose is very low. Common foods implicated in EPEC outbreaks are raw beef and chicken, although any food exposed to faecal contamination is strongly suspect. EPEC outbreaks most often affect infants, especially those who are bottle fed, suggesting that contaminated water is used to rehydrate milk powder feeds in underdeveloped countries.
The distinction of EPEC from other groups of pathogenic E. coli isolated from patient’s stool involves serological and cell culture assays. Serotyping, although useful, is not strict for EPEC. The isolation and identification of E. coli in foods follow standard enrichment and biochemical procedures. Serotyping of isolates to distinguish EPEC is laborious and requires high quality, specific antisera. The total analysis may require 7 to 14 days.
Enterotoxigenic (ETEC)
Enterotoxigenic (ETEC) produces a heat labile (LT) or heat stable (ST) toxin. Both may be produced by the same organism. They are responsible for cases of paediatric diarrhoea, severe cholera-like illness, and traveller’s diarrhoea. The symptoms consist of watery diarrhoea, abdominal cramps, low-grade fever, nausea and malaise. A relatively large dose (100 to 10 billion bacteria) is required to establish an infection. Infants and travellers to underdeveloped countries are most at risk of infection. ETEC is not considered as a serious foodborne disease hazard in countries with high sanitary standards. Contamination of water with human sewage may lead to contamination of foods. Infected food handlers may also contaminate foods. These organisms are infrequently isolated from diary products.
LT is a 86000 dalton protein and is structurally and functionally very similar to cholera toxin. ST, of which there are several types, are small polypeptides ranging in size from 18 to 50 amino acids. They are structurally related and some ST genes reside on a transposon. At least half the cases of traveller’s diarrhoea are caused by ETEC that elaborate only 1 toxin.
During the acute phase of infection, large numbers of organisms are excreted in faeces. These strains are differentiated from nontoxigenic E. coli present in the bowel by a variety of in vitro, immunochemical, tissue culture or NA probe tests designed to detect either the toxins or genes that encode for these toxins. The diagnosis can be completed in 3 days. With the availability of a gene probe method, foods can be analysed directly for the presence of enterotoxigenic E. coli, and the analysis can be completed in 3 days. Alternative methods which involve enrichment and plating of samples for isolation of E. coli and confirmation as toxigenic strains require at least 7 days.
Enteroinvasive (EIEC)
Enteroinvasive (EIEC) - pathogenicity is due to invasion of the gut mucosa. They give rise to a dysentery-like illness. The EIEC strains responsible for this illness are closely related to Shigella: they resemble Shigella in their pathogenic mechanisms and the kind of clinical illness they produce. Moreover, like Shigella, they are non-motile and lactose-negative. They cross-react with certain Shigella O Ags. Their pathogenicity is conferred by a large 140 Md plasmid. This plasmid is similar but not identical to the plasmids seen in Shigella species. The infective dose of EIEC is thought to be as few as 10 organisms (same as Shigella). It is currently unknown what foods may harbour EIEC, but any food contaminated with human faeces directly or indirectly through contaminated water could cause disease. Outbreaks have been associated with hamburger meat and unpasteurised milk.
The signs and symptoms of the disease are similar to that caused by Shigella dysenterie. The dysentery occurs 12 to 72 hours following the ingestion of contaminated food. The disease is characterised by abdominal cramps, diarrhoea, vomiting, fever, chills, and malaise. The disease may be complicated by haemolytic-uraemic syndrome (HUS)
The culturing of the organism from the stools of infected individuals and the demonstration of invasiveness of isolates in tissue culture in a suitable animal model is necessary to diagnose dysentery caused by this organism. More recently, genetic probes for the invasiveness genes of both EIEC and Shigella spp. have been developed. Foods are examined as stool cultures. Detection of this organism in foods is extremely difficult because undetectable levels may cause illness.
Enterohaemorrhagic (EHEC)
Enterohaemorrhagic (EHEC) is responsible for bloody diarrhoea and colitis. It differs from bacillary dysentery in that fever is not prominent and the bloody discharges are copious rather than scanty. This diarrhoea syndrome is distinct from the bacilllary dysentery caused by Shigella and EIEC. E. coli O157:H7 is the causative organism and it cause disease through the production of large quantities of one or more related toxins. Two toxins appear to be important. Toxin 1 is identical to the toxin produced by Shigella and is known as Shiga toxin. Toxin 2 appears to be related to severe disease.
Haemorrhagic colitis is the name of the disease caused by E. coli O157:H7. The illness is characterised by severe abdominal cramps and diarrhoea which is initially watery but becomes grossly bloody. Occasionally vomiting occurs. The illness is usually self-limited and lasts for an average of 8 days. Some individuals exhibit watery diarrhoea only. Some victims, particularly the very young, develop haemolytic uraemic syndrome (HUS) characterised by renal failure and haemolytic anaemia. Up to 15% of haemorrhagic colitis victims may develop HUS. In the elderly, HUS plus two other symptoms, fever and neurological symptoms constitute thrombotic thrombocytopenix purpura (TTP). This illness can have a mortality rate in the elderly as high as 50%. The infective dose is unknown but may be similar to that of Shigella (i.e. as few as 10 organisms). All people are believed to be susceptible to haemorrhagic colitis but larger outbreaks have occurred in institutional settings.
Haemorrhagic colitis is diagnosed by the isolation of E. coli O157:H7 from diarrhoeal stools. Alternatively, the stools can be tested directly for the vertoxins. E. coli O157:H7 will form colonies on agar media that are selective for E. coli. However, the high temperature growth procedure normally performed to eliminate background organisms before plating cannot be used because of the inability of these organisms to grow at temperatures of 45oC that support the growth of most E. coli. The use of molecular methods to detect verotoxins 1 and 2 is most sensitive method available.
Undercooked or raw hamburger (ground beef) has been implicated in nearly all documented outbreaks and in other sporadic cases. Raw milk has also been implicated in other outbreaks. These are currently the only two demonstrated food causes of disease, but other meats may contain E. coli O157:H7.
Laboratory Diagnosis
In testing water supplies, it is important to distinguish E. coli, which is an index of faecal contamination, from Enterobacter which is widely found in plants. Four metabolic tests (indole, methyl red, Vogts-Proskauer, and citrate utilization) collectively referred to as the IMVIC tests were originally used to distinguish Enterobacter from E. coli. Only the latter produces indole in media containing tryptophan. The methyl red test distinguishes heavy and light production of acid, because this indicator shifts from yellow to red below pH 4.5, and in glucose-peptone broth cultures incubated for 48 hours, only the mixed acid fermentation produces enough acid to turn the indicator red. The Voges-Proskauer reaction is a colour test for acetoin, a product of butylene glycol type of fermentation. Citrate can serve as the sole carbon source for Enterobacter but not for E. coli.
The shigella species are the principal agents of bacterial dysentery. The disease consists of fever, abdominal cramps, and bloody diarrhoea with mucus. As few as 100 organisms can lead to infection and hence they are the most communicable bacterial agents of diarrhoeal disease. In developed countries bacillary dysentery is primarily a disease of children from 6 months to 10 years of age. The genus Shigella and Escherichia are much closer in DNA homology than other species. All shigella are non-motile and lactose-negative and they do not produce gas from fermentable carbohydrates. The genus Shigella is subdivided into four groups on the basis of their biochemical and serological reactions.
Group A - Sh. ysenteriae, 10 serotypes, do not ferment mannitol
Group B - Sh. Flexneri, 6 serotypes, mannitol fermenting
Group C- Sh. Boydii, 15 serotypes, mannitol fermenting
Group D - Sh. Sonnei, one serotype, mannitol fermenting
Pathogenesis
Shigellosis is acquired by ingestion. Because of its high infectivity, person to person spread is of great significance although food and water do play a role. The penetration of epithelial cells and subsequent intracellular multiplication of the microorganism depends on a plasmid of approximately 140 Md, which is present in all virulent Shigella stains. Some strains produce a Shiga toxin which is very similar to the verotoxin produced by E. coli O157:H7. It causes paralysis when injected into small animals and is cytotoxic to cell cultures. It is composed of an enzymatically active A subunit (32000 daltons) and several identical receptor-binding B subunits (7700 daltons). The toxin inactivates the mamalian 60S ribosomal subunit.
The incubation period is between 12 to 50 hours. Symptoms consist of abdominal pain, cramps, diarrhoea, fever, vomiting, blood, pus or mucus in stools. Severe infections result in mucosal ulceration, rectal bleeding and dehydration: the fatality may be as high as 10-15% with some strains. Reiter’s disease, reactive arthritis, haemolytic uraemic syndrome are possible sequelae.
Epidemiology
Sh. Dysentery type 1 is of great public health importance because it causes especially severe infections and may occur in explosive epidemics. Large epidemics had been reported from many parts of the world including Central America, Central Africa, and the Indian subcontinent. These organisms harbour an R plasmid encoding resistance to several antibiotics. Although there are more than 30 recognized Shigella serotypes, only a few predominate in any geographic area. Sh. Sonnei (which causes a relatively mild disease) predominate in industrialized countries.
Associated foods include salads, raw vegetables, milk and diary products, and poultry. Contamination of these foods is usually through the faecal-oral route. Faecally contaminated water and unsanitary handling by food handlers are the most common causes of contamination. Sh. Sonnei is usually involved in food borne outbreaks, whereas Sh. dysenterie is usually associated with contaminated water. Sh. flexneri is now thought to be in large part sexually transmitted.
Laboratory diagnosis
Shigella are easily identifiable in the clinical laboratory. In the first few days of illness, the stool contains large numbers of organisms. When shigellosis is suspected, a faecal sample should be cultured on differential media that inhibit the growth of gram-positive organisms and that also distinguish between most pathogenic and nonpathogenic enteric species (e.g. MacConkey agar). Suspect colonies are picked to other differential meida and subjected to a battery of biochemical tests. Those which are identified as Shigella may be further characterized by type-specific antisera.
Organisms are difficult to demonstrate in foods because methods are not developed or are insensitive. A nucleic acid probe to the virulence plasmid has been developed by the FDA and is currently under field test.
Treatment
As with all diarrhoeal disease, the cardinal consideration is to maintain fluid and electrolyte balance. Because the disease is self-limiting, most experts consider antibiotic treatment only for the young. It reduces the average duration of the illness from 5 to 3 dyas and reduces the excretion of viable organisms in the stool. Shigella frequently acquire R plasmids which render them resistant to many common antibiotics. Where the sensitivity is known, ampicillin or tetracyclin is usully effective.
Members of the genus salmonella are ubiquitous pathogens found in humans, livestock, wild mammals, birds, reptiles, and even insects. Antigenic analysis has distinguished more than 1500 serotypes. About 10 serotypes make up most of the human isolates in a given year. A single serotype, S. typhimurium, is the most frequently isolated cause of Salmonella gastroenteritis. It also causes disease in many animal species. Other common human serotypes are S. infantis, S. heildelberg. The clinical pattern of salmonella disease can be divided into gastroenteritis, enteric fever (typhoid), bacteraemia, and the asymptomatic carrier state.
Salmonella is a rod-shaped gram-negative motile bacterium. There is widespread occurrence in animals, especially in poultry and swine. Environmental sources include water, soil, insects, factory surfaces, kitchen surfaces, animal faeces, raw meats, raw poultry, and raw seafoods etc.
Gastroenteritis
Salmonella gastroenteritis usually follows the ingestion of food or drinking water contaminated by faces and accounts for 15% of foodborne infection in the U.S. Typically, the illness begins 12 to 48 hours after the ingestion and consists of nausea and vomiting, with abdominal pain and diarrhoea. Fever is present in about half the patients and a mild headache may be present. Diarrhoea persists as the prominent symptom for 3 or 4 days. Salmonellosis may be complicated by reactive arthritis and Reiter’s syndrome. The infective dose is small: as few as 15-20 cells. The pathogenesis is due to the invasion of the GI tract by the organism. There is evidence that an enterotoxin may be produced.
Associated foods include raw meats, eggs, milk and diary products, shrimps, yeast, coconut, sauces and salad dressing, cake mixes, cream-filled desserts and toppings, peanut butter and chocolate. Various salmonella species have long been isolated from the outside of egg shells. The present situation with S. Enteriditis is complicated by the presence of the organism inside the egg. Therefore, raw eggs may cause salmonella infection. Foods other than eggs have also caused outbreaks of S. Enteriditis disease. It is estimated that 2 to 4 million cases of salmonellosis occur in the US annually.
All age groups are susceptible, but symptoms are most severe in the elderly, infants, and the infirm. AIDS patients suffer salmonellosis frequently (20 fold greater than the general population) and suffer from recurrent episodes.
Enteric fever
Enteric fever is caused by S. typhi. The bacilli enters the body via the Peyer’s patches and causes septicaemia. The first 7 to 10 days of the infection is usually asymptomatic. A high fever then develops with splenomegaly. Rose spots appear on the abdomen. Complications of typhoid fever include intestinal perforation and haemorrhage. Clinical improvement of untreated patients usually begins in the third week following infection. Typhoid fever is a severe diseases. When untreated, the average duration of fever is 30 days with a mortality of 20%; an additional 10% suffer a relapse of fever, intestinal haemorrhage or peritonitis. Because typhoid is a systemic disease, in the early stages, the organism should be cultured from the blood and not the stool. When the disease becomes established, both blood and stool may be positive, as is urine in 25% of cases. During the convalescent phase 4 to 5 weeks after infection, the blood will return to sterility but the stool may remain culture positive in half the patients. More than ¾ of patients will have high titres against O and H Ags. Those who recover may continue to excrete the organism for long periods of time. These chronic carriers serve as important reservoirs of infection.
Extraintestinal Manifestations
The acute gastroenteritis caused by many Salmonella serotypes is also associated with transient bacteraemia. In humans, S. choleraesuis often presents as a focal infection without any obvious GI manifestations. In persons with sickle cell anaemia, skeletal infection is common. S. choleraesuis and S. typhimurium gastroenteritis may be complicated by endocarditis, especially in older patients with plaque or aneurysm, or meningitis in patients under 2 years of age.
Carriers
About one-half of infected persons continue to excrete salmonellae 1 month after the symptoms have disappeared and 1 in 20 persons still do 5 months later. An unknown fraction of people become carriers after asymptomatic infection; the median carriage rate of Salmonella among healthy persons in developed countries is 0.13%.
Laboratory Diagnosis
Salmonella can be isolated on any of the common enteric media. Historically, rising titres of antibodies against Salmonella O and H Ags (Widal test) are used to diagnose typhoid. Methods have been developed for the detection of salmonella in foods.